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VOLUME 151, SUPPLEMENT 1
57th Pharmacological Days
(Czech and Slovak Pharmacological Meeting)
Olomouc, September 12–14, 2007
Scientific Committee
Pavel Anzenbacher, Chairman (Olomouc)
Vladimír Geršl (Hradec Králové)
Miloš Kršiak (Prague)
Jaroslav Květina (Hradec Králové)
František Perlík (Prague)
Jitka Ulrichová (Olomouc)
Karel Urbánek (Olomouc)
Organizing Committee
Vilím Šimánek, Chairman (Olomouc)
Pavel Anzenbacher (Olomouc)
Jitka Hýbnerová (Olomouc)
Jaroslav Matal (Olomouc)
Bohdana Řeháková (Olomouc)
Michal Šiller (Olomouc)
Rostislav Večeřa (Olomouc)
Kateřina Valentová (Olomouc)
PALACKÝ UNIVERSITY, OLOMOUC
2007
DEAR READER:
The Supplement 1 of Biomedical Papers, Vol. 151 (2007) contains contributions presented at the 57th Pharmacological
Days, in other words, at the 57th Czech and Slovak Pharmacological Meeting. This meeting takes place in Olomouc,
on September 12–14, 2007.
Palacky University at Olomouc is pleased to host this event which represents a traditional gathering of pharmacologists, toxicologists and scientists interested in fields overlapping with these two broadly defined fields of biomedical
sciences. It is also a good tradition that this meeting is a truly Czecho-Slovakian one with participants coming from
both sides of the friendly borders between Czech and Slovak Republics.
Olomouc is, in many aspects, an almost ideal place for meetings of scientific communities. It is a city with the
second oldest University in the Czech Republic (after Charles University of Prague), founded in 1573 which is also
known for their famous former students, e.g. Albrecht Eusebius Valdstejn (Wallenstein) or Gregor Mendel. Its location,
not much distant from Prague, Bratislava, Cracow, Vienna or Budapest makes Olomouc a lively city full of students
and scholars coming from all places of the world.
We believe that the 57th Pharmacological Days will offer a good opportunity for mutual exchange of ideas and for
meeting of friends and coworkers.
Vilím Šimánek
Chairman, Organizing Committee
Pavel Anzenbacher
Chairman, Scientific Committee
Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2007, 151 (Supplement 1)
3
THE INCREASED MYOCARDIAL COENZYME Q CONTENT
IN THE DIABETIC-HYPERCHOLESTEROLAEMIC RATS IS REDUCED
BY SIMVASTATIN THERAPY
Adriana Adameovaa, Zuzana Sumbalovab, Pavel Sveca, Jarmila Kucharskab,
Magdalena Kuzelovaa
a
Department of Pharmacology and Toxicology, Faculty of Pharmacy, Comenius University, Odbojarov 10, 832 32 Bratislava,
Slovak Republic
b
Pharmacobiochemical Laboratory, Faculty of Medicine, Comenius University, Hlboka 7, 811 05 Bratislava, Slovak
Republic
e-mail: [email protected]
Key words: Simvastatin/Coenzyme Q/Diabetes mellitus/Hypercholesterolaemia
BACKGROUND: Coenzyme Q, an endogenous lipid
soluble antioxidant, participates in a transfer of electrons
in mitochondrial respiratory chain resulting in a production of energy. Besides these important functions, coenzyme Q has also an essential role in the maintenance of
another powerful antioxidant such as tocopherol1. The
changed levels of coenzyme Q promoting number of both
neural and cardiovascular diseases have been postulated
in a presence of pathological conditions, namely diabetes
mellitus and hypercholesterolaemia1,2. Moreover, statin
therapy may also influence coenzyme Q content via an
inhibition of mevalonate production, a common precursor
Table 1. An influence of simvastatin on the levels of glucose, total cholesterol in plasma and the myocardial concentrations of alpha tocopherol and lipid peroxides measured as thiobarbituric acid reactive substances in the diabetichypercholesterolaemic rats.
Plasma
(mmol.L–1)
Group
GLU
C
Myocardium
(nmol.g–1)
TCHOL
11.2 ± 0.7
1.5 ± 0.1
aaa
DM-HCH
35.8 ± 1.1
DM-HCH+Simvastatin
15.0 ± 1.5 bbb
3.1 ± 0.2
aaa
2.5 ± 0.1
ALPHA TOC
TBARS
46.1 ± 4.1
77.3 ± 7.8
47.1 ± 5.2
75.2 ± 2.1
46.1 ± 3.7
68.8 ± 6.3
The results are expressed as means ± SEM for n = 5–8 measurements. aaaP < 0.01 diabetic-hypercholesterolaemic
animals vs. control rats; bbbP < 0.01 simvastatin-treated diabetic-hypercholesterolaemic animals vs. non- treated diabetichypercholesterolaemic rats
1B
CoQ9
1A
180
18
150
15
CoQ10
nmol/g
nmol/g
a
120
90
b
12
9
60
6
30
3
0
0
C
DM-HCH
DM-HCH +
Simvastatin
C
DM-HCH
DM-HCH +
Simvastatin
Fig. 1. The myocardial concentrations of coenzyme Q9 (1A) and Q10 (1B) after an administration of simvastatin in
the diabetic-hypercholesterolaemic rats. The results are expressed as means ± SEM for n = 5–8 measurements.
a
P < 0.05 diabetic-hypercholesterolaemic animals vs. control rats; bP < 0.05 simvastatin-treated diabetic-hypercholesterolaemic animals vs. non- treated diabetic-hypercholesterolaemic rats.
4
of cholesterol and coenzyme Q synthesis3. The goal of this
study was (1) to identify the levels of coenzyme Q10, Q9
(homologue present in rats) and alpha tocopherol levels
in the myocardium of the diabetic-hypercholesterolaemic
rats and (2) to find out an influence of simvastatin administered to the diabetic-hypercholesterolaemic rats on the
mentioned levels.
METHODS: The study was performed on male Wistar
rats, which were randomly divided in the following experimental group: control (C), diabetic-hypercholesterolaemic (DM-HCH) and diabetic-hypercholesterolaemic
rats treated by simvastatin (DM-HCH+Simvastatin).
A 10-day lasting pathological state of simultaneously occurring experimental diabetes and hypercholesteroleamia was induced by a single intraperitoneal application
of streptozotocin (80 mg/kg) and a fat-cholesterol diet
(20g/day). Simvastatin (10 mg/kg) was administered to
the diabetic-hypercholesterolaemic rats as a component
of the fat-cholesterol diet. Coenzyme Q9, (CoQ9), coenzyme Q10 (CoQ10) and alpha-tocopherol (alpha toc) were
determined in the myocardium by the method of highperformance liquid chromatography. The myocardial concentrations of lipid peroxides measured as thiobarbituric
acid reactive substances (TBARS) were determined by the
reaction with thiobarbituric acid spectrophotometrically
at 532 nm.
RESULTS: The development of 10-day experimental
diabetes and hypercholesterolaemia was confirmed by the
increased levels of glucose (GLU) and total cholesterol
levels (TCHOL) in the plasma. Simvastatin with a slight
influence on the plasma cholesterol levels, reduced significantly the plasma glucose concentrations (Table 1).
In the diabetic-hypercholesterolaemic rats, the myocardial coenzyme Q9 content had a tendency to be increased
(Fig. 1A) and the levels of CoQ10 in the myocardium were
significantly higher as compared with the control group
(Fig. 1B; P < 0,05). Simvastatin therapy in the diabetichypercholesterolaemic animals significantly reduced the
myocardial CoQ10 levels (P < 0.05) without a significant influence on coenzyme Q9 levels (Fig. 1A and B). The levels
of alpha tocopherol did not differ among all experimental
groups. On the other hand, the concentrations of lipid
peroxides, had a tendency to be decreased with simvastatin treatment (Table 1).
CONCLUSIONS: The increased content of coenzyme
Q in the myocardium of the animals with experimental
diabetes and hypercholesterolaemia indicates an acute
defence reaction to an oxidative stress induced by these
pathological conditions. Although simvastatin restored
the myocardial coenzyme Q content to the controls, lipoperoxidation was slighty reduced.
ACKNOWLEDGEMENT
Financial support by the grants UK19/2007, VEGA SR
1/4296/07 and 1/3442/06 is gratefully acknowledged.
REFERENCES
1. Turunen M, Olsson J, Dallner G. Metabolism and function of coenzyme Q. Biochim Biophys Acta 2004;1660:171–99.
2. Kucharska J, Braunova Z, Ulicna O, Zlatos L, Gvozdjakova A.
Deficit of coenzyme Q in heart and liver mitochondria of rats with
streptozotocin-induced diabetes. Physiol Res 2000; 49:411–8.
3. Hargreaves IP, Duncan AJ, Heales SJ, Land JM. The effect of
HMG-CoA reductase inhibitors on coenzyme Q10: possible biochemical/clinical implications. Drug Saf 2005; 28:659–76.
EXPRESSION OF CAVEOLIN-1 IN THE HEART OF RATS WITH
ISOPROTERENOL-INDUCED CARDIAC HYPERTROPHY
Zuzana Bajuszova, Peter Krenek, Jan Klimas, Miroslava Kroslakova, Jana Plandorova,
Dana Kucerova, Jan Kyselovic
Slovak Republic
Key words: Cardiac hypertrophy/Isoproterenol/Endothelial nitric oxide synthase/Caveolin-1
BACKGROUND: Myocardial hypertrophy is a risk factor for the development of arrhythmias, ischemic heart
disease and heart failure and regression of hypertrophy by
treatment is associated with a decrease of cardiovascular
risk1,2. Caveolins are structural proteins of the caveolae,
small invaginations of the plasma membrane enriched
in sphingolipids and cholesterol. Three isoforms of caveolins exist, caveolin-1 being expressed in endothelial
cells, smooth muscle cells and fibroblasts. The scaffolding domain of caveolin-1 mediates anchoring of many
proteins in the caveolae and has a regulatory function.
Certain components of signaling pathways relevant for
cardiac hypertrophy are localized in the caveolae, e.g. β1
and β2 adrenergic receptors, endothelin ETA receptors,
PDGF and EGF receptors, L-type of the calcium channel,
non-receptor tyrosine kinases components of the MAPK
pathway and others3. Inhibition of these cascades by caveolin-1 could in theory lead to a decreased production of
growth factors for cardiac myocytes. Caveolin-1 knockout
mice have cardiac hypertrophy associated with fibrosis
and hyperactivation of the MAPK pathway in cardiac
fibroblasts4. Caveolin-1 expression is decreased in some
5
Table 1. Biometrical data of isoproterenol-treated rats.
Control
Isoproterenol
% difference
Body weight (g)
327 ± 19
293 ± 26*
– 10
Left ventricle (g)
0.67 ± 0.05
0.89 ± 0.09*
+ 33
Right ventricle (g)
0.15 ± 0.03
0.21 ± 0.04*
+ 40
Left atrium (g)
0.03 ± 0.01
0.04 ± 0.01*
+ 34
Right atrium (g)
0.04 ± 0.01
0.05 ± 0.01*
+ 34
* P < 0.05 vs. Control
models of cardiac hypertrophy5,6. We decided to study the
expression of caveolin-1 in isoproterenol-induced cardiac
hypertrophy, which is a reliable model of pathological
cardiac hypertrophy associated with fibrosis7.
METHODS: We used male 16-week old Wistar rats.
To induce cardiac hypertrophy, we administered isoproterenol at the dose 5 mg/kg/day intraperitoneally for eight
days. Blood pressure and heart rate were measured by
the tail-cuff plethysmographical method. Rats were sacrificed using CO2 asphyxiation 24 hours after the last dose
of isoproterenol. The heart was dissected into atria and
ventricles and weighed. Protein homogenates were analyzed by immunoblotting analysis with antibodies against
caveolin-1 and actin, which was used as a standard.
RESULTS: Mortality in the isoproterenol group was
about 40 percent. After eight days of isoproterenol administration, basal systolic blood pressure and heart rate
were decreased (systolic blood pressure 110 ± 3 vs. 126
± 3 mmHg; heart rate 342 ± 8 vs. 366 ± 6 beats/minute,
both P < 0.05 vs Control). The weight of all parts of the
heart increased by about 30 % (P < 0.05 vs Control), see
Table 1. Caveolin-1 expression normalized to actin was
not changed by isoproterenol treatment in the atria, but
it tended to be decreased in the left ventricles (–10 %,
P = 0.065) and right ventricles (–14 %, P = 0.067).
CONCLUSIONS: We confirmed development of cardiac hypertrophy after isoproterenol administration in the
rat. We found a trend towards a decreased expression of
caveolin-1 in the cardiac ventricles, but not in the atria.
Further studies are necessary to determine whether the
decreased caveolin-1 expression would not be more pronounced if the rats were sacrificed within a shorter time
after isoproterenol administration.
ACKNOWLEDGEMENT
Financial support by the grants UK/26/2007 and FaF
UK/22/2007 is gratefully acknowledged.
REFERENCES
1. Kannel WB. Left ventricular hypertrophy as a risk factor: the
Framingham experience. J Hypertens Suppl 1991; 9:S3–8; discussion S8–9.
2. Verdecchia P, Angeli F, Borgioni C, Gattobigio R, de Simone G,
Devereux RB, Porcellati C. Changes in cardiovascular risk by reduction of left ventricular mass in hypertension: a meta-analysis. Am
J Hypertens 2003; 16:895–9.
3. Razani B, Woodman S, Lisanti MP. Caveolae: From cell biology
to animal physiology. Pharmacol Rev 2002; 54:431–67.
4. Cohen AW, Park DS, Woodman SE, Williams TM, Chandra M,
Shirani J, Souza AP, Kitsis RN, Russell RG, Weiss LM, Tang
B, Jelicks LA, Factor SM, Shtutin V, Tanowitz HB, Lisanti MP.
Caveolin-1 null mice develop cardiac hypertrophy with hyperactivation of p42/44 MAP kinase in cardiac fibroblasts. Am J Physiol
Cell Physiol 2003; 84:C457–74.
5. Piech A, Massart PE, Dessy C, Feron O, Havaux X, Morel N,
Vanoverschelde JL, Donckier J, Balligand JL. Decreased expression of myocardial eNOS and caveolin in dogs with hypertrophic
cardiomyopathy. Am J Physiol Heart Circ Physiol 2002; 282:
H219–31.
6. Piech A, Dessy C, Havaux X, Feron O, Balligand JL. Differential
regulation of nitric oxide synthases and their allosteric regulators
in heart and vessels of hypertensive rats. Cardiovasc Res 2003;
57:456–67.
7. Szabo J, Csaky L, Szegi J. Experimental cardiac hypertrophy induced by isoproterenol in the rat. Acta Physiol Acad Sci Hung
1975; 46:281–5.
6
RAPID LOSS OF VASCULOPROTECTIVE EFFECT FOLLOWING
THE WITHDRAWAL OF CALCIUM CHANNEL BLOCKER NIFEDIPINE
IN SPONTANEOUSLY HYPERTENSIVE RATS
Zuzana Bajuszova, Vaclav Vaja, Peter Ochodnicky, Peter Krenek, Jan Klimas, Jan Kyselovic
Department of Pharmacology and Toxicology, Faculty of Pharmacy, Comenius University, Bratislava, Slovak Republic
Key words: Vascular remodelling/Hypertension/Endothelial dysfunction/Calcium channel blockers/Nifedipine
BACKGROUND: In addition to potent antihypertensive effect, long-term therapy with calcium channel blockers (CCBs) is associated with morphologic and functional
beneficial changes in peripheral vasculature both in humans and experimental animals1,2. Reported reversal of
vascular remodeling include reduction in wall or media
thickness, increase in lumen diameter2,3 or improvement of
endothelium-dependent relaxation4. However, much less
is known about the kinetics of these vascular changes and
their potential duration after withdrawal of these drugs,
a problem frequently encountered in a clinical setting. In
the present study we hypothetised that even short-term
administration of CCBs might reduce both functional and
morphological vascular remodeling present in a model of
Table 1. Systolic blood pressure and morphometric parameters of the aorta isolated from either control or experimental animals treated with indicated CCBs for 7 days.
WKY
SHR
SHR
diltiazem
SHR
nifedipine
SHR
verapamil
122 ± 2
196 ± 4*
168 ± 8#
158 ± 4#
196 ± 7#
73.0 ± 0.6
94.5 ± 1.0*
84.1 ± 1.5#
85.6 ± 0.6#
83.9 ± 0.5#
0.42 ± 0.01
0.52 ± 0.01*
0.47 ± 0.01#
0.46 ± 0.01#
0.49 ± 0.01
43.1 ± 1.1
56.3 ± 1.8*
49.5 ± 0.6#
49.5 ± 1.4#
49.7 ± 1.1#
Parameter
Systolic BP
(mmHg)
Wall thickness
(μm)
Cross-sectional area
(mm2)
Media/lumen ratio
(μm/mm)
BP – blood pressure, average ± SEM, n = 6 per group, *p < 0.05 vs WKY, # p < 0,05 vs SHR
130
Wall thickness (μm)
*
¥
110
#
¥
#
90
70
50
WKY
SHR
SHR+NIF
SHR+NIF
after 24h
SHR+NIF
after 72h
SHR+NIF
after 120h
Fig. 1. Rapid disappearance of short-term nifedipine-induced reduction in aortic wall thickness after withdrawal of
the drug. SHR+NIF- SHR rats treated 7 days with nifedipine (1 mg/kg, s.c. twice daily), SHR+NIF after 24 h,
72h, 120h – SHR rats treated with nifedipine and sacrificed after 24, 72, 120 hours, respectively, following the
last dose of nifedipine, *p < 0.05 vs WKY, # p < 0,05 vs SHR, ¥ p < 0,05 vs SHR+NIF.
spontaneous hypertension, a spontaneously hypertensive
rat (SHR). To this end we analyzed the vasculoprotective
effect of three CCBs differing in selectivity to vascular/
cardiac calcium channels in aorta of SHR. Additionally,
we studied potential presence of these changes after withdrawal of short-term administration of vasculoselective
CCB, nifedipine.
METHODS: The morphometrical (wall thickness,
cross-sectional area, media-to-lumen ratio) and functional
(KCl- or noradrenaline-induced contraction, endothelium-dependent acetylcholine-induced relaxation) characteristics of isolated aorta were measured in 14 week-old
control WKY rats, SHR and SHR rats treated either with
diltiazem (5 mg/kg) or nifedipine (1 mg/kg) or verapamil
(4 mg/kg) twice daily by subcutaneous injection for 7 days
(n = 6 each group). Systolic blood pressure was monitored
daily by using non-invasive tail-cuff method. In the nifedipine arm, the animals were additionally sacrificed 24, 72
and 120 hours following the last dose of nifedipine.
RESULTS: Elevated systolic blood pressure in SHRs
was significantly reduced by 7-day administration of
nifedipine and diltiazem, but not verapamil (Table 1).
However, the regression of vascular hypertrophy, as evidenced by decrease in aortic wall thickness, cross-sectional area and media/lumen ratio was observed in all
three treated groups (Table 1). Similarly, premedication
of SHR with all three CCB improved endothelial dysfunction observed in SHR, the effect being most pronounced
in nifedipine-treated animals. Three days after withdrawal of nifedipine morphologic parameters, such as aortic
wall thickness (Fig. 1) returned to pre-treatment values.
7
Similarly, benefitial effect of nifedipine on endothelial
function displayed similar pattern after withdrawal.
CONCLUSIONS: Short-term vasculoprotective effect
of CCB against hypertensive remodelling does not seem to
depend on their vascular/cardiac channel selectivity and
is not necessarily associated with blood pressure reduction. Neverthless, vasculoselective CCB nifedipine might
provide the most pronounced benefit, which is however
maintained only for a short period following the withdrawal of the drug. Present results show that morphological
and functional changes induced by CCB are more rapid
than previously thought and even a very short withdrawal
might reactivate hypertensive vascular remodelling.
ACKNOWLEDGEMENT
Financial support by the grant of Comenius University
2006 is gratefully acknowledged.
REFERENCES
1. Schiffrin EL. Structure and function of small arteries of essential
hypertensive patients following chronic treatment with once-a-day
nifedipine. Cardiology 1997; 88 Suppl 3:20–6.
2. Saleh FH, Jurjus AR. A comparative study of morphological changes in spontaneously hypertensive rats and normotensive Wistar
Kyoto rats treated with an angiotensin-converting enzyme inhibitor
or a calcium-channel blocker. J Pathol 2001; 193:415–20.
3. Li JS, Schiffrin EL. Effect of calcium channel blockade or angiotensin-converting enzyme inhibition on structure of coronary,
renal, and other small arteries in spontaneously hypertensive rats.
J Cardiovasc Pharmacol 1996; 28:68–74.
4. Krenek P, Salomone S, Kyselovic J, Wibo M, Morel N, Godfraind
T. Lacidipine prevents endothelial dysfunction in salt-loaded strokeprone hypertensive rats. Hypertension 2001; 37:1124–28.
EFFECTS OF PRENYLATED ISOFLAVONES OSAJIN AND POMIFERIN IN THE
CONDITIONS OF ALLOXAN INDUCED DIABETES MELLITUS (PILOT STUDY)
Lenka Bartosikovaa, Jiri Necasa, Tomas Bartosikb, Martin Pavlikb, Galina Kuzminaa, Jiri Luzaa
a
Department of Physiology, Faculty of Medicine and Dentistry, Palacky University, Hnevotinska 3, 775 15 Olomouc, Czech
Republic
b
Department of Anaesthesiology and Intensive Care, St. Anne’s University Hospital Brno, Pekarska 53, 656 91 Brno, Czech
Republic
Key words: Prenylated isoflavones/Diabetes mellitus/Antioxidants
BACKGROUND: Osajin and pomiferin belong to the
group of prenylated isoflavones. The compounds were
isolated from infructences of Maclura pomifera (Raf.)
Schneid. (Moraceae) and their purity were HPLC-proved1.
The aim of the study was to monitor antidiabetic and
antioxidative effect of prenylated isoflavones osajin and
pomiferin in the conditions of alloxan induced diabetes
mellitus in experiment in vivo.
METHODS: The animals were divided by random
selection into 3 groups (n =7). The treated groups were
given osajin or pomiferin via intragastric sond in doses of
10 mg/kg in Avicel, the placebo diabetic group was given
only the solution of Avicel. The last group was intact.
Selected laboratory parameters (glucose, urea, cholesterol, antioxidative enzymes, total antioxidative capacity,
malondialdehyde in serum; diuresis, total glucose and
protein losses through urine) were determined in all animals. Kidney tissue and pancreas samples were taken for
histopathological analysis.
RESULTS: A statistically significant decrease of blood
glucose level in both treated groups compared to the placebo groups was found. An increase of the glutathione
8
Table 1a. Monitored biochemical parameters expressed as X ± SD.
Blood glucose
(mmol.L–1)
SOD
[U.mL–1]
GSHPx
[μkat.L–1]
AOC
[mmol.L–1]
MDA
[mmol.L–1]
Treated group pomiferin
8.62 ± 1.43
**
207.15 ± 10.31
1245.13 ± 89.31
**
0.85 ± 0.05
*
1.29 ± 0.49
**
Treated group osajin
9.12 ± 0.93
••
202.10 ± 5.64
1193.20 ± 94.06
•
0.94 ± 0.05
••
1.28 ± 0.49
••
Placebo group
20.35 ± 0.45
204.05 ± 8.74
1079.32 ± 79.91
0.77 ± 0.08
3.40 ± 0.93
4.92 ± 0.72
58.82 ± 2.76
1821.60 ± 55.07
1.20 ± 0.06
1.01 ± 0.05
Intact group
Table 1b. Monitored biochemical parameters expressed as X ± SD.
Diuresis
(mL/day)
Urine glucose
(mmol.L–1)
Protein losses through
ureine (g.L–1l)
Treated group pomiferin
14.50 ± 0.35
**
1.17 ± 0.45
**
0.83 ± 0.37
**
Treated group osajin
16.50 ± 0.25
••
2.19 ± 0.45
••
0.93 ± 0.37
••
Placebo group
27.65 ± 1.50
3.49 ± 0.63
1.36 ± 0.35
Intact group
12.45 ± 0.45
1.13 ± 0.54
0.43 ± 0.16
** p ≤ 0,01 treated with pomiferin vs placebo
* p ≤ 0,05 treated with pomiferin vs placebo
peroxidase catalytic activity, total antioxidative capacity
and a decrease of malondialdehyde level in the treated
groups compared to the placebo group were statistically
significant (see Table 1a). A statistically highly significant
decrease of diuresis, glucose and protein losses through
urine was identified in the treated groups compared to the
placebo group (see Table 1b). The superoxide dismutase
catalytic activity, urea and cholesterol levels involved nonsignificant changes. Histopathological changes of kidney
tissue and pancreas were evaluated as minimum and nonsignificant in both treated groups and in placebo group
too.
CONCLUSIONS: The results of biochemical examination show antidiabetic and antioxidative effects of
prenylated isoflavones osajin and pomiferin. The results
•• p ≤ 0,01 treated with osajin vs placebo
• p ≤ 0,05 treated with osajin vs placebo
of histopathological examination correlate with them
partially only. The changes in kidneys that can be evaluated histopathologically depend on the duration of diabetes and on the fact if and how diabetes was treated.
Histopathological changes in pancreas are not usually
microscopically symptomatic and findings are not consistent2.
REFERENCES
1. Janostikova E, Bartosikova L, Necas J, Jurica J, Florian T, Bartosik
T, Klusakova J, Suchy V, Liskova M, Frydrych M. Effects of pomiferin premedication on the antioxidant status of rats with ischemiareperfused kidney. Acta Vet Czech 2005; 74:557–64.
2. Rosai J. Ackerman’s Surgical Pathology. Mosby: St. Louis; 1996.
p. 2732.
9
IN VIVO MODEL OF TACHYCARDIA – COMPARISON OF BRADYCARDIC EFFECT
BETWEEN ENANTIOMERS OF NEWLY SYNTHESIZED ULTRASHORT-ACTING
BETA-BLOCKERS
Ladislava Bartosovaa, Katerina Holanovaa, Klara Horkaa, Marek Frydrycha, Vera Strnadovaa,
Radka Opatrilovab, Petr Mokryb, Pavel Suchya
a
Department of Human Pharmacology and Toxicology, and
Department of Chemical Drugs, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences (UVPS),
Palackeho 1-3, Brno, Czech Republic
b
Key words: Ultrashort-acting betalytics/Bradycardic effect/Experiment in vivo/Model of tachycardia/Enantiomers
BACKGROUND: Ultrashort-acting beta blocking
agents still form only a small fraction of a large group of
beta-adrenolytics. In the Czech Republic, the only registered drug with ultrashort-acting beta blocker is Brevibloc
with esmolol hydrochloride1. The tested compounds are
originals that have been made by the Department of
Chemical Drugs at the Faculty of Pharmacy of the UVPS
Brno. They are functional analogues of aryloxy aminopropanols with carbamate substitution of the aromatic core
and an ester bond incorporated into the linking chain.
They are modified in the aliphatic section of the molecule
by a linear alkyl chain – the 44Bu compound by the n-butyl
and the 444 compound by the tertiary butyl2. On the basis
of chemical structure and pilot pharmacokinetic study
444 and 44Bu compounds may be assumed to have a betablocking rapid action3. Enantiomers usually take part in
the final pharmacological as well as adverse effects of the
potential drug to a different degree, because different 3D
molecule structure results in the different affinity to drug
targets. The aim of the study was to evaluate bradycardic
Table 1. Statistical evaluation of heart rate changes.
Time
(min)
COMPARISON
44Bu x 444
Rac
S
0
Against control group
R
*
Rac
Rac
S
S
R
R
44Bu
444
44Bu
444
44Bu
444
*
**
*
0.5
*
**
**
**
1
*
**
**
*
2
*
*
*
*
3
*
*
*
*
4
**
**
**
5
**
*
*
6
**
*
**
*
8
**
*
**
*
10
**
*
**
*
12
**
*
*
*
14
*
*
*
*
16
*
*
*
*
18
*
*
**
*
20
**
*
**
**
25
**
*
**
**
0 – the time of i.v. administration of tested substance; * p < 0,05; **p < 0,01
**
Percentage of heart rate change (%)
Percentage of heart rate change (%)
10
Racemate
R-isomer
S-isomer
Control
Fig. 1. Time line of heart rate changes (in %) following administration of tested compounds.
effect of R- and S- enantiomers 44Bu and 444 compounds
on a model of isoprenaline-induced tachycardia.
METHODS: Experiment was proven in vivo on 59
laboratory rats. Both substances were tested in dose
1.5 mg/kg. The control group was administered vehiculum
(saline). Methodology of the experiment was approved
and monitored by the local Ethical Committee for Animal
Welfare at the UVPS Brno. The general anaesthesia was
induced by the administration of 1% ketamin (Narkamon®
inj. Spofa) and 2% xylazin (Rometar® inj. Spofa), the
model of tachycardia induced by subcutaneous administration of 4 μg/kg isoprenaline (Isuprel® inj. Abbott).
The tested compounds were administered i.v. to the vena
jugularis as a bolus dose. The heart rate was monitored
with ECG SEIVA Praktik Veterinary. The first measurement was made at the following the onset of general anaesthesia, the second measurement 10 min after the s.c.
administration of isoprenaline. That value corresponded
to the initial level of tachycardia (100 %). Student’s unpaired t-test and Mann-Whitney test for confirmation were
used to determine the statistical significance heart rate
changes. (Unistat 5.1).
RESULTS: A graphical representation of the results
– heart rate changes along the time line is shown in Fig. 1.
Results of statistical analysis are given in Table 1.
CONCLUSIONS: After administration of S-enantiomer 444, the heart rate decreases significantly more
than after administration R-enantiomer 444. Bradycardic
effect of 444 racemate gives rise to S-enantiomer effect
in the first instance. There are no significant differences
between bradycardic effects of both enantiomers 44Bu for
all that the racemates of both compounds 44Bu and 444
displayed the same bradycardic effect. Bradycardic effect
is given either by blockade of β-receptors or by inhibiton
of ion membrane currents (prolongation of PQ interval)
or by both way. Although 44Bu and 444 compounds are
structurally similar, it seems, that their bradycardic effect
is given by different rate of these ways.
AKNOWLEDGEMENTS
This study was supported by grants GA CR
No. 305/06/0863 and IGA MZ CR No. NR9126-3/2006.
11
REFERENCES
1. Wiest D. Esmolol. A review of its therapeutic efficacy and pharmacokinetic characteristics. Clin Pharmacokinet 1995; 28:190.
2. Mokry P, Zemanova M, Csollei J, Racanska E, Tumova I. Synthesis
and pharmacological evaluation of novel potential ultrashort-acting
β-blockers. Pharmazie 2003; 58:18–21.
3. Cevelova V, Bartosova L, Opatrilova R. Sledování plazmatických
koncentrací ultrakrátkých betalytik, Student’s scientific conference,
Faculty of Pharmacy, UPVS Brno; 2007.
STRUCTURE-FUNCTION STUDIES OF DIMERIC METABOTROPIC
GLUTAMATE RECEPTOR
Jaroslav Blahosa,b, Jean-Philippe Pinb, Veronika Hlavackovaa, Laurent Prezeaub
a
Department of Pharmacology, 2nd Medical School of Charles University in Prague, Czech Republic
Department of Molecular Pharmacology, Institute of Molecular Genetics, Czech Academy of Science, Prague, Czech
Republic
c
Department of Molecular Pharmacology, Laboratory of Functional Genomic, CNRS Unite Propre de Recherche 2580,
Montpellier, France
b
Key words: Glutamate receptor/Dimerization/G-protein coupling/Allosteric modulators
BACKGROUND: The metabotropic glutamate (mGlu)
receptors are G-protein-coupled receptors (GPCRs) that
exist and function as homodimers composed of two identical subunits covalently bound in the extracellular domain.
The relevance of dimerization of these receptors in respect
to activation of the transmembrane heptahelical domain
(HD) of each subunit upon full receptor activation is of
particular interest.
METHODS: We constructed several chimerical receptors in which quality control of GABAb receptor
heterodimerization and cell-surface expression was used.
This allowed us to measure G-protein coupling efficacy of
dimeric mGlu receptors in which only one subunit bears
specific mutations.
RESULTS: Single mutation in the third intracellular
loop is known to fully prevent G-protein activation when
present in both subunits. We show, that when present
only in a single subunit per dimer, this mutation decreases
coupling efficacy. In contrast when a single HD has engineered binding site for allosteric inhibitor and is blocked
in its inactive state by such compound, no decrease in
receptor activity is observed. In a receptor dimer in which
the same subunit binds the negative modulator and is also
mutated in its i3 loop, the compound enhances agonistinduced activity. We propose, that this phenomenon reflects a ‘better’ activation of the adjacent HD. Our data
are in agreement with a model in which a single HD is
turned on upon activation of homodimeric receptors.
Complementary with these results are data obtained using positive allosteric modulators in experiments with
similar settings. Here the most interesting finding is that
occupation of a single HD is enough for full enhancement effect.
CONCLUSIONS: Situation of asymmetric functioning of mGlu receptors corresponds to findings on GABAb
receptor activation process.
ACKNOWLEDGEMENT
This work was supported by Grant Agency of the Czech
Republic (204/05/0920), and Projet EUREKA E! 3302.
12
ANALYSIS OF ANTIGLAUCOMATICS AND COMPARISON OF THE EFFECTS
OF PROSTAGLANDIN ANALOGS IN ELDERLY PATIENTS
Renata Blechova
Department of Pharmacology and Toxicology, Faculty of Pharmacy, VFU Brno, 612 42 Brno, Czech Republic
e-mail: blechovar @vfu.cz
Key words: Glaucoma/Intraocular pressure/Prostaglandin analogs/Elderly patients
BACKGROUND: Glaucoma is an optic neuropathy
associated with retinal ganglion cell death that results in
visual field loss. Elevated intraocular pressure (IOP) is
a primary risk factor for the disease and a prime target for
therapy1. Glaucoma is much more common among older
people. One is six times more likely to get glaucoma when
over 60 years old2. The options in the management of glaucoma have significantly expanded in the last decade. To
the early therapy staples of pilocarpine and timolol have
been added drugs in three classes of topical medication:
alfa agonists, carbonic anhydrase inhibitors and, perhaps
most importantly, prostaglandin analogs. In view of all
risk factors possibly influencing the prognosis of glaucoma, it is clear that the treatment should be prescribed
on an individual basis with respect to life quality3.
METHODS AND RESULTS: Observational study,
prospective data audit: Altogether 159 patients with
a diagnosis of ocular hypertension or chronic glaucoma.
Part A is composed of 127 patients by analyzing trends
in glaucoma medication. Patients are ranged in age of
69.5 ± 7.4. This group is determinated to actual frequency of prescriptions in effective topical medications and
to check how guidelines are applicated in conditions of
common clinical practice. Part B is composed of 32 newly
diagnosed patients, 20 females and 12 males. Patients are
ranged in age of 65.4 ± 5.0. and were instructed to instill
one drop medication in each eye in the evening (between
7 PM and 9 PM). Our previously reported 6-month study
compares three prostaglandin analogs. Patients received
bimatoprost (n = 12) or latanoprost (n = 14) or travoprost (n = 6) once daily in the evening for 6 months. Visit
were at prestudy, baseline (day 0), week 1, and months 1,
3, and 6. Analysis of prescriptions (Part A) included 51
% of Prostaglandin analogs, 45 % of Beta- blockers, 3 %
of topical Carbonic anhydrase inhibitors, 1 % of topical
alfa 2 – Selective adrenergic agonists. The study (Part
B) compares the hypotensive lipids at the current time,
however, the preponderance of evidence indicates that
bimatoprost provides greater IOP (intraocular pressure)
lowering than latanoprost and travoprost. There was no
statistically significant difference in change of visual acuity between three treatment groups. Conjunctival hyperemia, eyelash growth and itching were significantly more
common in patients treated with bimatoprost than with
latanoprost and travoprost.
CONCLUSIONS: A potent topical prostaglandin analogs reduce baseline ocular tensions by an average of 25 %
(range 15 % to 30 %). Sign of ocular discomfort and adverse reactions occur significantly more frequently with bimatoprost. Thus systemic side effects are unlikely present
in monotherapy prostaglandins that should be considered
as a therapy of choice for patients with contraindications
in beta-blockers or who cannot tolerate medications which
alter pupil function.
REFERENCES
1. The AGIS Investigators. The advanced glaucoma intervention
study (AGIS) 7: the relationship between control of intraocular
pressure and visual field deterioration. Am J Ophthalmol 2000;
130:429–40.
2. Flammer J, Gasser P, Prünte Ch, Yao K. The probable involvement
of factors other than intraocular pressure in the pathogenesis of
glaucoma. Pharmacology of Glaucoma. Baltimore: William and
Wilkins; 1992. p. 273–83.
3. Goldberg I. Relationship between intraocular pressure and preservation of visual field in glaucoma. Surv Ophthalmol 2003; 48:3–7.
13
DOSE-DEPENDENCE STUDY OF A NOVEL IRON CHELATOR PCTH IN A MODEL
OF CATECHOLAMINE CARDIOTOXICITY
Zuzana Bobrovovaa, Radomir Hrdinaa, Premysl Mladenkaa, Mojmir Hubla, Jaroslava Vavrovab,
Magdalena Holeckovab, Vladimir Palickab
a
Department of Pharmacology and Toxicology, Charles University in Prague, Faculty of Pharmacy in Hradec Kralove,
Czech Republic
b
Institute of Clinical Biochemistry and Diagnostics, Charles University in Prague, Faculty of Medicine in Hradec Kralove,
Czech Republic
Key words: Cardioprotective effect/2-Pyridylcarboxaldehyde-2-thiophenecarboxyl hydrazone/Myocardial infarction/Reactive
oxygen species
BACKGROUND: Coronary heart disease (CHD) is
a significant health problem in developed countries1. It
is well known that production of reactive oxygen species
catalyzed by ferrous atoms plays important role in the
pathogenesis of CHD (ref.2). CHD is accompanied by
elevated levels of catecholamines which can generate ROS
as well3. A synthetic catecholamine isoprenaline (ISO) is
frequently used in an experimental model of myocardial
impairment for testing potential cardioprotective drugs4.
Based on the pathogenesis, iron chelators may be useful
as myocardium protective drugs. 2-Pyridylcarboxaldehyde2-thiophenecarboxyl hydrazone (PCTH) is a novel iron
chelator with documented effectivity in vitro5. The goal of
this study was to evaluate the possible protective effect of
PCTH in the model of acute catecholamine cardiotoxicity
in rats and to establish dose-response relationship.
METHODS: Male Wistar rats, average body weight
360 g, were used in this study at standard laboratory conditions and under the supervision of the Ethical Committee
of the Charles University in Prague, Faculty of Pharmacy
in Hradec Králové (Czech Republic).
Animals were randomly divided into 6 groups: control group (KP, 7 animals) – solvent only (propyleneglycol solution 3 ml.kg–1 i.v.); isoprenaline group (KPI,
16 animals) – propyleneglycol i.v. 5 minutes before 100
mg.kg–1 s.c. isoprenaline (Sigma-Aldrich, USA); PCTH
groups: P20 (7 animals), (P10, 6 animals) – received either 20.4 mg.kg–1 or 10.2 mg.kg–1 i.v. PCTH in 20% propyleneglycol solution; groups of combination PCTH and
ISO – PI20 (7 animals), PI10 (6 animals) – rats received
PCTH 20.4 and 10.2 mg.kg–1, respectively, before application of isoprenaline (in the same dose as KPI group).
24 hours after drug(s) administration, animals were anesthetized with urethane (1.2 g.kg–1 i.p.; Sigma-Aldrich,
USA). Functional variables were measured by use of the
apparatus Cardiosys® (Experimentria Ltd, Hungary) with
software Cardiosys V 1.1. At the end of experiment blood
was withdrawn from the abdominal aorta. Serum cTnT
was determined by electrochemiluniscence immunoassay
(Elecsys 2010, Roche Diagnostics). Data are expressed
as means ± SEM. Groups were compared by two tailed
T-test, Pearson correlation analysis was used to describe
relation between variables using GraphPad Prism version 4.00 for Windows, GraphPad Software, (San Diego,
California, USA). Statistical significance: p ≤ 0.05.
RESULTS: ISO caused 31% mortality. The higher dose
of PCTH totally hindered the mortality however the lower
dose did not show any prevention. In the control and
chelators groups no mortality was observed. In agreement
with mortality rate, superior dose of PCTH significantly
reduced increases in cardiac troponin T (cTnT) and wet
ventricles weight index caused by ISO while low dose of
PCTH did not inhibit either. Similarly, in the control and
chelators groups, only negligible concentrations of cTnT
were measured and wet ventricles weight indices in these
groups were not statistically different. The results concerning haemodynamic parameters are somehow controversial
and no statistical difference between the control and ISO
group was found neither in double product nor in cardiac
index. Especially, the raise of cardiac index in P10 group
has to be further examinated.
CONCLUSIONS: It is evident that higher concentration of PCTH is more protective but due to some controversial results, particularly related to heart haemodynamic
function, new studies will be necessary for better elucidation of dose-response of PCTH.
REFERENCES
1. Janero DR, Hreniuk D, Sharif HM. Hydrogen peroxide-induced
oxidative stress to the mammalian heart-muscle cell (cardiomyocyte): lethal peroxidative membrane injury. J Cell Physiol 1991;
149:347–64.
2. Rona G. Catecholamine cardiotoxicity. J Mol Cell Cardiol 1985;
17:291–306.
3. Rump AF, Klaus W. Evidence for norepinephrine cardiotoxicity
mediated by superoxide anion radicals in isolated rabbit hearts.
Naunyn Schmiedebergs Arch Pharmacol 1994; 349:295–300.
4. Verheugt FWA. Unstable coronary syndromes. Early phase of
acute myocardial infarction. In: Crawford MH, DiMarco JP, eds.
Cardiology. London: Mosby 2001.
5. Wong CS, Kwok JC, Richardson DR. PCTH: a novel orally active
chelator of the aroylhydrazone class that induces iron excretion
from mice. Biochim Biophys Acta. 2004 Dec 24; 1739:70–80.
14
RETINOIDS, REXINOIDS AND THEIR COGNATE NUCLEAR RECEPTORS:
CHARACTER AND THEIR ROLE IN THERAPY
OF SELECTED MALIGNANT DISEASES
Julius Brtko
Institute of Experimental Endocrinology, Slovak Academy of Sciences, Vlarska 3, 833 06 Bratislava, Slovak Republic
Key words: Retinoids/Rexinoids/Nuclear receptors/Therapeutic effects
BACKGROUND: Retinoids, rexinoids and their biologically active derivatives are involved in a complex arrangement of physiological and developmental responses
in many tissues of higher vertebrates. Both retinoids and
rexinoids are either natural or synthetic compounds related to retinoic acids that act through interaction with
two basic types of nuclear receptors: retinoic acid receptors (RARα, RARβ and RARγ) and retinoid X receptors
(RXRα, RXRβ and RXRγ) as retinoid-inducible transcription factors.
RESULTS: Thus, the retinoid receptors are considered to be ligand-activated, DNA-binding, trans-acting,
transcription-modulating proteins involved in a general
molecular mechanism responsible for transcriptional re-
sponses in target genes. They exert both beneficial and
detrimental activity; they have tumour-suppressive activity but on the other hand they are teratogenic. Retinoids
inhibit carcinogenesis, suppress premalignant epithelial
lesions and tumour growth and invasion in a variety of
tissues.
CONCLUSIONS: Natural and synthetic retinoids
have therapeutical effects due to their antiproliferative
and apoptosis-inducing effects. They are known to cause
redifferentiation or to prevent further dedifferentiation of
various neoplastic tissues.
ACKNOWLEDGEMENT
Supported in part by the grant of VEGA No. 2/5017/5.
INFLUENCE OF AZITHROMYCIN ON HEPATOBILIARY AND RENAL EXCRETION
OF METHOTREXATE IN RATS
Jolana Cermanovaa, Stanislav Micudaa, Eva Brcakovaa, Leos Fuksaa, Milos Hrocha,
Jaroslav Chladeka Jitka Hajkovaa, Halka Lotkovab, Jirina Martinkovaa, Zuzana Cervinkovab,
Frantisek Staudc
a
Department of Pharmacology, and
Department of Physiology, Charles University in Prague, Faculty of Medicine in Hradec Kralove, Czech Republic
c
Czech Republic
b
Key words: Azithromycin/Methotrexate/Mrp2
BACKGROUND: Azithromycin (AZT), a 15-ring
member macrolide antibiotic, is eliminated from the organism mainly in unchanged form via biliary excretion
and intestinal secretion, whereas urinary excretion is the
minor elimination route in humans1. The active excretion
of AZT in these organs is mediated by multidrug resistance-associated protein 2 (Mrp2) and P-glycoprotein. AZT
has consequently been shown to produce inhibition of the
hepatobiliary excretion of drugs that are substrates for these
transporters2. Methotrexate (MTX) is an antifolate drug,
whose clinical application is hampered by potentially toxic
drug-drug interactions with many commonly used drugs,
e.g., nonsteroidal anti-inflammatory drugs. Main mecha-
nism of these interactions is the inhibition of MTX elimination through blockade of transport proteins for organic
anions in the liver and kidney. Within these interactions,
inhibition of Mrp2 has been shown to produce altered
biliary excretion of MTX (ref.3) with significant changes
in its plasma concentrations. The aim of the present study
was to investigate potential Mrp2-mediated influence of
azithromycin on the excretion of MTX. Potency of AZT
to inhibit hepatocellular efflux of MTX was tested in vitro
using primary rat hepatocytes. Thereafter, in vivo clearance
study was performed in rats where influence of AZT on
pharmacokinetics of MTX was tested during steady-state
of MTX plasma concentrations.
METHODS: Hepatocytes were isolated from male
Wistar rats (270–330 g) using a collagenase perfusion
technique and plated onto culture dishes. Efflux experiments were performed 24 h after plating the cells. For
MTX efflux studies, cells were rinsed twice with 2.0 mL
of warm standard HBSS and incubated in 2.0 mL of the
same buffer for 10 min at 37 °C. Cells were then incubated
in 2.0 mL of 10 μM MTX dissolved in standard HBSS for
30 min and subsequently washed four times with 2.0 ml
of ice-cold standard HBSS to remove extracellular MTX.
Hepatocytes then were incubated with either standard or
Ca2+-free HBSS for 30 min. At the end of the incubation
period, a sample was taken from the extracellular medium
to determine efflux by measuring MTX concentrations.
Male Wistar rats (n = 4 in each group) weighing 270
to 330 g were obtained from BioTest Ltd. (Konarovice,
Czech Republic). Rats under sodium pentobarbital anesthesia (50 mg/kg) received bolus of MTX (10 mg/kg) into
the right jugular vein which was followed by a constant
infusion of MTX solution (0.65 mM in 2.5% mannitol)
at a rate of 15 ml/h.kg–1 to attain steady-state plasma concentration (Css). After a 60-min infusion, bile and urine
samples were collected at 20-min interval for 40 min. After
a 100-min infusion, AZT (40 mg/kg) or isotonic saline
was administered intravenously. Bile and urine samples
were thereafter collected at 20-min intervals from 160 to
220 min. Blood samples were collected at the midpoints
of the bile collection periods. Plasma was obtained by
centrifugations of blood samples.
The concentrations of AZT and MTX in plasma, urine
and bile were determined by previously described high
performance liquid chromatography (HPLC) method4.
Concentrations of creatinine in plasma, and urine were
measured on Cobas Integra® 800 (Roche Diagnostics,
MTX concentration in efflux
medium (μM)
15
25
20
15
10
IC50 = 69.7 μM
= 52.2 mg/L
5
0
-3
-2
-1
0
1
2
3
4
5
Azithromycin (μM)
Fig. 1. Influence of azithromycin on methotrexate efflux
from primary rat hepatocytes.
Mannheim, Germany) according to manufacturer’s instructions.
Total plasma clearance (CLTot) of MTX was estimated by dividing the constant infusion rate of MTX by the
steady-state concentration in plasma (Css). Biliary and
renal clearance (CLBile and CLR) of MTX during each
collection period was calculated by dividing the respective excretion rate by Css determined for that collection
period. Glomerular filtration rate (GFR) was evaluated
as clearance of endogenous creatinine.
RESULTS: Fig. 1 shows the azithromycin induced inhibition of the MTX efflux from isolated rat hepatocytes
in a concentration-dependent manner. The IC50 value was
52.2 mg.L–1 (69.7 μM). Table 1 summarizes the effects
of AZT (40 mg/kg) on the biliary and renal excretion
of MTX under steady-state conditions obtained by the
Table 1. Renal and hepatic elimination of MTX in control and AZT (40 mg/kg) administered rats.
Control
AZT
Urine flow rate (μl/min)
13.2 ± 1.5
10.6 ± 1.2
Bile flow rate (μl/min)
16.1 ± 0.7
17.0 ± 0.8
Urinary excretion rate (nmol/ml.kg–1)
75.5 ± 5.5
48.4 ± 1.9**
Biliary excretion rate (nmol/ml.kg–1)
131 ± 8.1
98.3 ± 2.6*
12.7 ± 0.7
12.5 ± 0.3
Plasma Css (μM)
CLR (ml/min.kg–1)
6.1 ± 0.6
4.0 ± 0.1*
CLBile (ml/min.kg–1)
10.1 ± 1.0
8.0 ± 0.3
CLTot (ml/min.kg–1)
13.1 ± 0.8
13.3 ± 0.2
GFR (ml/min.kg–1)
5.1 ± 0.9
4.4 ± 0.5
AZT (rats administered with azithromycin at 100 min of MTX infusion)
Values are means ± SEM (n = 4). Differences between means were evaluated using the unpaired t-test. A difference of
P < 0.05 was considered statistically significant; *P < 0.05, **P < 0.01.
16
continuous infusion. AZT produced reduction of biliary
excretion, urinary excretion and renal clearance to 75 %,
65 % and 66 % of control animals, respectively. However,
the total clearance of MTX remained unchanged after
AZT. Concentrations of AZT in plasma during final
20 min of experiment were 1.6 ± 0.3 mg.L–1.
CONCLUSIONS: The present study is the first to report that azithromycin, a substrate for Mrp2, inhibited
the in vitro and in vivo biliary as well as renal excretion
of high-dose MTX. In contrast to results of hepatocyte
in vitro study, the in vivo inhibition of MTX elimination
occurred within the clinically achievable azithromycin
concentrations. Therefore, the interaction between azithromycin and methotrexate can not be excluded in clinical settings.
ACKNOWLEDGEMENT
Financial support by the grants No. MSM 0021620820
and No. 1P05OC061 – COST B25.001 is gratefully acknowledged.
REFERENCES
1. Sugie M, Asakura E, Zhao YL et al. Possible involvement of the
drug transporters P glycoprotein and multidrug resistance-associated protein Mrp2 in disposition of azithromycin. Antimicrob
Agents Chemother 2004; 48:80914.
2. Asakura E, Nakayama H, Sugie M et al. Azithromycin reverses
anticancer drug resistance and modifies hepatobiliary excretion
of doxorubicin in rats. Eur J Pharmacol 2004; 484:333–9.
3. Ueda K, Kato Y, Komatsu K, Sugiyama Y. Inhibition of biliary
excretion of methotrexate by probenecid in rats: quantitative prediction of interaction from in vitro data. J Pharmacol Exp Ther 2001;
297:1036–43.
4. Chladek J, Grim J, Martinkova J et al. Pharmacokinetics and
pharmacodynamics of low-dose methotrexate in the treatment of
psoriasis. Br J Clin Pharmacol 2002; 54:147–56.
STUDY OF THE EXPRESSION AND FUNCTION OF BCRP IN RAT PLACENTA
ON 12TH AND 21ST GESTATION DAY
Lenka Cygalova, Martina Ceckova, Petr Pavek, Antonin Libra, Frantisek Staud
Department of Pharmacology and Toxicology, Charles University in Prague,
Faculty of Pharmacy in Hradec Kralove, Heyrovskeho 1203, 50005 Hradec Kralove, Czech Republic
Key words: Breast cancer resistance protein/Placenta/Drug transport/ABC drug efflux transporters
BACKGROUND: Breast cancer resistance protein
(BCRP/ABCG2) is a member of the ABC transporter superfamily (ATP binding cassettes)1 that recognizes a variety of chemically unrelated compounds. BCRP transporter
contributes to the multidrug resistance of tumor cells and
has also been shown to be expressed in various human
physiological tissues such as the small intestine, colon,
liver, brain capillaries or placenta, where it is believed to
affect disposition of several drugs and xenobiotics2, 3. The
expression of Bcrp in mouse placenta4 as well as its contribution to the protection of fetus in Bcrp knockout mice
was demonstrated5. Recently we have demonstrated that
Bcrp reduces passage of its potentially toxic substrates
from mother to fetus and even removes the drug already
Fig. 1. (a) Functional activity of Bcrp in rat placenta on gestation day 12 and 21 – ratio of [3H]cimetidine concentration in fetus over maternal plasma at 60 min after the beginning of infusion, ***p < 0.001; (b) mRNA
expression of the rat Bcrp in rat placenta on gestation day 12 and 21.
present in fetal circulation6. In this study we aimed to
evaluate the protective role of placental BCRP to the fetus
during gestation. The expression pattern and functional
activity of Bcrp were studied on the 12th gestation day
(gd), when the rat chorioallantoic placenta is fully developed and begins to fulfil its physiological role7 and at the
end of gestation, on the term 21st gd.
METHODS: To observe a functional activity of Bcrp
in rat placenta, the infusion of pregnant Wistar rats was
used. The animals of gestation day 12 and 21 (n = 4–5)
were infused with cimetidine (including trace amount of
radiolabeled [3H]cimetidine), a BCRP model substrate.
After the attainment of steady state the animals were sacrificed. 1-3 randomly selected fetuses of each animal were
sampled and their radioactivity measured. The amount
of drug that crossed the placenta was expressed as the
[3H]cimetidine concentration ratio in fetus over maternal
plasma at 60 min after infusion was started. 1-3 placentas
of each animal were randomly chosen and real-time RTPCR was employed to investigate the Bcrp expression at
mRNA level.
RESULTS: The amount of drug that penetrated across
the placenta to the fetus was measured at the end of each
infusion. Ratio of fetal cimetidine concentration to maternal plasma concentration was almost 10 times higher
for fetuses on gd 12 compared with fetuses on term 21st
gd, the average values reached 1.180 and 0.114 respectively (Fig. 1a). These data could indicate poor functional
activity of placental Bcrp at the early phases of gestation. However, the real-time RT-PCR did not reveal any
remarkable differences in Bcrp expression at mRNA level
between gd 12 and 21 (Fig. 1b).
CONCLUSIONS: Our study revealed high penetration of cimetidine to the fetus at early phase of gestation
(gd 12); the amount of drug that penetrated to the fetus
decreased by 10 fold from gd 12 to gd 21, although no dif-
17
ference in the Bcrp expressions on mRNA level between
gd 12 and 21 were observed. This discrepancy could be
explained by protection of fetus through its own detoxicating mechanisms at the end of gestation. Verification of
this theory will be the subject of our further studies.
ACKNOWLEDGEMENT
This work was supported by Grants No. 103/2006/C/FaF
and 119007 C 2007 FaF of the Grant Agency of the Charles
University in Prague, Czech Republic.
REFERENCES
1. Doyle LA, Yang W, Abruzzo LV, Krogmann T, Gao Y, Rishi AK,
Ross DD. A multidrug resistance transporter from human MCF-7
breast cancer cells. Proc Natl Acad Sci USA 1998; 95:15665–70.
2. Staud F, Pavek P. Breast cancer resistance protein (BCRP/ABCG2).
Int J Biochem Cell Biol 2005; 37:720–5.
3. Maliepaard M, Scheffer GL, Faneyte IF, van Gastelen MA,
Pijnenborg AC, Schinkel AH, van De Vijver MJ, Scheper RJ,
Schellens JH. Subcellular localization and distribution of the breast
cancer resistance protein transporter in normal human tissues.
Cancer Res 2001; 61:3458–64.
4. Wang H, Wu X, Hudkins K, Mikheev A, Zhang H, Gupta A,
Unadkat JD, Mao Q. Expression of the breast cancer resistance
protein (Bcrp1/Abcg2) in tissues from pregnant mice: effects of
pregnancy and correlations with nuclear receptors. Am J Physiol
Endocrinol Metab 2006; 291:E1295–304.
5. Jonker JW, Buitelaar M, Wagenaar E, Van Der Valk MA, Scheffer
GL, Scheper RJ, Plosch T, Kuipers F, Elferink RP, Rosing H,
Beijnen JH, Schinkel AH. The breast cancer resistance protein
protects against a major chlorophyll-derived dietary phototoxin and
protoporphyria. Proc Natl Acad Sci U S A 2002; 99:15649–54.
6. Staud F, Vackova Z, Pospechova K, Pavek P, Ceckova M, Libra A,
Cygalova L, Nachtigal P, Fendrich Z. Expression and transport
activity of breast cancer resistance protein (Bcrp/Abcg2) in dually
perfused rat placenta and HRP-1 cell line. J Pharmacol Exp Ther
2006.
7. Enders AC, Blankenship TN. Comparative placental structure. Adv
Drug Deliv Rev 1999; 38:3–15.
COMPARATIVE STUDY OF THE EFFECTS OF NATURAL ANTIOXIDANTS
ON ACUTE PARACETAMOL HEPATOTOXICITY
Pavla Cerna, Dana Kotyzova, Vladislav Eybl
Department of Pharmacology and Toxicology, Faculty of Medicine in Pilsen, Charles University in Prague, 301 66 Pilsen,
Czech Republic
Key words: Paracetamol/Hepatotoxicity/Glutathione/Antioxidants/Aminotransferases
BACKGROUND: Paracetamol (N-acetyl-4-aminophenol, APAP) is an analgesic agent causing hepatic failure
when taken in overdose. Hepatotoxicity is attributed to its
highly reactive metabolite N-acetyl-p-benzoquinoneimine
(NAPQI) generated by microsomal enzymes of P450 family. NAPQI is detoxified by glutathione (GSH). Once GSH
level is depleted by large amount of NAPQI occurring
in overdose, centrilobular hepatic necrosis results1. The
relationship between serum aminotransferase levels and
paracetamol-induced liver necrosis is known2. Protective
effects of natural antioxidants curcumin3, quercetin4 and
silymarin5 against paracetamol hepatotoxicity were studied in various conditions however not intensively. Also no
comparative study of these compounds was performed.
In our experiment in rats, the effects of widely used
natural antioxidants curcumin, quercetin and silymarin
18
on paracetamol-induced liver injury were compared. Our
study is focused on the protective effect of these natural
antioxidants in this intoxication. It is important also for
the study of eventual interactions of such compounds with
hepatotoxic drugs.
METHODS: Male Wistar rats (SPF, Velaz Prague)
weighing 310±40 g were used in the experiment. The
experiment protocol was approved by the local Animal
Care and Use Committee. Animals were randomly divided into 8 groups (N = 10/7) with free access to diet
and drinking water. The group I serve as a control receiving 0.5% methylcellulose. The animals of groups II, III,
IV and V received paracetamol (900 mg/kg). At 48 h,
24 h and 2 h before paracetamol application, the animals
were pretreated with curcumin (50 mg/kg; group III),
quercetin (15 mg/kg; group IV) or silymarin (175 mg/kg;
group V). The animals of the group VI, VII and VIII received only curcumin, quercetin and silymarin at the same
doses as above. All studied substances, dissolved in 0.5%
methylcellulose, were administered by gastrical gavage in
amount of 0.5 ml/100 g b.w. At 24th h after paracetamol
administration, the animals were decapitated and serum
and liver tissue were collected for analyses. The levels
of reduced glutathione (GSH) and lipid peroxidation in
liver homogenates were measured according to ref.6, 7. The
serum levels of alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT) and lactate dehydrogenase
(LDH) were determined using commercial kits (DiaSys
Table 1. The effect of antioxidant pretreatment on serum levels of ALAT, ASAT and LDH in rats exposed
to paracetamol (APAP).
ASAT
[U.L–1]
LDH
[U.L–1]
178.7±38.7
1970±409
112.4±24.0 ***
234.2±60.5 *
2080±435
10
67.8±14.0 # # #
163.9±26.0 # #
1965±302
APAP + quercetin
10
71.1±15.5 # # #
160.0±30.4 # #
1787±371
APAP + silymarin
10
68.9±19.0 # # #
158.6±21.1 # #
1587±317 #
Curcumin
7
43.4±5.5 *
128.0±12.9 **
1285±232 **
Quercetin
7
43.8±7.6 *
135.9±17.2 *
1293±278 *
Silymarin
7
48.4±5.0
126.4±21.4 **
1168±253 ***
Group
N
ALAT
[U.L–1]
Control
10
50.6±5.0
APAP
10
APAP + curcumin
Results are expressed as mean ± SD; *** significantly different from control group at p < 0.001; ** p < 0.01;
* p < 0.05; # # # significantly different from APAP group at p < 0.001; # # p < 0.005, # p < 0.05.
6
5
**
**
curcumin
quercetin
**
μmol GSH/g
4
*
3
2
1
0
control
APAP
APAP
+curc.
APAP
+quer.
APAP
+silym.
silymarin
Fig. 1. The effect of antioxidant pretreatment on hepatic glutathione level in rats exposed to paracetamol (APAP).
Results are expressed as mean ± SD. N = 7–10. * Significantly different from control group at p < 0.05;
** p < 0.001.
Diagnostic Systems, Germany). Statistical evaluation was
done by non-parametrical Student’s t-test.
RESULTS: The administration of APAP induced
a depletion of GSH. The levels of GSH in APAP groups
pretreated with curcumin, quercetin and silymarin did
not differ from control group. In the groups treated with
antioxidants alone, an increase of the GSH was found
(see Fig. 1.). The level of lipid peroxidation increased by
APAP (p < 0.005) was not altered by curcumin or quercetin pretreatment and was increased by silymarin pretreatment (p < 0.05) (not shown). The application of APAP
significantly enhanced the level of ALAT (p < 0.001) and
ASAT (p < 0.05) and did not change the LDH level. In
the APAP groups pretreated with curcumin, quercetin and
silymarin, the ALAT level (p < 0.001) and ASAT level
(p < 0.005) were lower in comparison to APAP only-intoxicated group. All three antioxidants curcumin, quercetin
and silymarin administered alone significantly decreased
the level of both aminotranferases (ALAT, ASAT) as well
as the level of LDH (see Table 1.)
CONCLUSIONS: A protective effect of curcumin,
quercetin and silymarin against acute paracetamol hepatotoxicity was demonstrated in acute experiment in rats.
Paracetamol-induced liver damage was expressed by
the elevation of serum aminotransferases levels, which
are reliable markers of liver injury. All studied antioxidants significantly ameliorated the increase of serum
19
aminotransferases and correspondingly caused enhancement of hepatic glutathione content. Further studies for
understanding of mechanisms of hepatoprotective action
of natural compounds are considered.
ACKNOWLEDGEMENT
This study was supported by the Grant Agency of Czech
Republic (305/05/0344).
REFERENCES
1. Newsome PN, Plevris JN, Nelson LJ, Hayes PC. Animal models
of fulminant hepatic failure: A critical evaluation. Liver Transpl
2000; 6:21–31.
2. Dixon MF, Fulkner MJ, Walker BE, Kelleher J, Losowsky MS.
Serum transaminase levels after experimental paracetamol-induced
necrosis. Gut 1975; 16:800–7.
3. Donatus IA, Sardjoko, Vermeulen NP. Cytotoxic and cytoprotective
activities of curcumin. Effects on paracetamol-induced cytotoxicity,
lipid peroxidation and glutathione depletion in rat hepatocytes.
Biochem Pharmacol 1990; 39:1869–75.
4. Gilani AH, Janbaz KH, Shah BH. Quercetin exhibits hepatoprotective activity in rats. Biochem Soc Trans 1997; 25:S619.
5. Muriel P, Garciapina T, Perez-Alvarez V, Mourelle M. Silymarin
protects against paracetamol-induced lipid peroxidation and liver
damage. J Appl Toxicol 1992; 12:439–42.
6. Sedlak J, Lindsay RH. Estimation of total, proteinbound and nonprotein sulfhydryl groups in tissue with Ellman’s reagent. Anal
Biochem 1968; 25:192–205.
7. Mihara M, Uchiyama M. Determination of malondialdehyde precursor in tissues by thiobarbituric acid test. Anal Biochem 1978;
86:271–8.
DPD MUTATION (IVS14+1G>A) IN RELATION TO THE TOXICITY IN PATIENTS
RECEIVING 5-FLUOROURACIL-BASED CHEMOTHERAPY
Martina Cizmarikovaa, Maria Wagnerovab, Viera Habalovac, Anton Kohuta, Alexander Bercb,
Igor Andrasinab, Ladislav Mirossaya
a
Department of Pharmacology, Faculty of Medicine, P. J. Safarik University, 040 66 Kosice, Slovak Republic
East Oncology Institute a.s., Department of Radiotherapy and Oncology, 040 01 Kosice, Slovak Republic
c
Department of Biology, Faculty of Medicine, P. J. Safarik University, 040 66 Kosice, Slovak Republic
b
Key words: Dihydropyrimidine dehydrogenase/Toxicity/5-Fluorouracil/Colorectal cancer/Breast cancer
BACKGROUND: 5-Fluorouracil (5-FU)-based therapy
is considered standard therapy in the treatment of many
malignancies, including colorectal and breast cancers.
The main mechanism of 5-FU action is the inhibition
of the thymidylate synthase through its active metabolite 5-fluoro-2-deoxyuridine. This prevents the only de
novo source of thymidine, which is essential in the DNA
synthesis. Dihydropyrimidine dehydrogenase (DPD) is
one of the several factors influencing intracellular 5-FU
levels. DPD is the initial and rate-limiting enzyme in the
catabolism of 5-FU. It is suggested that patients with
a partial deficiency of this enzyme are at the risk from
developing severe toxicity1,2. Seventeen variant alleles of
DPD have been identified, one of them being by far the
most frequent one; it consists of a splice-site mutation
IVS14+1G >A. The present study was conducted to study
interrelationship of this DPD mutation and toxicity in
patients treated with 5-FU-based chemotherapy for colorectal and breast cancers.
METHODS: Mutation IVS14+1G>A was analysed
in 103 patients treated with 5-FU-based chemotherapy
(sixty-four patients with colorectal cancer and thirty-nine
patients with breast cancer). DNA was extracted from
blood lymphocytes using standard methods. The PCRRFLP assay was used for genotyping (Fig. 1). PCR amplification of exon 14 and its flanking 5’ donor intronic
region was carried out using the following primer sets:
NDEA: 5’-ATCAGGACATTGTGACATATGTTTC–3’ and
20
NDEB: 5’-CTTGTTTTAGATGTTAAATCACACATA–3’.
Restriction analysis of the PCR products was performed
using restriction endonuclease NdeI. The obtained fragments were separated by electrophoresis in 2.5 % agarose
gel after overnight incubation at 37 °C and analysed after
staining with ethidium bromide. PCR product was also
Exon 14 (165 bp)
layed into gel as a positive control for the digestion efficiency of NdeI.
RESULTS: Toxicity was classified according to WHO
criteria. Statistical correlation was not possible to realize
because all DNA samples from patients studied for DPD
mutation were homozygous for wild allele. Nevertheless
C
5´
3´
198 bp
CATATG –––––––––––––––– CGTATG
a)
17 bp
b)
PCR product
181 bp
CA TATG ––––––––––––– CGTATG
Wild-Type
NdeI
17 bp
c)
154 bp
CA TATG ––––––––––––– CA
NdeI
TATG
27 bp
IVS14+1G>A
NdeI
Fig. 1. PCR-RFLP genotyping of the IVS14+1G>A mutation in the DPD gene2
a) The undigested PCR product of the wild-type DPD gene (198 bp).
b) After digestion of the wild-type DPD gene with NdeI, two fragments will be produced (17 bp, 181 bp).
c) The PCR product of a mutant DPD allele containing the IVS14+1G>A mutation will produce three fragments after digestion with NdeI (17 bp, 154 bp, 27 bp).
Table 1. The occurrence of the toxicity in patients treated with 5-FU-based chemotherapy.
Type
of toxicity
Colorectal cancer
The occurence of toxicity
n
%
Breast cancer
The occurence of toxicity
n
%
Anemia
9
14.1
4
10.3
Leukopenia
18
28.1
7
17.9
Neutropenia
8
12.5
11
28.2
Trombocytopenia
1
1.6
2
5.1
Nauzea/vomiting
17
26.6
25
64.1
Diarrhea
22
34.4
0
0.0
Renal
0
0.0
1
2.6
Hepatic
5
7.8
3
7.7
Cutaneous
1
1.6
0
0.0
Mucous
10
15.6
0
0.0
Alopecia
3
4.7
38
97.4
Constipation
1
1.6
0
0.0
Neurological
1
1.6
1
2.6
Note: The toxicity may be affected by other cytostatics in some cases (especially in patients with breast cancer treated
with combined chemotherapy).
we have observed, that toxicity of 5-FU-based chemotherapy was found in despite of absence the splice-site mutation
IVS14+1G>A (Table 1).
CONCLUSIONS: This study demonstrated that the
(IVS14+1G>A) mutation genotyping in DPD could not
be of help in predicting therapeutic toxicity to 5-FUbased chemotherapy. The obtained result also confirmed,
that DPD mutation is infrequent in Slovak population.
Complete or near-complete enzyme deficiency occurs in
one patient out of 10 000 according to the literature1.
21
ACKNOWLEDGEMENT
Supported by the grant VEGA SR 1/2282/05 and research project MZ SR 2005/46-VOUKE-01 from Ministry
of Health of the Slovak Republic.
REFERENCES
1. Robert J, Le Morvan V, Smith D, Porquier P, Bonnet J. Predicting
drug response and toxicity based on gene polymorphisms. Crit Rev
Oncol Hematol 2005; 54:171–96.
2. Van Kuilenburg AB, Muller EW, Haasjes J, Meinsma R, Zoetekouw
L, Waterham HR et al. Lethal Outcome of a Patient with a Complete Dihydropyrimidine Dehydrogenase (DPD) Deficiency after
Administration of 5-Fluorouracil: Frequency of the Common
IVS1411G>A Mutation Causing DPD Deficiency. Clin Cancer
Res 2001; 7:1149–53.
DO POTENTIAL INTERACTIONS INCREASE THE PROBABILITY
FOR A HOSPITALISATION FROM CARDIOVASCULAR REASONS IN HIGH RISK
CARDIOVASCULAR PATIENTS IN SLOVAKIA?
Tatiana Foltanovaa, Martin Thurzob, Ingrid Tumovaa, Jan Lietavab, Pavel Sveca
a
Slovak Republic
b
2nd Department of Internal Medicine. Comenius University, Faculty of Medicine, Mickiewiczova 13, 813 69 Bratislava,
Slovak Republic
Key words: High cardiovascular risk/Potential interactions/Cardiovascular morbidity
n of events
BACKGROUND: The pharmacotherapy can harm
every patient it is intended to help if clinically significant
drug interactions occur1. Drug interactions result in an
undesirable modification of the action of one or more
administered agents2. The effect of potential interactions
of moderate and major severity on cardiovascular (CV)
morbidity in a long term trial in Slovakia wasn’t studied
yet.
METHODS: Therapy of 849 high-risk ischemic heart
disease (IHD – 100 %) patients (defined as age over
55 yrs, symptomatic IHD [defined as myocardial infarction (MI – 45.47 %), unstable angina pectoris (UAP
200
180
160
140
120
100
80
60
40
20
0
1
2
3
4
5
year
chf
cardiac
ua
stroke
other CV events
Fig. 1. Differences in cardiovascular morbidity and mortality (chf – congestive heart failure, cardiac – arrhythmias
and other disturbances of heart rate, other CV events – MI, bypass, transient ischemic attack).
22
Table 1. Characteristics of the study population.
year (2000 – 2005)
0
1
2
3
4
5
849
824
770
73
686
639
178
155
176
174
169
193
0–5
0–6
0–4
0–5
0–3
0–5
186
171
199
171
137
138
0–5
0–4
0–4
0–4
0–3
0–3
% of pts with inter 3
20.97
18.81
22.86
23.84
24.64
30.20
% of pts with inter 4
21.91
20.75
25.84
23.42
19.97
21.60
CV hospital
172
102
111
118
91
other hospital
207
198
185
162
130
mortality
25
54
40
44
47
NS
NS
NS
01.61**
NS
nb of patients
nb of inter 3
nb of inter 4
OR inter 3, CV hosp
OR inter 4, CV hosp
1.08–2.40
1.64*
1.76**
1.02–2.64
1.10–2.82
NS
NS
1.54*
0.99-2.38
OR – odds ratio, ** p < 0.01, * p < 0.05
– 7.30 %)], stroke – 17.79 %, peripheral vascular disease
– 11.31 %, diabetes mellitus (DM – 62.45 %) with at least
one more risk factor (arterial hypertension (79.88 %) or
hypercholesterolemia (66.08 %) or smoking (45.94 %)
was analyzed during five years. Pharmacotherapy, with
special concern on CV drugs, has been evaluated according to structured drug related interview with patients and
subsequent cross-control with their medical records. For
analysis only regular prescribed medications were used,
defined as taken daily or in regular intervals. According to
Tatro et al.3 potential interactions of moderate and major
severity were studied. The medications combination of
moderate severity (grade 3) may lead to changes in the
therapy, but it is possible to control by individual dosage
or plasma concentration monitoring. The medications
combination of major severity (grade 4) may result in
serious clinical consequences in terms of adverse drug
reaction or therapeutic failure and it is difficult to control
by individual dosage.
Statistical analyses were performed using SPSS 10.0 for
Windows statistical program. The data were analyzed
using cross tabulations, chi-square tests. The significance
of the results is presented as P-values.
RESULTS: High risk cohort (849 pts) was monitored
for five years (2000–2005). During the study CV morbidity was observed in 61.96 % (562 pts). Most frequent
reason for a hospitalization was congestive heart failure
(CHF 245 times, 40–64), although patients with CHF
(NYHA >I) were not included (Fig. 1). Potential interactions of moderate severity were present in almost one
half of the patients 40.40 % (343 pts). Their effect on CV
morbidity was not significant except the fourth year (1.61
(1.08–2.40), p < 0.01). Potential interactions of major severity were present in almost one third 35.92 % (305 pts).
Their effect on CV morbidity was significant except the
third and fourth year. Patients with drug interaction had
higher odds for hospitalisation from CV reasons. The
highest odds ratio was in second year (OR = 1.76 (1.10
– 2.82), p < 0.01). The mortality in this year was also the
highest one (54 pts, (25–54) (Table 1).
CONCLUSIONS: Potential interactions of major severity significantly increased the probability for a hospitalization from CV reasons in high risk CV patients.
ACKNOWLEDGEMENT
Financial support by the grants FaF UK/017/2004
– G 203, UK/260/2005 is gratefully acknowledged.
REFERENCES
1. Balkrishnan R, Furberg CD. Developing an optimal approach to
global drug safety. J Intern Med 2001; 250:271–9.
2. Juurlink DN, Mamdani M, Kopp A, Laupacis A, Redelmeier DA.
Drug-drug interactions among elderly patients hospitalized for drug
toxicity. JAMA 2003; 289:1652–8.
3. Tatro DS. Drug Interaction Facts 2004. Facts and Comparisons,
2004. p. 1648
23
PHARMACOLOGICAL INFLUENCE OF UTERINE SMOOTH MUSCLE REACTIVITY
Sona Franovaa, Frantisek Janicekb, Gabriela Nosalovaa, Martina Sutovskaa, Jozef Visnovskyb
a
Department of Pharmacology, Jessenius Faculty of Medicine, Comenius University, Martin, Slovak Republic
Clinic of Obstetrics and Gynecology, Jessenius Faculty of Medicine and Martin University Hospital, Martin, Slovak
Republic
b
Key words: Uterine smooth muscle/Uterotonic/Uterolytic effects
BACKGROUND: Regulation of uterine smooth
muscle contraction represents the complicated complex
process influenced by many uterotonic and uterolytic
mediators. Besides, contractility of the uterus is under
control of estrogen and progesterone and varies with the
menstrual cycle and pregnancy. During the sexual cycle
estrogen dominance during the follicular phase enhances
uterotonic-hormone stimulated contractility in the uterus.
Progesterone dominance during the latter phase of the cycle and during pregnancy1 leads to a decrease in contractile response of the uterine smooth muscle to uterotonic
agents. The aim of study was to give the preview of more
important results related to: 1) Uterine smooth muscle
reactivity to uterotonic (oxytocin, PGF2α, ET-1, bradykinin
and Ang II) uterolytic (potassium channel openers) factors in experimentally induced follicular and luteal phase
of sexual cycle in rabbits. 2) Smooth muscle reactivity
of human non-pregnant myometrium to uterotonic mediators oxytocin, PGF2α, ET-1, and bradykinin. 3) Uterorelaxant effect of PDE IV-selective inhibitor rolipram in
monotherapy and during simultaneous administration
with salbutamol on oxytocin induced contractions in vitro
in pregnant myometrium.
METHODS: The aim of the study was to create the
experimental model for evaluation of different mediator
efficacy on rabbit uterine smooth muscle reactivity in
in vitro conditions. The proliferative phase (17β-estradiol,
1 mg/kg i.m., for 7 days) and secretory phase (progesterone 2 mg/kg s.c., for 7 days, 17β-estradiol, 1 mg/kg i.m.,
for last 3 days) of sexual cycle was induced by exogenous
administration of sex hormones after ovariectomy in rabbits. The amplitude of contraction (mN) and frequency
of contraction to oxytocin (10–9–10–7 mol.L–1), PGF2α
(10–10–10–7 mol.L–1), bradykinin (10–10–10–7 mol.L1) ET1 (10–10–10–7 mol.L–1) and angiotensin II (10–10 mol.L–1)
were used as evaluated parameters2.
RESULTS: We observed that estrogen dominance,
during proliferative phase, caused increase of contractile
amplitude to all used mediators. However, we did not
follow the changes in frequency of contraction. On the
contrary, gestagen ascendance caused the decline of contractile amplitude, the increase of contractile frequency
induced by PGF2α and ET-1 and reduction of bradykinin
elicited contraction frequency. Angiotensin II add to organ bath increased the amplitude of contraction, but this
effect was independent of sex hormone levels. The role of
potassium channels in regulation of myometrial reactivity
is in present time actual problem from experimental and
therapeutical point of view3. In our experiments we investigated the participation of ligand-sensitive large conductance BKCa and KATP potassium channels in uterine smooth
muscle reactivity during different stage of experimentally
induced sexual cycle in ovariectomized rabbits. The effect
of KATP opener pinacidil (10–5 mol.L–1) and inhibitor of this
channel glibenclamide (10–6 mol.L–1), opener of BKCa2+ –
NS1619 (10–6 mol.L–1) and inhibitor tetraethylammonium
(10–4 mol.L–1) pretreatment on isolated myometrial strips
reactivity to oxytocin (10–6 mol.L–1) was investigated. Our
findings showed that activity of myometrial potassium
channels is influenced by levels of estrogens and gestagens. The BKCa and KATP openers had not significant effect
on amplitude of uterine contraction during proliferative
phase of cycle. The gestagens probably increase the activity of BKCa and KATP channels during secretory phase.
Pretreatment with pinacidil and NS1619 caused the decline of contraction of myometrial strips obtained during
secretory phase cycle and this effect was inhibited with
selective channel blockers.
Reactivity of human myometrial strips, obtained from
the pre-menopausal woman undergoing total hysterectomy for benign gynaecological indications was evaluated
by in vitro method. Our results showed the increased
amplitude of uterine smooth muscle strips to contractile mediators oxytocin (10–9-10–7 mol.L–1), PGF2α (10–10–
10–7 mol.L–1), bradykinin (10–10–10–7 mol.L1) and ET-1
(10–10–10–7 mol.L–1) during follicular phase of the cycle.
Our findings support the idea that estrogens might have
a positive influence on the expression of various types of
receptors (FP, OTR, BK2R and ETAR) and thus promotes
the contractility to uterotonic agents4. On the contrary,
minimal myometrial response to oxytocin, PGF2α, ET-1,
and bradykinin were observed during luteal phase when
progesterone levels are increased.
The effect of rolipram, the prototype of PDE IV selective inhibitor, on oxytocin induced contractions of human
term myometrial strips was studied, and compared with
salbutamol, β2-adrenergic agonist, in monotherapy and
with their simultaneous application. Human myometrium
was obtained from 15 pregnant women in term, that delivered by the caesarian section. The myometrial smooth
muscle strips were pretreated in organ bath with selected
substances 10 minutes before oxytocin induced contraction. The mean peak amplitude of contraction (mN) of
the myometrial smooth muscle strips to the doses of oxy-
24
tocin (10–6 mmol.L–1) with organ-bath pretreatment with
rolipram (10–4 mmol.L–1) or salbutamol (10–4 mmol.Ll1)
and simultaneous administration of rolipram and salbutamol (both 10–4 mmol.Ll–1), was used as a parameter of
myometrial reactivity. Monotherapy with rolipram showed
decrease in the contractile amplitude of oxytocin induced
contraction to 57.3 %, salbutamol monotherapy resulted
in amplitudinal decrease to 67.9 %, and combination of
both rolipram and salbutamol in their simultaneous administration decreased the oxytocin induced amplitude
to 40 %.
CONCLUSIONS: Our results demonstrate that endothelin-1, bradykinin and angiotensin II play the role
in stimulation of myometrial contractility. Myometrial
response to endothelin-1, bradykinin is functionally dependent upon amount of level of its specific binding receptor in the uterine tissue, which is dependent probably
utterly on levels of estrogens. The uterotonic activity of
angiotensin II was independent of sex hormone levels.
Potassium channel activity is the important factor regulated uterolytic process of uterine smooth muscle. The
BKCa and KATP openers significantly lowered the amplitude
of uterine contraction during luteal phase of cycle. The
results showed that except for oxytocin and PG2α the other
mediators bradykinin and ET-1 play the role in regulation
of contractile activity of human myometrium during men-
strual cycle. This experimental model will be used for the
pharmacological influence of myometrial reactivity and
could be helpful in the preventing of preterm uterine contractions and to relieve dysmenorrhea. Our study proves
the theory of the enhanced efficiency of β2-adrenergic agonist5, when administered together with PDE IV inhibitor,
moreover we have shown that rolipram in monotherapy
has more profound effect on oxytocin induced contractions than salbutamol alone.
REFERENCES
1. Wu JJ, Geimonen E, Andersen J. Increase expression of estrogen
receptor beta in human uterine smooth muscle at term. Eur J
Endocrinol 2000; 142:92–6.
2. Accorsi-Mendonça D, Correa FMA, Anselmo-Franci JA, Paiva
TB, Oliveira AM. Angiotensin Actions on the Isolated Rat Uterus
during the Estrous Cycle: Influence of Resting Membrane Potential
and Uterine Morphology. Pharmacology 2002; 65:162–9.
3. Khan RN, Matharoo-Ball B, Arulkumaran S, Ashford ML. Potassium
channels in the human myometrium. Exp Physiol 2001; 86:255–64.
Word RA, Kamm KE, Casey ML. Contractile effects of prostaglandins, oxytocin, and endothelin-1 in human myometrium in vitro:
refractoriness of myometrial tissue of pregnant women to prostaglandins E2 and F2 alpha. J Clin Endocrin 1992; 75:1027–32.
4. Schmitz T, Souil E, Herve R, Nicco C, Batteux F, Germain G,
Cabrol D, Evain-Brion D, Leroy MJ, Mehats M. PDE4 Inhibition
Prevents Preterm Delivery Induced by an Intrauterine Inflammation.
J Immunol 2007; 178: 1115–21.
HYPOTENSIVE EFFECT OF R AND S ISOMERS OF NEW COMPOUND 444
Marek Frydrycha, Ladislava Bartosovaa, Klara Horkaa, Josef Krcmara, Petr Mokryb
a
Department of Human Pharmacology and Toxicology, Faculty of Pharmacy, University of Veterinary and Pharmaceutical
Sciences, 612 42 Brno, Czech Republic
b
Department of Chemical Drugs, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences, 612 42 Brno,
Czech Republic
Key words: Hypotensive effect/Plasma esterase/Ester functional group/Compound 444
BACKGROUND: The ultrashort-acting beta blockers
are parenteral agents that can be rapidly titrated in special
clinical settings where immediate beta adrenergic blockade is warranted1. They are used in patients with unstable
angina, myocardial infarction, atrial fibrillation or flutter
and supraventricular tachycardia2. Two isomers of compound 444 – R and S, synthesized at the Department of
Chemical Drugs of the Faculty of Pharmacy of Veterinary
and Pharmaceutical Sciences in Brno, developed as ultrashort-acting beta blockers were tested in this experimental work. The advantage of this isomers should be in
the immediate onset of action after administration, possibility of dose titration, very short duration of action and
rapid offset of the action3. The synthesized compounds are
derivatives of arylcarbonyloxyaminopropanoles containing metabolically unstable ester functional group which is
rapidly hydrolyzed by esterases in red blood cells, plasma
and liver esterases.
METHODS: The aim of this work was to pharmacologically evaluate the effect of R and S isomers of compound 444 by way of non invasive method on systolic
blood pressure in rats. The experiment was performed
in vivo with 34 male Wistar laboratory rats. The tested
agents were administered at the doses of 2.0 mg/kg and
2.5 mg/kg of body mass. The tested group was divided
into 5 subgroups: Group 1 (n = 7) was administered
the dose of 2.0 mg/kg of isomer R, Group 2 (n = 7) the
dose of 2.5 mg/kg of isomer R, Group 3 (n = 7) the dose
of 2.0 mg/kg of isomer S, Group 4 (n = 7) the dose of
2.5 mg/kg of isomer S and Group 5 (n = 6) placebo. The
tested doses were administered into vena jugularis and
the values of systolic blood pressure were monitored for
20 minutes following the administration. For systolic
blood pressure non invasive monitoring a “Non-Invasive
Blood Pressure Monitor” by Columbus Instruments company was used. The tested doses were compared with placebo.
RESULTS: The dose of 2.0 mg/kg of 444R caused statistically significant decrease (p < 0.05) in systolic blood
pressure compared to the placebo 9 minutes after administration, dose of 2.5 mg/kg of 444R 11 minutes, dose of
2.0 mg/kg of 444S 11 minutes and the dose of 2.5 mg/kg
of 444S 12 minutes.
CONCLUSIONS: All the tested doses of isomers
R and S caused in trial by way of non-invasive method
hypotensive effect which started immediately after administration and the hypothesis of ultra-short action was
verified.
25
ACKNOWLEDGEMENT
Financial support by the grant GA CR Nr. 305/06/0863
is gratefully acknowledged.
REFERENCES
1. Frishman WH, Murthy S, Strom JA. Ultra-short-acting beta-adrenergic blockers. Med Clin North Am 1998; 72:359–72.
2. Barbier GH, Shettigar UR, Appunn DO. Clinical rationale for
the use of an ultra-short acting beta-blocker:esmolol. Int J Clin
Pharmacol Ther 1995; 33:212–8.
ANALYTICAL SUPPORT OF HYPERTENSION TREATMENT. DIURETICS
Vilma Habrdova, Viktor Voriseka, Jiri Ceralb, Karel Macekc, Zuzana Svetlikovad, Pavel Zivnya,
Vladimir Palickaa
a
Institute of Clinical Biochemistry and Diagnostics, Medical Faculty of Charles University and University Hospital,
500 05 Hradec Kralove, Czech Republic
b
Department of Internal Medicine, University Hospital, 500 05 Hradec Kralove, Czech Republic
c
Center of Clinical Pharmacology, University Hospital, 500 05 Hradec Kralove, Czech Republic
d
Faculty of Pharmacy, Charles University, 500 05 Hradec Kralove, Czech Republic
Key words: LC/MS/MS /Hydrochlorothiazide/Chlortalidone/Serum/Compliance
BACKGROUND: A simple, sensitive and specific
LC/MS/MS method was developed and validated for the
quantitation of hydrochlorothiazide (HCTZ) and chlorthalidone (CT) in human serum. The presented method
is a useful tool for monitoring compliance in the therapy
of hypertension and is currently being used to assay serum
samples of patients.
METHODS: Serum samples (1 mL) were extracted
using a mixture of ethyl acetate and dichloromethane.
Evaporated extracts were reconstituted in 200 μl of mobile
phase (0.2 % formic acid and acetonitrile). The chromatographic separation was performed on an Agilent Zorbax
XDB-C8 column with gradient at a flow rate of 0.2 mL/
min. Detection was accomplished on a LTQ linear ion
trap MS using electrospray ionization. The MS system
was operated in the SRM mode.
RESULTS: The assay exhibited a linear range 0.5 –
200 ng.mL–1 for HCTZ and 1.25 – 500 ng.mL–1 for CT in
human serum. The validation criteria for intra-assay precision (RSD ≤ 12 %), inter-day precision (RSD ≤ 15 %),
and accuracy (less than ±15 %) were fulfilled for both
diuretics. Typical concentrations found in patient samples
ranged from 6 – 498 ng.mL–1 for HCTZ and 50 ng.mL–1
for CT.
CONCLUSIONS: An LC/MS/MS assay was developed and validated for the determination of low concentrations of diuretics in human serum. The method can be
used to quantify hydrochlorothiazide and chlorthalidone
in human serum for studies in therapeutic drug monitoring (TDM) and monitoring of patients’ compliance.
26
BENEFICIAL EFFECT OF PYCNOGENOL® ON IMPAIRED CONTRACTILITY OF
LEFT VENTRICLE IN EXPERIMENTAL DIABETES MELLITUS
Barbora Hamakova, Elena Priesolova, Diana Yaghi, Stanislava Jankyova, Jana Kmecova,
Jan Kyselovic, Stefan Matyas, Jan Klimas
Key words: Diabetes mellitus/Left ventricular function/Pycnogenol/Streptozotocin/eNOS
diabetic patients1,2. We tested whether antioxidative effect
of Pycnogenol® can affect also the left ventricular dysfunction in experimental model of diabetes mellitus II. type.
METHODS: Wistar rats were treated with streptozotocin (STZ, 3x25 mg/kg, i.p., in 24-h intervals) to induce
BACKGROUND: Antioxidants are attractive substances widely used as adjuvant therapy in many pathological conditions. Positive effects of pine maritime
extract Pycnogenol® were shown in diabetes mellitus-induced microangiomyopathy and lowering of glucose in
Table 1. Body weight (BW), heart weight (HW), systolic and diastolic blood pressure (sBP and dBP) in control
(CON), streptozotocin (STZ) and streptozotocin+Pycnogenol®-treated (STZ + PYC) rats.
CON
STZ
STZ+PYC
8
7
7
Body weight (g)
404 ± 11
250 ± 12*
287 ± 12*†
Heart weight (mg)
963 ± 24
748 ± 26
841 ± 31*†
Heart/body weight (mg/g)
2.39 ± 0.09
3.02 ± 0.10*
2.94 ± 0.07*
arterial sBP (mmHg)
105 ± 5
108 ± 4
103 ± 6
arterial dBP (mmHg)
67 ± 3
n
88 ± 3*
77 ± 3*†
average ± SEM, * p<0.05 vs. CON, †p<0,05 vs. STZ
Fig. 1. Left ventricular pressure (LVP), rate of contraction (dP/dtmax) and relaxation (dP/dtmin) by in vivo
catheterization (average ± SEM, *p < 0.05 vs. CON, †p < 0.05 vs. STZ).
diabetes. Thereafter, Pycnogenol® (50 mg/kg/d, p.o.)
was administered eight weeks in the STZ+PYC group.
Controls received vehicle (CON). Systolic and diastolic
blood pressure (sBP and dBP) were measured invasively
in a.carotis and left ventricular pressure (LVP), rate of
contraction (dP/dtmax) and relaxation (dP/dtmin) were
studied using left ventricular catheterization under tribromethanol anaesthesia.
RESULTS: Streptozotocin led to significant elevation
of dBP and decrease of left ventricular function (Table 1,
Fig. 1). Pycnogenol® reduced increased dBP after streptozotocin and restored left ventricular function.
CONCLUSIONS: We conclude that Pycnogenol® had
positive effect on left ventricular impairment in experimental diabetes mellitus.
27
ACKNOWLEDGEMENT
Financial support by the grant of VEGA 2/5129/25 is
gratefully acknowledged.
REFERENCES
1. Cesarone MR, Belcaro G, Rohdewald P, Pellegrini L, Ledda A,
Vinciguerra G, Ricci A, Gizzi G, Ippolito E, Fano F, Dugall M,
Cipollone G, Acerbi G, Cacchio M, Del Boccio G, Di Renzo A,
Stuard S, Corsi M. Improvement of diabetic microangiopathy with
pycnogenol: A prospective, controlled study. Angiology 2006;
57:431–6.
2. Liu X, Zhou HJ, Rohdewald P. French maritime pine bark extract
Pycnogenol dose-dependently lowers glucose in type 2 diabetic
patients. Diabetes Care 2004; 27:839.
CHANGES IN ENDOTHELIAL FUNCTION AFTER SHORT-TERM EXPERIMENTAL
DIABETES, ADMINISTRATION OF FAT-CHOLESTEROL DIET AND THEIR
COMBINATION IN RATS
Anna Harcarova, Miroslava Kroslakova, Zuzana Bajuszova, Adriana Adameova,
Peter Krenek, Jan Kyselovic, Magdalena Kuzelova
Slovak Republic
Key words: Diabetes/Hypercholesterolemia/eNOS/Endothelial function/Caveolin-1
BACKGROUND: In endothelial cells, endothelial nitric oxide synthase (eNOS) produces NO, responsible for
both vascular tone and structure regulation1. Endothelial
dysfunction is a well established response to cardiovascular risk factors, such as diabetes mellitus and hypercholesterolaemia2,3. Hypercholesterolaemia has been described
to be associated with an upregulation of eNOS in en-
dothelial cells that can be considered compensatory4,5.
Administration of cholesterol to animals has been shown
to decrease eNOS enzyme activity by inducing the eNOS
inhibiting protein caveolin (cav-1)6. On the other hand in
some studies short-term experimental diabetes has been
recognised to induce eNOS expression in rat aorta7 and
myocardium8. Influence of the combination of these two
A
B
cav-1/actin
*
350
cav-1/actin (control=100 %)
eNOS/actin (control=100 %)
eNOS/actin
300
250
200
150
100
50
0
C
DM
FCH
DM-HCH
*
160
120
80
40
0
C
DM
FCH
DM-HC
Fig. 1. Changes in eNOS and cav-1 expression in rat aorta under the influence of short-term experimental diabetes
and fat-cholesterol diet (P<0.05 vs. FCH).
28
Table 1. Relaxation of rat aorta isolated from rats with short-term experimental diabetes and fat-cholesterol
diet to acetylcholine and sodium nitroprusside.
Relaxation of aorta to acetylcholine
log concentration
(mol.L–1)
-9.0
-8.5
-8.0
-7.5
-7.0
-6.5
-6.0
-5.5
-5.0
% tone remaining
C (n=6)
100.0 ± 0.0
100.0 ± 0.0
99.8 ± 0.2
92.9 ± 2.2
76.9 ± 7.0
52.9 ± 7.2
32.0 ± 4.3
26.2 ± 4.2
23.7 ± 4.2
DM (n=6)
100.0 ± 0.0
100.0 ± 0.0
99.8 ± 0.2
98.3 ± 0.5
85.8 ± 4.3
63.1 ± 5.6
44.3 ± 5.1
33.6 ± 3.0
31.8 ± 2.3
FCH (n=6)
100.0 ± 0.0
100.0 ± 0.0
99.6 ± 0.4
96.5 ± 1.7
75.1 ± 5.7
52.8 ± 6.8
36.3 ± 6.3
30.8 ± 7.2
28.3 ± 5.5
DM-HCH (n=6)
100.0 ± 0.0
100.0 ± 0.0
99.3 ± 0.5
96.2 ± 1.4
79.5 ± 3.4
59.6 ± 3.3
47.3 ± 2.9**
40.1 ± 3.6*
40.1 ± 3.6*
Relaxation of aorta to sodium nitroprusside
log concentration
(mol.L–1)
-9.0
-8.5
-8.0
-7.5
-7.0
-6.5
-6.0
-5.5
-5.0
% tone remaining
C (n=6)
99.8 ± 0.2
94.6 ± 0.7
77.8 ± 1.6
48.1 ± 2.3
17.9 ± 1.8
6.2 ± 0.4
2.8 ± 0.4
0.0 ± 0.0
0.0 ± 0.0
DM (n=6)
100.0 ± 0.0
95.2 ± 0.4
76.8 ± 0.6
44.5 ± 0.8
14.7 ± 0.3
5.2 ± 0.1
1.4 ± 0.1
0.0 ± 0.0
0.0 ± 0.0
FCH (n=6)
100.0 ± 0.0
94.7 ± 1.1
79.7 ± 2.7
56.9 ± 5.3
25.7 ± 5.4
5.5 ± 1.2
2.1 ± 0.8
0.0 ± 0.0
0.0 ± 0.0
DM-HCH (n=6)
100.0 ± 0.0
92.4 ± 2.0
74.4 ± 6.2
48.9 ± 6.4
20.3 ± 3.8
7.5 ± 0.9
3.7 ± 0.7
1.5 ± 0.6#
0.0 ± 0.0
**P < 0.01 vs. C, *P < 0.05 vs. C, #P < 0.05 vs. C, DM, FCH
cardiovascular risk factors on eNOS expression has not
been studied yet. We decided to study influence of shortterm experimental diabetes, administration of fat-cholesterol diet and their combination on endothelial function,
expression of eNOS and its allosteric inhibitor cav-1 in
left ventricle and aorta of rats.
METHODS: Male Wistar rats were divided into
4 groups labeled as control (C), diabetic (DM), group
fed with fat-cholesterol diet (FCH) and diabetic-hypercholesterolaemic group (DM-HCH). Experimental diabetes was induced by a single application of streptozotocin
(80 mg/kg, i.p.). Fat-cholesterol diet was administered
within 5 days. eNOS and cav-1 expression was detected by
the Western blot method. Endothelial function was evaluated according to relaxation of norepinephrine-precontracted isolated aorta after administration of acetylcholine
and sodium nitroprusside in different concentrations.
RESULTS: Glucose plasma level in DM group was
21.40 ± 1.77 mmol.L–1 (P<0.001 vs. C). Cholesterol levels
were 1.51 ± 0.07 mmol.L–1 in plasma, 4.99 ± 0.66 mg/g
in liver of FCH group (NS vs. C, NS vs. DM) and 8.65
± 2.96 mmol.L–1 in plasma, 6.28 ± 0.86 mg/g in liver in
DM-HCH group (P<0.05 vs. DM). Changes in eNOS
and cav-1 expression were not registered in the left ventricle of any group of animals. In aorta of diabetic animals,
we observed a tendency of eNOS and cav-1 expression
to increase in comparison with the control group. As
shown in Fig. 1, eNOS expression increased twofold in
DM-HCH animals in comparison with the control ones
and FCH group (P < 0.05), expression of cav-1 increased
significantly by 42 % in comparison with FCH group
(P<0.05). Ability of isolated aorta to relax to acetylcholine decreased in C, FCH, DM and DM-HCH groups
(Table 1). Decreased ability of aorta to relax was significant in DM-HCH animals according to control group
(P<0.05). Relaxation of aorta to sodium nitroprusside
was not influenced by short-term experimental diabetes
and fat-cholesterol diet.
CONCLUSIONS: Neither short-term experimental diabetes nor administration of the fat-cholesterol diet alone
caused changes in eNOS nor cav-1 proteins expression in
rat left ventricle and aorta. Expression of eNOS and cav-1
was increased by the combination of both cardiovascular
risk factors in the aorta of diabetic-hypercholesterolaemic
animals. Relaxation of isolated aorta to acetylcholine and
exogenous NO donor sodium nitroprusside in diabetichypercholesterolemic rats corresponded to a worsened
endothelial function. Although elevated eNOS expression
was observed, increased cav-1 expression may be responsible for this undesirable reaction.
ACKNOWLEDGEMENT
Financial support by the grants UK/26/2006,
UK/46/2007, VEGA 1/4296/07 is gratefully acknowledged.
REFERENCES
1. Desjardins F, Balligand JL. Nitric oxide-dependent endothelial function and cardiovascular disease. Acta Clin Belg 2006; 61:326–34.
2. Hadi HA, Carr CS, Al Suwaidi J. Endothelial dysfunction: cardiovascular risk factors, therapy, and outcome. Vasc Health Risk
Manag 2005; 1:183–98.
3. Yetik-Anacak G, Catravas JD. Nitric oxide and the endothelium:
history and impact on cardiovascular disease. Vascul Pharmacol
2006; 45:268–76.
4. Huige L, Wallerath T, Münzel T, Försterman U. Regulation of endothelial-type NO synthase expression in pathophysiology and in
response to drugs. Nitric Oxide 2002; 7:149–64.
29
5. Feron O, Dessy C, Moniotte S, Desager JP, Balligand JL.
Hypercholesterolemia decreases nitric oxide production by promoting the interaction of caveolin and endothelial nitric oxide
synthase. J Clin Invest 1999; 103:897–905.
6. Coppinger RJ, Baum CL. Cholesterol-dependent regulation of nitric oxide production: potential role in atherosclerosis. Nutr Rev
1999; 57:279–82.
7. Oyadomari S, Gotoh T, Aoyagi K, Araki E, Shichiri M, Mori M.
Coinduction of endothelial nitric oxide synthase and arginine
recycling enzymes in aorta of diabetic rats. Nitric Oxide 2001;
5:252–60.
8. Ma G, Al-Shabrawey M, Johnson JA, Datar R, Tawfik HE, Guo
D, Caldwell RB, Caldwell RW. Protection against myocardial
ischemia/reperfusion injury by short-term diabetes: enhancement
of VEGF formation, capillary density, and activation of cell survival signaling. Naunyn Schmiedberger’s Arch Pharmacol 2006;
373:415–427.
ARGININE METABOLISM IN A RAT MODEL OF BRONCHIAL ASTHMA
Zuzana Havlinova, Jaroslav Chladek, Stanislav Micuda, Jana Prokopova, Jirina Martinkova
Department of Pharmacology, Faculty of Medicine, Charles University, Simkova 870, 500 38 Hradec Kralove, Czech Republic
Key words: Arginine metabolites/Arginase/ iNOS/Rat/Allergic airway inflammation
BACKGROUND: Airway inflammation in asthma is
associated with an enhanced expression of inducible nitric
oxide synthase (iNOS) and arginase (AG). Arginine is
a substrate for both enzymes. Involvement of L-arginine
metabolism via iNOS and AG in asthma pathogenesis
is a subject of intensive research. The goal of this work
was to develop the methods for evaluation of iNOS and
arginase expression and activity in the airways and to validate these methods using the repeated-antigen-exposure
model in rats.
METHODS: All experimental protocols were approved
by the Institutional Animal Care and Use Committee of
the Faculty of Medicine in Hradec Kralove.
Sensitization of rats. Male Wistar rats: (Biolab, Konarovice) that were 6–8 weeks of age and weighed around
250–280 g were used for the study. The rats were actively
sensitized on Days 1 and 8 against ovalbumin (OVA)
by subcutaneous (s.c.) injection of sterile normal saline
(0.15 mL) containing 0.2 mg OVA (grade II; Sigma, St.
Louis, MO, USA) and 8 mg aluminum hydroxide (Sigma).
The animals were used 14 to 16 days after the first injection. Sensitized rats were challenged twice within 24 h
by intratracheal administration of 1 mg OVA in 0.2 ml
sterile saline. At 6 h after the second i.t. administration,
2 ml of blood was taken under ether anesthesia from
the abdominal aorta and the rats were exsanguinated.
Bronchoalveolar lavage (BAL) was performed via the
tracheal cannula using 2 × 5 ml of cold saline. The lavage fluid was centrifuged at 1000 × g for 10 min at 4 oC
and the supernatant stored at –80 oC. The cell pellet was
resuspended in 1.0 mL sterile saline with 0.2 % rat serum.
The total cell count was determined by adding trypan blue
stain and then counting the cells under a light microscope.
The lung were removed and immediately frozen in liquid
nitrogen and stored at −80 oC until further use.
Experimental groups: Controls-saline: s.c. administration of Al(OH)3, intratracheal administration of saline;
Saline-OVA: sensitized and OVA-challenged rats, i.p. administration of saline at –1 h, 6 h a 23 h since the first
challenge; NAME–OVA: sensitized and OVA-challenged
rats, i.p. administration of 100 mg/kg L-NAME at –1 h,
6 h a 23 h since the first challenge; AG-OVA: sensitized
and OVA-challenged rats, i.p. administration of 30 mg/kg
aminoguanidine at –1 h, 6 h a 23 h since the first challenge; DEX-OVA: sensitized and OVA-challenged rats,
oral administration of 1mg/kg dexamethasone at –24 h,
0 h a 24 h since the first challenge.
Western blot analysis of arginase I: Lung tissue was
thawed and then homogenized in 50 mM Tris HCL buffer
(pH 7.4) containing 1 nM PMSF, 1 mM DTT, 1% Tritone
X-100, 10 μg/mL pepstatin A, 10 μg/mL aprotinin and
10 μg/mL leupeptin. Aliquots of the supernatants were
used for SDS-PAGE electrophoresis (10 % SDS, 15 mA,
200 V). The proteins were transferred to nitrocellulose
membranes, and the membranes were blocked in phosphate saline buffer containing 5 % nonfat dried milk. The
membranes were incubated for 1 h with the primary arginase I antibody (diluted 1:1000, BD Transduction Lab.)
and then washed with phosphate saline buffer (pH 7.4).
The secondary antibody was peroxidase-labelled antimouse antibody (GE Healthcare), which was diluted
1 : 5 000 before use. Immunoblots were developed using
30
Table 1. Body and lung weights, leucocyte counts and results for nitrite (NI) and nitrate (NA), arginine (ARG),
monomethylarginine (NMMA) and dimethylarginine (symetrical SDMA, asymetrical ADMA) concentrations in
bronchoalveolar lavage and plasma.
Controlssaline
SalineOVA
NAME-OVA
AGOVA
DEXOVA
Body weight (g)
Lung weght (g)
276 (5.5)
2.6 (0.09)
259 (6.6)
2.8 (0.2)
264 (11)
3.1 (0.2)
262 (7)
2.9 (0.2)
255 (6)
2.5 (0.09)
Leu in plasma (×109 L–1)
5.5 (0.7)
6.3 (0.7)
6.7 (1.1)
5.9 (0.6)
6.4 (0.5)
0.22 (0.05)
0.20 (0.20)
0.36 (0.26)
0.66 (0.21)
0.64 (0.13)
1.38 (0.33)
18.6 (2.67)
156 (12.4)
0.83 (0.22)
0.66 (0.17)
0.26 (0.07)
0.61 (0.02)
1.94 (0.08)
0.57 (0.25)
2.1 (0.4)
1.44 (0.15)
29.3 (8.1)
170 (15)
1.26 (0.17)
0.87 (0.04)
0.36 (0.04)
0.67 (0.02)
3.17 (0.4)e
1.9 (0.3)h
6.6 (1.6)
1.52 (0.06)
5.8 (0.49)a
203 (10)
1.2 (0.1)
1.05 (0.07)
0.38 (0.04)
0.61 (0.015)
2.49 (0.24)
2.5 (0.2)h
8.9 (1.3)h
1.71 (0.05)
23.1 (3.3)
192 (14.7)
1.06 (0.16)
0.83 (0.02)
0.34 (0.02)
0.7 (0.04)
1.12 (0.15)f
1.7 (0.3)
6.1 (2.0)
1.81 (0.28)
8.0 (0.6)b
106 (6.6)c
0.49 (0.06)
0.58 (0.05)d
0.23 (0.01)
0.67(0.018)
0.62 (0.15)g
1.3 (0.2)
2.4 (0.6)
Leu in BAL (×109 L–1)
–1
NI in plasma (μmol.L )
NA in plasma(μmol.L–1)
ARG in plasma (μmol.L–1)
NMMA in plasma (μmol.L–1)
ADMA in plasma (μmol.L–1)
SDMA in plasma (μmol.L–1)
NI in BAL (μmol.L–1)
NA in BAL (μmol.L–1)
ARG in BAL (μmol.L–1)
NMMA in BAL (nmol.L–1)
a, b
p < 0.05 vs. Saline-OVA, c p < 0.05 vs. NAME-OVA and AG-OVA, d p < 0.05 vs. NAME-OVA, ep < 0.05 vs. ControlsSaline, f p < 0.05 vs. Saline-OVA and NAME-OVA, g p < 0.05 vs. Controls-saline and Saline-OVA and NAME-OVA,
h
p < 0.05 vs. Controls-saline
Arginase I activity in lung homogenates
140
900
Activity in % of Control-saline
Optical density in % of Control-saline
Arginase I expresion in lung homogenates
120
100
80
60
40
20
0
800
700
600
500
400
300
200
100
0
Saline-OVA
NAME-OVA
AG-OVA
DEX-OVA
Saline-OVA
NAME-OVA
AG-OVA
DEX-OVA
Fig. 1. Western blot of arginase I and arginase I activity in cytosolic fractions of lung homogenates.
the enhanced chemiluminiscence kit (Pierce) according
to the manufacturer’s instructions. Arginase activity was
measured as a rate of arginine conversion to urea by using the urea reagent (Sigma-Aldrich, St. Louis, Missouri,
USA). Arginine and its metabolites (NMMA -N-monomethylarginine, ADMA – asymetrical dimethylarginine,
SDMA – symetrical dimethylarginine) were assayed by
liquid chromatography after derivatisation with o-phthaldialdehyde-mercaptopropionic acid1.
RESULTS: Sensitized and OVA-challenged animals
(OVA-Saline) showed trends towards increased concentrations of total protein in BAL, nitrates in BAL and plasma,
arginine in BAL and plasma and leucocyte counts in BAL
(Table 1). However, only nitrates and arginine in BAL
were significantly increased above the levels observed
in control animals (Controls-Saline). Administration of
L-NAME and AG (nonspecific inhibitors of iNOS) as
well as of DEX reduced nitrate concentrations in BAL
and plasma of OVA-challenged animals. The effect of
iNOS inhibitors on arginine concentrations in BAL
and plasma of sensitized and OVA-challenged animals
differed from that of dexamethasone. L-NAME caused
further increase, aminoguanidine left the concentrations
unchanged while dexamethasone significantly reduced
arginine concentrations. There were only subtle changes
between experimental groups in expression of arginase
I in lung homogenates (Fig. 1). Measurement of arginase
activity in lung homogenates revealed an interesting effect
of aminoguanidine, which augmented the activity by some
not yet identified mechanism (Fig. 1).
CONCLUSION: We have developed a rat model suitable for the study of L-arginine metabolism and transport
in allergic airway inflammation. Data obtained during
this pilot study suggest that changes in concentrations
of arginine and methylated metabolites in BAL parallel
those in the plasma. The putative effects of inhibitors of
iNOS (decreased NO formation) and of dexamethasone
(decreased NO formation and arginine availability in the
31
airways) were clearly demonstrated. However, further refinement of the modell with regard to allergen challenge
and measurement of NOS expression and activity is mandatory.
ACKNOWLEDGEMENT
The study was supported by a grant from the Czech
Ministry of Education and Youth MSM 0021620820.
REFERENCES
1. Teerlink T, Nijveldt RJ, de Jong S, van Leeuwen PA. Determination
of arginine, asymmetric dimethylarginine, and symmetric dimethylarginine in human plasma and other biological samples by
high-performance liquid chromatography. Anal Biochem 2002;
303:131–7.
READMINISTRATION OF NEWLY SYNTHESIZED ULTRASHORT-ACTING BETA
BLOCKER 444 – PILOT STUDY
Klara Horkaa, Ladislava Bartosovaa, Marek Frydrycha, Pavel Suchya
a
Department of Human Pharmacology and Toxicology, Faculty of Pharmacy, University of Veterinary and Pharmaceutical
Sciences, Palackeho 1–3, Brno, Czech Republic
Key words: Ultrashort-acting betalytics/Readministration/Experiment in vivo
BACKGROUND: The tested compound 444 is an original that has been made by the Department of Chemical
Drugs at the Faculty of Pharmacy of the UVPS Brno. It
is functional analogue of aryloxyaminopropanol modified
in the aliphatic section of the molecule by the tertiary
butyl1. On the basis of chemical structure we supposed
that 444 compound is an ultrashort-acting beta-blocker.
Pharmacological effects of ultrashort-acting beta-blockers
Fig. 1. Differences of the bradycardic effect of the readministrated drug in the each measured time interval.
32
last only several minutes, and their intravenous administration for indicated patients in critical care situations is
much safer than the use of longeracting beta-adrenergic
blocking agents2, 3. In previous experiments we confirmed
that compound 444 has a significant bradycardic effect4.
The aim of the study was to find out, whether bradycardic
effect of 444 is the same after readministration compared
with the first administration or not.
METHODS AND RESULTS: An experiment was
proven in vivo on 14 laboratory rats. The substance was
tested in dose of 2.5 mg·kg–1. A time interval between
administrations lasted one hour. The methodology of
the experiment was approved and monitored by the local Ethical Committee for Animal Welfare at the UVPS
Brno.
The general anaesthesia was induced by the
administration of 1% ketamin (Narkamon® inj. Spofa)
and 2% xylazin (Rometar® inj. Spofa). During the whole
experimental time the animals were kept in the general
anaesthesia. The tested compound was administered i.v.
to the vena jugularis as a bolus dose. The heart rate was
monitored with ECG SEIVA Praktik Veterinary. The first
measurement was always made immediately before the
administration and its value was marked with 100 %. To
determine the statisticly significant heart rate changes
among individual administrations the Mann-Whitney Utest was used (Unistat 5.1).
A graphical presentation of the results – heart rate
changes along the time line is shown in Fig. 1. Results of
statistic analysis are given in Table 1.
CONCLUSIONS: Each readministration caused
a more delayed onset of action of the compound 444,
while the bradycardic effect increased. Statistical significance of the heart rate changes was confirmed between
the first and the second administrations and between the
first and the third one. The first administration caused
a maximum decrease of 17.9 % in the heart rate in the
20th minute, the second administration of 18.8 % in the
12th minute and the third administration of 34.9 % in the
10th minute compared with the baseline. We supposed
that bradycardic effect increased after readministration of
compound 444 in dose 2.5 mg.kg–1 due to sensitisation of
a drug targets. We didn’t confirm the idea, that readministration of 444 compound induces tachyphylaxis.
Table 1. Statistical resume of the experiment
(Mann-Whitney U-test).
Time interval
[min]
0
0.5
1
2
3
4
5
6
8
10
12
14
16
18
20
25
1st adm.
x
2nd adm.
*
*
***
***
***
***
***
***
***
***
***
***
***
***
-
1st adm.
x
3rddm.
*
**
**
**
**
**
***
**
**
*
*
**
**
-
2nd adm.
x
3rd adm.
-
Note: 1st adm. = the first administration;
2nd adm. = the second administration,
3rd adm. = the third administration;
0 = the moment of i. v. drug administration of tested
substance;
p < 0.05 * ; p < 0.01 ** ; p < 0.001 ***
ACKNOWLEDGEMENT
This study was supported by grants GA CR No.
305/06/0863 and IGA MZ CR No. NR9126-3/2006.
REFERENCES
2. Reilly CS, Wood M, Koshakji RP, Wood AJ. Ultra-short acting
beta-blockade: a comparison with conventional beta- blockade. Clin
Pharmacol Ther 1985; 38:579–85.
3. Gray RJ. Managing critically ill patients with esmolol. Chest 1988;
93:398–403.
4. Bartosova L, Frydrych M, Vaculova G, Berankova M, Bebarova
M. et al. Ultrashort Bradycardic Effect of Newly Synthesized
Compounds. Acta Vet Brno 2006; 75:183–96.
33
THE EFFICACY AND TOLERABILITY OF ESCITALOPRAM DURING 6 MONTHS
OF OUTPATIENT’S TREATMENT
Radovan Hrubya,b, Igor Ondrejkab , Gabriela Nosalovaa, Sabina Hrubac, Valeria Kerna jr.a,b
a
Institute of Pharmacology, Jessenius Faculty of Medicine, Martin, Slovak Republic
Clinic of Psychiatry, Jessenius Faculty of Medicine, Martin, Slovak Republic
c
Private Outpatient Psychiatry Unit, Martin, Slovak Republic
b
Key words: Escitalopram/Efficacy/Tolerability/Outpatient/Treatment
BACKGROUND: The long-life prevalence of depressive disease is 5–15 % in men and 9–26 % in women1. High
seriousness and occurrence of depressive diseases lead
to the progressive development of new antidepressants.
Nowadays, antidepressants of first choice are selective serotonine reuptake inhibitors (SSRI). Application of SSRI
in clinical practice made a significant progress in treatment of depressive disease. Clear advantages of SSRI’s
are selective impact on the serotonine system, absence of
adverse effects due to influence on other neurotransmitter’s receptors, simple dosage management and very good
efficacy2. A typical representative of SSRI is citalopram. It
is a racemate of S- and R-enantiomers. Pharmacological
studies indicate, the antidepressant’s action of citalopram
is mostly associated with the action of S-enantiomer, escitalopram3. So, the next step in the development of modern
antidepressants was an agent formed by the S-enantiomer,
without inhibiting influence of R-citalopram. Some data
suggest escitalopram (S-enantiomer) proves better clinical
efficacy as citalopram4. Many modern pharmacological
studies usually prefer to probe pharmacological properties
of new agents only for several weeks. Our clinical study
followed efficacy and tolerability of escitalopram during
6 months of outpatient’s treatment.
METHODS: A total of 18 patients were suffering
from depressive epizode, reccurent depressive disorder
or anxious-depressive disorder (according to ICD-10) and
undertaken our open-label prospective study. The aim of
the study was to evaluate efficacy and tolerability of escitalopram in depressed nonpsychotic outpatients treated
in psychiatry care with escitalopram in monotherapy
during 6 months. All of 12 females and 6 males with an
average age of 36.2 years were treated with escitalopram
in dose of 10 mg daily. During 6 months of clinical trial
an assessment of tolerability and efficacy of escitalopram was performed repeatedly. The efficacy of escitalopram/intensity of depression was assessed by MADRS
(Montgomery-Asberg Depressive Rating Scale) during
the 1st visit of patient (baseline assessment-MADRS 0)
and then after the 1st, 3rd and 6th month (MADRS 1, 3,
6) of outpatient’s treatment. The tolerability of escitalopram was assessed by SARS (Structured Adverse Effects
Rating Scale) also at the same time periods (SARS 0, 1,
3, 6). Change of total MADRS score indicates the efficacy
of agent and the treatment response is characterized as
50% reduction of MADRS total score at the beginning of
treatment. Changes of total SARS score during treatment
indicate the tolerability of agent (lower the total score,
better is tolerability of an agent). Total SARS score at
baseline assessment (SARS 0) is always 0, because it is
the time of first application of an agent. Obtained data
were consequently processed by comparative analysis, using Wilcoxon’s pair test.
RESULTS: We have observed a significant decrease
in total MADRS score after 1st month of treatment
(MADRS 0 vs MADRS 1, P = 0.038), after the 3rd
(MADRS 0 vs MADRS 3, P = 0.0019) and also 6th month
(MADRS 0 vs MADRS 6, P = 0.0002). There were no significant differences in MADRS 1 vs MADRS 3, MADRS
1 vs MADRS 6 and MADRS 3 vs MADRS 6. The treatment response (more than 50% decrease in total MADRS
score) scored 6 patients (33.3 %) after first month, 10 patients (55.6 %) after 3 months and 12 patients (66.7 %)
after 6 months. Values of total SARS score remained at
very low levels after the 1st and also 3rd and 6th month
of treatment. The highest SARS score was observed after
1st month of treatment with significant decrease after 3rd
(SARS 1 vs SARS 3, P=0.0431) month and 6th month
(SARS 1 vs SARS 6, P=0.117). There were no significant
differences between SARS 3 vs SARS 6. Total SARS score
after 6th month showed 0 in all patients, which means
there were no adverse effects reffered.
CONCLUSIONS: The treatment with escitalopram
during 6 months has lead to the significant decrease of
depression intensity in study sample (Fig. 1). The treatment response was observed in 66.7 % patients after six
months of therapy. There was a very low occurrence of
adverse effects during treatment, with it’s significant decrease after 3rd and also 6th month. The efficacy and
very good tolerability of escitalopram have been already
validated in several placebo-controlled studies5–7. Our data
confirmed efficacy and very good tolerability of escitalopram monotherapy during 6 months in nonpsychotic
depressed outpatients. Unipolar major depression is the
most common reason of invalidity all over the world and
affective disorders usually occur for the first time in early
adulthood (25–32 years) and affect approximately one
fifth of population8. Antidepressants cause clearly better
conditions in 65–70 % of patients, but complete recovery
is observed only in 40–50 % of patients9. This facts should
34
B o x & W h is k e r P lo t
28
24
20
16
12
8
4
0
MAD R S 0
MAD R S 1
MAD R S 3
MAD R S 6
Min -Ma x
2 5 % -7 5 %
Me d ia n va lu e
Fig. 1. Decrease in MADRS total score during 6 months of escitalopram monotherapy.
Axis Y – MADRS total score
Axis X – MADRS 0 (baseline assessment), MADRS 1 – after 1st month,
MADRS 3 – after 3rd month, MADRS 6 – after 6th month
be the reason to keep on advancement of new antidepressant agents with new mechanisms of action. Application
of escitalopram in clinical practice is the next step to improve depressed patient’s conditions.
REFERENCES
1. Seifertová D, Bareš M, Kopeček M. Léčba farmakorezistentní deprese. Farmakoterapie 2005; 6.589–94.
2. Švestka J. SSRI léky prvé volby. Praha: Maxdorf s.r.o.;1998.
3. Hyttel J, Bogeso KP, Perregaard J, Sanchez C. The pharmacological
effect of citalopram reside in the (S)-(+)- and not the R-(-)- enantiomer. J Neural Transm 1992; 88:157–60.
4. Lepola U, Wade A, Andersen HF. Do equivalent doses of escitalopram and citalopram have similar efficacy? A pooled analysis of
two positive placebo-controlled studies in major depressive disorder. Int Clin Psychopharmacol 2004;19:149–55.
5. Llorca PM, Azorin JM, Despiegel N, Verpillat P. Efficacy of escitalopram in patients with severe depression: a pooled analysis. Int
J Clin Pract 2005; 59:268–75.
6. Burke WJ, Gergel I, Bose A. Fixed – dose trial of the single isomer SSRI escitalopram in depressed outpatients. J Clin Psychiatry
2002; 63:331–6.
7. Montgomery SA, Huusom AKT, Bothmer JA. Randomised study
Comparing Escitalopram with Venlafaxine XR in Primary Care
Patients with Major Depressive Disorder. Neuropsychology 2004;
50:57–64.
8. Kessler, R. C., Berglund, P., Demler, O. Lifetime prevalence and
age-of-onset distributions of DSM-IV disorders in the National
Comorbidity Survey Replication. Arch Gen Psychiatry 2005;
62:593–602.
9. American Psychiatric Association. Practice guidelines for major
depressive disorder in adults. Am J Psychiatry 1993;150 (Suppl.4):
S1–S26.
35
A RANDOMIZED CROSS-OVER STUDY OF FOLIC ACID SUPPLEMENTATION
DURING THE INITIAL PHASE OF PSORIASIS TREATMENT
WITH METHOTREXATE
Jaroslav Chladeka, Jaroslava Vaneckovab, Marie Simkovab, Milos Hrocha, Jirina Martinkovaa,
Jiri Grimc
a
Department of Pharmacology, Faculty of Medicine, Charles University, Simkova 870, 500 38 Hradec Kralove, Czech
Republic
b
Department of Dermatology, University Hospital, Hradec Kralove, Czech Republic
c
Department of Oncology, University Hospital, Hradec Kralove, Czech Republic
Key words: Psoriasis/Methotrexate/Pharmacokinetic/Folic acid
BACKGROUND: Major problem of systemic antipsoriatic therapy with oral methotrexate (MTX) is to attain
long-term efficacy with favorable toxicity profile. Folate
supplementation prevents excessive depletion of intracellular folates caused by MTX. According to a recent survey,
folic acid is prescribed using empirical rules regardless
of the intracellular folate status and levels of MTX polyglutamates1. Moreover, recommended weekly doses vary
between 5 to 75 mg of folic acid. We have undertaken
a randomized cross-over study to investigate the effects
of folic acid supplementation during the initial 32 weeks
of psoriasis treatment with MTX.
METHODS: Nineteen patients (13 M/6 F) aged
49±10 yr with moderate to severe chronic plaque psoriasis (The Psoriasis Activity and Severity Index, PASI
= 23.5±7.4) were enrolled in the study and randomly
assigned into two treatment arms. Patients in the arm
A received MTX and folic acid for initial 16 weeks
Table 1. Demographic characteristics of patients. Evaluation of disease activity using the Psoriasis area and severity index (PASI). Results of laboratory tests and of the analysis of methotrexate polyglutamates in red blood cells
(RBC MTXPG).
Study arm A
(MTX + FA WEEK 1-16)
Baseline
Week 16
Week 32
5F/5M
45 (SD 7)
84 (SD 15)
17 (SD 4)
-
Study arm B
(MTX + FA WEEK 17-32)
Baseline
Week 16
Week 32
1F/8M
53 (SD 11)
89 (SD 19)
16 (SD 4)
-
20.1 (2.1)
-
8.8 (1.3)
44.9 (10.9)
96.2 (10.6)
5.7 (1.2)
60.1 (12.8)
103 (10.9)
27.2 (2.1)a
-
5.1 (2.0)
82.6 (6.0)b
111 (9.8)
6.5 (2.7)
80.3 (7.1)
83.6 (10.7)
35.8 (6.5)
961 (173)
33.8 (2.3)
1090 (158)
16.6 (2.7)
739 (94)
20.8 (4.5)
731 (130)
15.1 (2.6)c
618 (91)a
29.8 (2.8)b
843 (130)
Homocysteine(μmol. L–1)
VRBC (fL)
10.1 (0.85)
91.2 (0.79)
9.6 (1.0)
92.8 (0.87)
10.4 (1.0)
93.0 (1.0)
10.9 (1.2)
90.2 (1.7)
13.0 (1.2)d
94.5 (1.8)
8.2 (0.52)
92.4 (1.9)
ALT (μkat.L–1)
0.57 (0.08)
0.59 (0.09)
0.83 (0.22)
0.66 (0.15)
0.89 (0.22)
0.69 (0.12)
AST (μkat.L )
0.53 (0.08)
0.48 (0.09)
0.56 (0.09)
0.51 (0.07)
0.58 (0.07)
0.55 (0.08)
–1
0.82 (0.31)
0.55 (0.18)
0.63 (0.17)
0.70 (0.20)
0.70 (0.16)
0.84 (0.35)
N
Age (yr)
Body weight (kg)
Dose (mg MTX/week)
PASI
on the scale 0-72
%-improvement
RBC MTXPG (nmol.L–1)
Folates
Plasma (nmol.L–1)
RBC (nmol.L–1)
–1
GMT (μkat.L )
Data are given as means (SEM) unless otherwise indicated. Between-arm comparison at the corresponding week:
p < 0.05, b p < 0.01, c p < 0.001, d p = 0.051.
a
(weeks 1–16) followed by MTX monotherapy for additional 16 weeks (weeks 17–32). In the arm B, the sequence of
treatments was the oposite. MTX was given once a week
in three oral doses separated by 12-h intervals. Folic acid
(20 mg/week) was administered in two single oral doses of
10 mg two and four days after MTX administration. The
weekly MTX dose was individualized as follows: One week
preceeding the study, the area under the concentrationtime curve of plasma MTX in the interval 0–8 h postdose
(AUC0–8h) was measured after a bolus dose of 10 mg and
the starting weekly dose was calculated in order to achieve
the target AUC0–8h of 1800 nmol.h.L–1. The grade of skin
impairment was evaluated using the Psoriasis Activity and
Severity Index (PASI) at baseline and at 4-week intervals.
Safety of therapy was evaluated by means of laboratory
tests (haemoglobin, total erythrocyte, leukocyte and platelet counts, bilirubin, aminotransferases, γ-glutamyltransferase, creatinine, homocysteine, plasma and red blood
cell folates) at 4-week intervals. Concentrations of plasma
MTX and that of MTXPGs in red blood cells were assayed
using liquid chromatography methods2,3.
RESULTS: The AUC0–8h after the test dose achieved
1120 ± 390 and 1390 ± 530 nmol.h.L–1 in the study arms
A and B, respectively. Dosing of MTX was comparable
between study arms (Table 1). MTXPGs gradually accumulated in RBC during the initial 16 weeks of therapy.
MTXPGs levels in RBC at weeks 16 and 32 were similar
in both study arms. Comparisons of patient characteristics at the baseline revealed that patients in the study
arm B had somewhat higher PASI than patients in the
arm A (Table 1). Folic acid supplementation caused
expected changes in plasma and RBC folate concentrations. Results of other laboratory tests were comparable
between study arms except for plasma homocysteine and
the mean corpuscular volume of the erythrocyte at the
week 16 which increased in the study arm B as a sign of
a decreased folate pool (the differences were marginaly
significant). Percent improvements in PASI in the course
of the study are shown in Fig. 1. At the week 16, the mean
percent improvement in PASI was almost 50 % less in
the study arm A compared to the arm B as a result of
folic acid supplementation. In the second phase of the
study, discontinuation of supplementation in study arm
Improvement in PASI (%)
36
100
90
80
NS
70
60
50
40
30
p<0.01
MTX + Folic acid (Week 1-16)
20
10
0
MTX + Folic acid (Week 17-32)
0
4
8
12
16
20
24
28
32
Week
Fig. 1. Percent improvement in Psoriasis Area and
Severity Index in each treatment arm. Data are
means (SEM).
A accelerated improvement in PASI while, at the same
time, folic acid administration resulted in only minor deterioration of PASI scores in the arm B.
CONCLUSIONS: Results of this study clearly show
that folic acid reduces the efficacy of MTX. Its use at the
start of psoriasis treatment with MTX its strongly discouraged. However, this does not necessarily indicate that
folic acid added later in the course of long-term treatment
decreases the benefit/risk ratio of MTX.
ACKNOWLEDGEMENT
The study was supported by a grant from the Czech
Ministry of Health no. NR7947-3/2004 and from the Czech
Ministry of Education and Youth MSM 0021620820.
REFERENCES
1. Boffa MJ. Methotrexate for psoriasis: current European practice.
A postal survey. J Eur Acad Dermatol Venereol 2005; 19:196–
202.
2. Chladek J, Grim J, Martinkova J, Simkova M, Vaneckova J,
Koudelkova V, Nozickova M. Pharmacokinetics and pharmacodynamics of low-dose methotrexate in the treatment of psoriasis. Br
J Clin Pharmacol 2002; 54:147–56.
3. Chladek J, Grim J, Martinkova J, Simkova M, Vaneckova J. Lowdose methotrexate pharmacokinetics and pharmacodynamics in
the therapy of severe psoriasis.Basic Clin Pharmacol Toxicol 2005;
96:247–8.
37
PYCNOGENOL® DECREASES ELEVATED LEVELS OF ENDOTHELIAL
NITRIC OXIDE SYNTHASE IN DIABETIC RATS
Stanislava Jankyovaa, Diana Yaghia, Jan Klimasa, Peter Kreneka, Jana Navarovab,
Stefan Matyasa
a
Department of Pharmacology and Toxicology, Faculty of Pharmacy UK, 83232 Bratislava, Slovak Republic
Institute of Experimental Pharmacology, Slovak Academy of Sciences, 841 04 Bratislava, Slovak Republic
b
Key words: Diabetes/eNOS/Caveolin/Hsp90/Pycnogenol®
BACKGROUND: Endothelial nitric oxide synthase
(eNOS) is one of three isoenzymes that converts L-arginine to L-citrulline. It is localized in caveolae of plasmalemmal membrane1 and plays a key role in a production of
NO, which has relaxing effects on vessel’s smooth muscle
cells2. According to the type of tissue, in caveolae are three
types of caveolines. Myocardial cells contain caveolin-3,
endothelial cells contain caveolin-1 (ref.3). The interaction of eNOS with several proteins, e.g. caveolin-1 (ref.4)
and heat shock protein 90 (Hsp90), has a various sequels.
Hsp90 is 90 kDa protein of heat shock proteins family,
which affects activity and function of other proteins by
changing their structure. Hsp90 binds eNOS and is incorporated in eNOS-complex with the help of agonists, which
stimulate production of eNOS. This coupling increases
the activity of eNOS (ref.5). Caveolin-1, which interacts
with eNOS, by this type of interaction, decreases the activity of eNOS (ref.6). Endothelial dependent vasodilatation, which is largely impaired in diabetes type 1 and 2,
has a direct impact on decreased NO-synthesis and also
causes increase of production free oxygen radicals. This
action can be prevented by increase in the level of eNOS
(ref.7). Kobayashi et all.8 found that in diabetic rats the
expression of endothelial NOS increased, though with
a time period this level of expression decreased. Ibrahim
at all.9, who followed the level of NO, observed decreased
expression of eNOS. These autors concluded9, that streptozotocin-induced diabetes in rats decreases level of NO
in aortas, thus it can be suggested existence of endothelial
dysfunction. The purpose of our study was to observe the
influence of streptozotocin-induced diabetes on expression
of eNOS, caveolin-1 and Hsp90, and to determine effects
of flavonoic extract from Pinus Maritima – Pycnogenol®
on expression of eNOS, its allosteric inhibitor caveolin-1
and Hsp90 in thoracic aorta of diabetic rats.
METHODS: In our study we used male Wistar rats,
which were randomized in three groups (n=8 in each
group). The first control group consisted of healthy rats.
The second group – diabetic was affected by mild type of
diabetes, which was induced by streptozotocin (25 mg/
kg i.p. in three days sequential). In the third group, the
diabetic animals were treated by Pycnogenol®, which had
been administered (50 mg/kg/day per os) to this group
two weeks after diabetes was induced. Treatment lasted
for eight weeks. During the experiment blood glucose was
measured, which was significantly increased in diabetic
rats compared to control group. After animals were sacrified, their organs weights were measured. Thoracic aortas
Fig. 1. Expression of endothelial NO synthase, caveolin-1 and Hsp90 in rats aortas
in control, diabetic and treated group (n = 6–8 in each group). The values
are expressed as mean ± SEM;
* (p < 0.05) diabetic group vs. control group.
38
were homogenized and amount of proteins was assessed
by use of Lowry method. The expressions of endothelial NO synthase, caveolin-1 and Hsp90 were assessed by
SDS-PAGE and Western blotting. After these measurements, densitometric analysis was performed. Statistical
interpretation was made with non-pair Student’s t-test. All
of the proteins were standardized on actine.
RESULTS: In diabetic rats, there was significantly increased the expression of endothelial NO synthase: 187
± 27 % compared to control group, in which expression
was 100 ± 21 % (p < 0.05). In Pycnogenol® treated animals expression of eNOS (138 ± 35 %) was lower than in
diabetic group, and this depression was not statistically
significant. The level of caveolin-1 in diabetic rats (102 ±
15 %) was comparable with that in control group. When
comparing the level of caveolin-1 between diabetic rats
and treated group, in treated group the level of caveolin-1
was decreased (81 ± 6 %). In both groups in diabetic and
treated one, we observed decreased level of Hsp90 expression (diabetic: 85 ± 4 %, treated: 94 ± 5 % against control
group). These changes were not statistically significant.
CONCLUSIONS: The expression of endothelial NO
synthase in diabetic rats significantly increased, however,
Pycnogenol® decreased this expression to the level of
control group. Levels of caveolin-1 and Hsp90 were not
significantly changed either in diabetic or in treated animals (Fig. 1).
ACKNOWLEDGEMENT
Financial support by the grant VEGA SR č.2/5129/25 is
REFERENCES
1. Shaul PW, Smart EJ, Robinson LJ, German Z, Yuhanna IS, Ying
Y, Anderson RGW, Michel T. Acylation Targets Endothelial Nitricoxide Synthase to Plasmalemmal Caveolae. J Biol Chem 1996; 271:
6518–22.
2. Johansen SJ, Harris KA, Rychly DJ and Ergul A. Oxidative stress
and the use of antioxidants in diabetes: Linking basic science to
clinical practice. Cardiovascular Diabetology 2005; 4:5.
3. Tang ZL, Scherer PE, Okamoto TO, Song K, Chu C, Kohtz
DS, Nishimoto I, Lodish HF, Lisanti MP. Molecular Cloning of
Caveolin-3, a novel member of the caveolin gene family expressed
predominantly in muscle. J Biol Chem 1996; 271:2255–61.
4. Feron O, Belhassen L, Kobzik L, Smith TW, Kelly RA and Michel
T. Endothelial nitric oxide synthase targeting to caveolae. Specific
interactions with caveolin isoforms in cardiac myocytes and endothelial cells. J Biol Chem 1996; 271:22810–4.
5. Garcia-Cardena G, Fan R, Shah V, Sorrentino R, Cirino G,
Papapetropoulos A, Sessa WC. Dynamic activation of endothelial
nitric oxide synthase by Hsp90. Nature 1998; 392(6678): 821–4.
6. GrattonDagger JP, Fontana J, O’Connor DS, Garcia-Cardena G,
McCabe TJ, Sessa WC. Reconstitution of an endothelial nric-oxide
synthase (eNOS), Hsp90, and caveolin-1 complex in citro. Evidence
that Hsp90 facilitates calmodulin stimulated displacement of eNOS
from caveolin-1. J Biol Chem 2000; 275:22268–72.
7. Triggle CR, Ding H, Anderson TJ and Pannirselvam M. The endothelium in health and disease: A discussion of the contribution
of non-nitric oxide endothelium-derived vasoactive mediators to
vascular homeostasis in normal vessels and in type II diabetes.
Molecular and Cellular Biochemistry 2004; 263:21–7.
8. Kobayashi T, Matsumoto T, Ooishi K and Kamata K. Differential
expression of {alpha}2D-adrenoceptor and eNOS in aortas from
early and later stages of diabetes in Goto-Kakizaki rats. Am J
Physiol Heart Circ Physiol 2004; 287:H135–48.
9. Ibrahim MA, Kanzakib T, Zamagataa SI, Satoha N and Uedaa
S. Effect of diabetes on aortic nitric oxide synthesis in spontaneously hypertensive rats; does captopril modulate this effect? Life
Sci 2005; 77:1003–14.
METHODS AND RESULTS OF PHARMACOKINETIC RESEARCH
AT THE DEPARTMENT OF PHARMACOLOGY, FACULTY OF MEDICINE,
MASARYK UNIVERSITY, BRNO, CZECH REPUBLIC
Jan Jurica
Department of Pharmacology, Faculty of Medicine, Masaryk University, Brno, Czech Republic
Key words: Research report/ Pharmacokinetics/Dpt. Pharmacology, Masaryk Univ. Brno
BACKGROUND: Research in the field of pharmacokinetics at the Department of Pharmacology, Faculty
of Medicine, Masaryk University in Brno is a part of all
granted research projects focused for a long time on drugs
influencing the CNS functions especially those biodegraded by the isoenzymes of the cytochrome P 450 (CYP).
METHODS: Both, pharmacokinetic studies in vivo
as well as in vitro model of isolated and perfused rat liver
is used. New HPLC and electrophoretic methods for determination of specific markers of metabolic activity for
selected CYP isoenzymes are developed1,2.
RESULTS: Recent results received during last 3 years
of rat studies both in vitro and in vivo showed stimulant in-
teraction potential of methamphetamine with isoenzymes
1A1/2, CYP2C6/11, CYP2D2 a CYP3A4/5 which are
involved in metabolism of clinically used drugs of many
classes3,4. Gender studies confirmed differential bioactivity and sensitivity to changes of isoenzymes analysed in
male and female rats5. There was also found significantly
important effect of St John‘s wort (Hypericum perforatum) extract LI 160 used in the indication of depression
on CYP2C6 cytochrome P-450 activity in perfused rat
liver6. In collaboration with psychiatrists clinical studies
evaluating patient‘s phenotype of metabolic enzymes for
treatment with psychotropic drugs are performed. Results
refer for instance to great importance of CYP2D6 isoen-
zyme relation to efficacy of antidepressants prescribed in
patients suffering from mental anorexia and sexual dysfunctions, and to some extent with gender differences7, 8.
CONCLUSIONS: The team devoted to pharmacokinetic research published Czech textbook on pharmacokinetic principles9.
REFERENCES
1. Jurica J, Dostalek M, Konecny J, Glatz Z, Hadasova E, Tomandl J.
HPLC determination of midazolam and its three hydroxy metabolites in perfusion medium and plasma from rats. J Chromatogr B
2007; doi: 10.1016/j.chromb.2007.02.034
2. KonecnyJ, Jurica J, Tomandl J, Glatz Z. Study of recombinant cytochrome P450 2C9 activity with diclofenac by micellar electrokinetic
capillary chromatography. Electrophoresis 2007; 28:1229–34.
3. Dostalek M, Hadasova E, Hanesova M, Pistovcakova J, Sulcova
A, Jurica J, Tomandl J, Linhart I. Effect of methamphetamine
on pharmacokinetics of dextromethorphan and midazolam. Eur J
Drug Metab Pharmacokinet 2005; 30:195–201.
39
4. Dostalek M, Pistovcakova J, Minarikova V, Jurica J, Tomandl J,
Hadasova E. Effect of acute and sub-chronic administration of
methamphetamine on activity of CYP2D in isolated perfused rat
liver. Acta Vet Brno 2005; 74:339–45.
5. Zahradnikova L, Zendulka O, Jurica J, Dostalek M, Hadasova E..
The influence of gender on metabolic activity of enzymatic system
of cytochrome P450 in the model of isolated perfused rat liver.
Homeostasis 2006; 44:128–30.
6. Dostalek M, Pistovcakova J, Jurica J, Tomandl J, Linhart I, Sulcova
A, Hadasova E. Effect of St John‘s wort (Hypericum perforatum)
on cytochrome P-450 activity in perfused rat liver. Life Sci 2005;
78: 239–44.
7. Zourkova A, Hadasova E, Pilarova L, Robes M, Ravcukova B.
Léčba antidepresivy: volba dle aktivity CYP2D6 a pohlaví. Česká
a Slovenská psychiatrie 2006; 102:183–8.
8. Zourkova A, Hadasova E, Pulkrabkova L, Ravcukova B, Kasparek
T. Clinical impact of CYP2D6 activity in long-term paroxetine treatment. New developments in anxiety disorder research. New York:
Nova Science Publishers 2006; pp. 177–98.
9. Dostalek M, Jurica J, Janostikov E, Zahradnikova L. Farmakokinetika. Praha: Grada; 2006. 220 pp.
EFFECT OF CARBOXYMETHYLATED PYRIDOINDOLES
ON FREE-RADICAL-INDUCED HEMOLYSIS OF RAT ERYTHROCYTES
Maria Juskova, Vladimir Snirc, Alena Gajdosikova, Andrej Gajdosik,
Ludmila Krizanova, Milan Stefek
Key words: Pyridoindoles/Antioxidants/Aldose reductase inhibitors/Hemolysis/ Oxidative stress
BACKGROUND: Although multiple biochemical pathways are likely to be responsible for the pathogenesis of
diabetic complications, substantial evidence suggests a key
role for the polyol pathway and oxidative stress1. Recently,
novel carboxymethylated pyridoindoles, analogues of the
efficient chain-breaking antioxidant stobadine2, were designed, synthesized and characterized as bifunctional compounds with joint antioxidant/aldose reductase inhibitory
activities with potential of antidiabetic agents3,4. Among
the novel compounds developed, (2-benzyl-2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indole-8-yl)-acetic acid (1) was
characterized as an aldose reductase inhibitor efficient
in micromolar region while its saturated analogue (2-benzyl-2,3,4,4a,5,9b-hexahydro-1H-pyrido[4,3-b]indole-8-yl)acetic acid (2) showed only marginal aldose reductase
inhibitory activity3,5. Critical property for the efficacy of
the novel aldose reductase inhibitors in vivo is how well
they penetrate into target tissues. In this study, the issue
was addressed by measuring their antioxidant activity in
the cellular systems of intact erythrocytes exposed to peroxyl radicals generated by thermal degradation of the
azoinitiator 2,2’-azobis(2-amidinopropane)hydrochloride
(AAPH) in vitro.
METHODS: The hemolysis studies were performed in
rat erythrocyte suspensions (2 mL) in isotonic PBS with
the hematocrit at 1.5 %. The compounds studied were add-
ed from stock solutions in isotonic PBS to the erythrocyte
suspensions. After 5 min preincubation, AAPH solution
was added to the final concentration of 30 mM and incubation continued at 37 °C up to 4 hours. The incubations
were terminated by cooling the suspensions in the ice bath
followed by centrifugation at 3000 rpm for 10 min. The
hemolysis degree was estimated by spectrophotometry of
the hemoglobin released into the supernatant as described
by Winterbourn6. The results were described as percent-
Table 1. AAPH–induced hemolysis of rat erythrocytes.
Effect of substituted pyridoindoles in comparison with
standard Trolox.
Compound
No inhibition
Compound 1
Trolox
Compound 2
Stobadine
Lag period (min)
88.6 ± 2.2 (6)
120.6 ± 0.5 (4)
143.5 (2)
144.4 ± 4.2 (4)
> 300 (2)
Results are mean values ± SD from number of experiments
in parentheses.
40
120
Hemolysis (%)
100
80
60
40
20
0
0
40
80
120
160
200
240
Time (min)
Fig. 1. Time course of hemolysis of rat erythrocytes induced by AAPH.
Erythrocyte suspensions (1.5%) were incubated with 30 mM AAPH
alone (solid circles) or in the presence of compound 1 (empty circles). Results are means ± SD values from at least 4 experiments.
age of hemolysis. Total hemolysis (100 %) was assigned
when isotonic PBS was replaced by hypotonic one.
RESULTS: As shown in Fig. 1, rat erythrocytes exposed to AAPH underwent progressive hemolysis; after
3 hours the degree of hemolysis approached 100 %. The
onset of AAPH-induced hemolysis was shifted from the
starting zero point by the time interval assigned as a lag
period. In the presence of the compounds studied the lag
period prolonged significantly (Table 1). The free radical-initiated hemolysis was inhibited by the compounds
studied with decreasing efficiency as follows: stobadine >
compound 2 ~ Trolox > compound 1.
CONCLUSIONS: The results presented here proved
in the system of intact erythrocytes the antioxidant activity of the novel carboxymethylated pyridoindoles developed as potential agents for multitarget pharmacology of
diabetic complications.
ACKNOWLEDGEMENT
Financial support by the grant VEGA 2/5005/26 and
APVV 51-017905 is gratefully acknowledged.
REFERENCES
1. Yabe-Nishimura C. Aldose reductase in glucose toxicity: a potential
target for the prevention of diabetic complications. Pharmacol Rev
1998; 50:21–33.
2. Horakova L, Stolc S. Antioxidant and pharmacodynamic effects of
pyridoindole stobadine. Gen Pharmacol 1998; 30:627–38.
3. Stefek M, Snirc V, Demopoulos V, Djoubissie P, Rackova L,
Majekova M, Karasu C. Carboxymethylated pyridoindoles, preparation and utilization in therapy as aldose reductase inhibitors
and antioxidants. Patent application PP 98–2005 registered on the
16.9.2005 at the Slovak Patent Agency.
4. Djoubissie P-O, Snirc V, Sotnikova R, Zurova J, Kyselova Z, Skalska
S, Gajdosik A, Javorkova V, Vlkovicova J, Vrbjar N, Stefek M. In
vitro inhibition of lens aldose reductase by (2-benzyl-2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indole-8-yl)-acetic acid in enzyme preparations isolated from diabetic rats. Gen Physiol Biophys 2006; 25:
415–25.
5. Stefek M, Djoubissie P-O, Snirc V, Rackova L, Majekova M,
Sotnikova R, Kyselova Z, Skalska S, Javorkova V, Vlkovicova J,
Vrbjar N, Demopoulos V, Karasu C. Carboxymethylated pyridoindoles as aldose reductase inhibitors and antioxidants of zwitterionic
nature. Diabetic Complications 2007; Role of Aldose Reductase
and Related Pathways, Kona, Hawaii, March 24–28, 2007. Program
and Abstracts, p. 61
6. Winterbourn CC. Oxidative reactions of hemoglobin. Meth
Enzymol 1990; 186:265–72.
41
NEGATIVE IMPACT OF ANTIPSYCHOTICS ON CERTAIN SOMATIC FUNCTIONS
Valeria Kerna jr.a,b, Gabriela Nosalovaa, Igor Ondrejkab, Radovan Hrubya,b
a
Institute of Pharmacology, Jessenius Faculty of Medicine in Martin, Slovak Republic
Clinic of Psychiatry, Jessenius Faculty of Medicine in Martin, Slovak Republic
b
Key words: Antipsychotics/Adverse effects/Laboratory screening
BACKGROUND: It is well known that antipsychotics
exert antipsychotic effect and succesfully suppress delusions, hallucinations, abnormal thoughts and bizarre
behaviour. However, they can be associated with several
adverse effects1. The aim of this retrospective study was to
compare levels of liver enzymes (AST, ALT, ALP, GGT),
total and conjugated bilirubin, TAG, total cholesterol, leucocytes, prolactin, fasting glucose and body weight at the
beginning of the antipsychotic therapy and after 4 weeks.
We also assessed its relation to the age and sex.
METHODS: We studied clinical records of 1184 psychiatric patients who were hospitalized at the Clinic of
Psychiatry in Martin during 2003–2006. Antipsychotic
therapy was administered to 340 patients (28.71 %) and
laboratory parameters were examined twice only in 128
patients (37.65 %). We investigated the changes of chosen
parameters, comparing starting levels (without treatment)
with those during the 4th week of treatment.
RESULTS: We have found out the significant increase in body weight, total cholesterol and GGT. The
significant decrease was noted in AST, total bilirubin and
conjugated bilirubin (Table 1). 69.1 % of patients exerted
hyperprolactinemia after 4 weeks. We found out negative correlations between age and percentage deviation in
AST (r = –0.376), ALT (–0.367), total bilirubin (–0.235)
and weight (–0.356). The age positively correlated with
total cholesterol (0.485), AST (0.274), ALT (0.468) and
GGT (0.266) values at the beginning of treatment. There
was positive correlation between age and fasting glucose
(0.468), total cholesterol (0.323) and GGT (0.349) measured after 4 weeks. Male and female did not differ significantly in AST, GGT, conjugated bilirubin and weight.
There was significant difference in percentage deviation
of total cholesterol, total bilirubin and leucocytes between
men and women (higher levels of cholesterol and lower
total bilirubine and leucocytes in men).
CONCLUSIONS: The side effects of atypical antipsychotics are being explored especially within the content of
metabolic syndrome2 and hyperprolactinemia3. Elevated
liver enzymes4 and leukopenia5 occur just occasionally.
There is no uniform algorithm for examination of the
laboratory parameters of the patients receiving antipsychotics in our country. The outcomes of the study point
out the usefulness of repeated screening of referred bio-
Table 1. Differences between 1. and 2. measurements
n
x
SD
p(Wilcoxon)
CH1
CH2
47
47
4.74
5.24
1.09
1.29
0.01
AST1
AST2
67
68
0.60
0.46
0.47
0.22
0.05
GMT1
GMT2
90
90
0.40
0.48
0.34
0.32
0.001
BIt1
BIt2
74
74
12.29
9.04
7.64
4.33
0.001
BIc1
BIc2
35
35
4.39
3.00
3.15
1.11
0.001
BW1
BW2
47
47
75.51
78.55
15.45
14.53
0.001
chemical, endocrine and haemathological parameters. It
also suggest that older patients are more likely to develope
metabolic syndrome. They usually exert higher increase
in fasting glucose and total cholesterol after administration of antipsychotics. Despite the hepatic lesion occurs
rarely, we have observed higher increases in GGT levels
according to growing age in study sample. The decline in
AST, total and conjugated bilirubin could be explained by
unhealthy lifestyle and noncompliance of patients in the
acute phase of illness. This parameters tend to improve
during the stay in hospital.
REFERENCES
1. Hoschl C, Libiger J, Svestka J. Psychiatrie. Praha: Tigis; 2004.
p. 696–708.
2. Goethe JW, Szarek BL, Caley CF, Woolley SB. Signs and symptoms associated with the metabolic syndrome in psychiatric inpatients receiving antipsychotics: a retrospective chart review. J Clin
Psychiatry 2007; 68:22–8.
3. Miller KK. Management of hyperprolactinemia in patients receiving antipsychotics. CNS Spectr 2004; 9(8 Suppl 7):28–32.
4. Dumortier et al. Hepatic tolerance of atypical antipsychotic drugs.
Encephale 2002; 28:542–51.
5. Thome J, Kopf D. Haemathologic abnormalities during treatment
with atypical antipsychotics. Psychiatr Prax 2005; 32:167–71.
42
ENALAPRILAT PREVENTS VASCULAR FUNCTION
IN DAUNORUBICIN-INDUCED CARDIOTOXICITY
Miroslava Kroslakova, Zuzana Bajuszova, Jana Kmecova, Peter Krenek,
Peter Ochodnicky, Jan Klimas
Key words: Daunorubicin/Cardiotoxicity/Enalaprilat/Vascular function/eNOS
BACKGROUND: Recently, different drugs and substances are studied to attenuate the cardiotoxic properties
of anthracyclines1. In clinical and experimental studies,
angiotensin-converting enzyme inhibitors (ACEi) a potent
antihypertensive drugs showed beneficial effects in anthracycline cardiotoxicity with improvement of cardiac
performance2. Besides inducing cardiotoxicity, anthracyclines also affect the vasculature resulting in impaired endothelial relaxation3. We hypothesized that co-treatment
with enalaprilat would rescue impaired relaxation after
administration of daunorubicin in rats.
METHODS: Wistar rats were treated for two weeks
with daunorubicin (DAU, 3mg/kg, i.p., dosage in 48 h
interval), enalaprilat (ENA, 5 mg/kg, i.p., dosage in 12 h
interval) or both (DAU+ENA). Controls (CON) received
the vehicle. Relaxation of isolated thoracic aorta in response to acetylcholine was evaluated in norepinephrineprecontracted aortic rings. Blood pressure (Table l) was
measured invasively in a.carotis and left ventricular function was studied using left ventricular catheterization.
RESULTS: In histological preparations, daunorubicintreated rats showed cardiac and renal injury, which was
associated with loss of heart weight and impaired left
ventricular function. Daunorubicin administration led to
a diminished relaxation to acetylcholine (pD2 = 6.30 ±
0.30, Emax = 90.2 ± 2.8) as compared to controls (pD2
= 7.10 ± 0.35, p<0.05, Emax = 88.3 ± 4.4). Treatment
with enalaprilat only did not affect relaxation (pD2 = 7.16
± 0.49, Emax = 94.2 ± 1.3). However, co-administration
of enalaprilat in daunorubicin-treated rats improved re-
Table 1. Body weight (BW), heart weight (HW), systolic and diastolic blood pressure (sBP and dBP)
in control (CON), enalaprilat (ENA), daunorubicin (DAU) and DAU+ENA-treated rats.
BW (g)
HW (g)
CON
255 ± 16
747 ± 72
120 ± 11
89 ± 13
ENA
245 ± 17
746 ± 63
102 ± 9*
66 ± 9*
DAU
176 ± 23*
112 ± 8
81 ± 8
100 ± 8*
67 ± 8*†
DAU+ENA
164 ± 11*
‡
sBP (mmHg)
567 ± 30*
‡
532 ± 52*
n = 10 in each group, average ± SD, * p < 0.05 vs. CON, † p < 0,05 vs. DAU, ‡ p < 0,05 vs. ENA
Fig. 1. Norepinephrine-induced contraction of aortic rings and relaxation to acetylcholine
(average ± SEM, n = 4/8 per group, *p < 0.05 vs. CON, † p < 0.05 vs. DAU).
dBP (mmHg)
laxation to acetylcholine in comparison to daunorubicin
alone (pD2 = 6.86 ± 0.41, p<0.05, Emax = 88.8 ± 3.8)
(Fig. 1).
CONCLUSIONS: We conclude that treatment with
enalaprilat normalizes endothelial dysfunction in daunorubicin therapy. This positive effect of ACE inhibition
could participate on the long-term improvement of chemotherapy-induced heart failure.
ACKNOWLEDGEMENT
Support by the grant of Slovak Society of Cardiology
43
REFERENCES
1. Mojzisova G, Mirossay L, Kucerova D, Kyselovic J, Mirossay A,
Mojzis J. Protective effect of selected flavonoids on in vitro daunorubicin-induced cardiotoxicity. Phytother Res 2006; 20:110–4.
2. Cardinale D, Colombo A, Sandri MT, Lamantia G, Colombo
N, Civelli M, Martinelli G, Veglia F, Fiorentini C, Cipolla CM.
Prevention of high-dose chemotherapy-induced cardiotoxicity in
high-risk patients by angiotensin-converting enzyme inhibition.
Circulation 2006; 114:2474–81.
3. Duquaine D, Hirsch GA, Chakrabarti A, Han Z, Kehrer C,
Brook R, Joseph J, Schott A, Kalyanaraman B, Vasquez-Vivar J,
Rajagopalan S. Rapid-onset endothelial dysfunction with adriamycin: evidence for a dysfunctional nitric oxide synthase. Vasc Med
2003; 8:101–7.
COMBINATION OF CLARITHROMYCIN AND FUROSEMIDE PROLONGS QT
INTERVAL AND INCREASES RISK OF TORSADES DE POINTES
Jana Kmecova, Katarina Mackovicova, Peter Vavrinec, Jana Ondrejkova, Peter Krenek,
Peter Ochodnicky
Key words: Torsades de pointes/Furosemide/Claritromycin/Arrhythmia/QT interval
BACKGROUND: Prolongation of QT interval caused
by certain drugs may induce life-threatening arrhythmia
such as Torsades de pointes (TdP)1. Its incidence is increased by disrupted ionic balance in organism2. Increased
beta-adrenergic stimulation contributes to induction of
ventricular tachycardias3 and also to development of
TdP (ref.4). In this work we investigated the influence of
macrolide antibiotic clarithromycin in combination with
diuretic furosemide on electrocardiographic parameters
and the occurence of TdP in Wistar rats. We also tried
to determine, whether one-week administration of both
drugs may influence the expression of rERG gene, which
encodes the expression of potassium channel involved in
regulation of repolarization and QT duration.
METHODS: During seven days, Wistar rats were
administered with claritromycin (CLA, 100 mg/kg, p.o.,
twice a day), furosemide (FUR, 100 mg/kg, p.o., twice
a day) or both (CLA+FUR). Controls (CON) were treated by vehicle. 12-lead ECG was recorded in rats under
general anaesthesia5. The same parameters were monitored during beta-adrenergic stimulation (isoproterenol
i.v., dose 5, 10, 15, 20, 30, 45 a 60 ng/min).
RESULTS: Using RT-PCR, we studied the expression of gene rERG in left ventricles. The duration
of QT interval was increased in groups in the order
CON<FUR<CLA<CLA+FUR. The duration of RR interval was increased in furosemide-treated groups (Table 1).
In CLA+FUR group, the occurrence of TdP was noted
Table 1. The changes of electrocardiographic
parameters caused by clarithromycin, furosemid,
or both in Wistar rats.
RR
QT (ms)
QTcn
CON
152 ± 6
83 ± 2
102 ± 1
FUR
178 ± 9*
103 ± 4*
110 ± 3*
CLA
163 ± 9
111 ± 4*
124 ± 3*
CLA+FUR
174 ± 7*
127 ± 5*†‡
137 ± 5*†‡
n = 10 in each group, average ± SEM, * p<0.05 vs. CON,
†p<0,05 vs. FUR, ‡p<0,05 vs. CLA
Fig. 1. ECG recording of TdP in rat from CLA+FUR group under beta-adrenergic stimulation.
44
during beta-adrenergic stimulation (Fig.1). In this group,
the expression of rERG was decreased in 30 % compared
to control (p < 0.05). In other experimental groups, the
expression of rERG did not change.
CONCLUSIONS: Co-administration of clarithromycin and furosemide increases the duration of QT interval.
Their combination can induce TdP, which occurrence
could be associated with decreased expression of rERG.
ACKNOWLEDGEMENT
Financial support by the grant UK/340/2007 is gratefully acknowledged.
REFERENCES
2.
3.
4.
5.
1. Haverkamp W, Breithardt G, Camm AJ, Janse MJ, Rosen MR,
Antzelevitch C et al. The potential for QT prolongation and pro-ar-
rhythmia by non-anti-arrhythmic drugs: clinical and regulatory implications. Report on a Policy Conference of the European Society
of Cardiology. Cardiovasc Res 2000; 47:219–33.
Tan HL, Hou CJ, Lauer MR, Sung RJ. Electrophysiologic mechanisms of the long QT interval syndromes and torsades de pointes.
Ann Intern Med 1995; 122:701–14.
Kirchhof P, Klimas J, Fabritz L, Zwiener M, Jones LR, Schafers
M, Hermann S, Boknik P, Schmitz W, Breithardt G, Kirchhefer
U, Neumann J. Stress and high heart rate provoke ventricular
tachycardia in mice expressing triadin. J Mol Cell Cardiol 2007;
42:962–71.
Nattel S, Quantz MA. Pharmacological response of quinidine induced early afterdepolarisations in canine cardiac Purkinje fibres:
insights into underlying ionic mechanisms. Cardiovasc Res 1988;
22:808–17.
Bacharova L, Kyselovic J, Klimas J. The initial stage of left ventricular hypertrophy in spontaneously hypertensive rats is manifested
by a decrease in the QRS amplitude/left ventricular mass ratio. Clin
Exp Hypertens 2004; 26:557–67.
EFFECTS OF CONSTITUENTS FROM ADHATODA VASICA
TESTED IN ISOLATED RAT TRACHEA
Martina Kottovaa, Marie Voprsalovaa, Jana Pourovaa, Jana Klecakovab, Ludek Jahodarb
a
Czech Republic
b
Department of Pharmaceutical Botany and Ecology, Charles University in Prague, Faculty of Pharmacy in Hradec Kralove,
Czech Republic
Key words: Vasicine/Vasicinone/Asthma/Quinazoline alkaloids
100
90
theophylline
80
vasicinone
70
% max. relaxation
BACKGROUND: The shrub Adhatoda vasica (Acanthaceae) has been most frequently used in traditional medicine for the treatment of respiratory diseases like cough,
asthma and colds. Vasicine and vasicinone are two main
alkaloids, whose bronchodilator activity was supposed.
The aim of this study was to evaluate effect of constituents Adhatoda vasica on the smooth muscle of the respiratory tract.
METHOD AND RESULTS: Quinazoline alkaloids
(vasicine and vasicinone) isolated from Adhatoda vasica
(Acanthaceae) and ethanolic extract of Adhatoda vasica
were used as tested substances. For the experiments, the
in vitro model of the rat-trachea was appllied. Male rats
(6 in each experimental group) were killed and the tracheas were removed. The trachea was cut into two parts
each containing 5-7 cartilaginous rings. The rings were
then cut open, opposite to the tracheal muscle. The tracheal rings were placed in a 50 mL organ bath (Schuller
bath, HSE Germany) containing Krebs-Henseleit solution
(37 °C, oxygenation by carbogene). First, the trachea was
precontracted by carbachol 10–5M. Then, the relaxant effects of cumulative concentrations (10–7 – 10–3M) of each
test-compound were examined and compared with the
maximal relaxation produced by theophylline anhydrous
(3x10–3M) at the end of each experiment. As a positive
control theophylline was used. The relaxant effect of each
concetration was expressed as a percentage change in pro-
vasicine
Adhatoda vasica
extract
60
50
40
30
20
10
0
-8
-7
-6
-5
-4
-3
-2
log c (mol/l)
Fig. 1. Relaxant effect of vasicine, vasicinone and extract
of Adhatoda vasica compared with theophylline.
portion to the maximal relaxation. The EC50 of the testcompound was calculated using the non-linear regression
method (GraphPad Software) – see Fig. 1, Tab. 1.
CONCLUSIONS: In this study, all tested constituents
from Adhatoda vasica showed smooth muscle relaxant activity in isolated rat tracheal rings. Adhatoda vasica extract and isolated vasicine had more relaxant effect than
theophylline. Vasicinone was less potent than that of vasicine (as well as theophylline). Adhatoda vasica extract
had stronger relaxant effect than summation of effects of
single alkaloids.
45
Table 1. ED50 (dosis effectiva media)
of tested substances.
The tested substance
ED50 (mol.L–1)
Adhatoda vasica
extract
1.5 x 10–4
Vasicine
3.0 x 10–4
Vasicinone
1.1 x 10–3
Theophylline
5.7 x 10–4
EFFECT OF A NEWLY SYNTHESIZED ARYLPROPANOLAMINES ON LIPOLYSIS
IN EPIDIDYMAL ADIPOSE TISSUE
Josef Krcmara, Hana Kotolovaa, Peter Kollara, Michal Karpiseka, Tomas Gonecb,
Ladislava Bartosovaa, Pavel Suchya
a
University of Veterinary and Pharmaceutical Science Brno, Faculty of Pharmacy, Department of Human Pharmacology
and Toxicology, Czech Republic
b
University of Veterinary and Pharmaceutical Science Brno, Faculty of Pharmacy, Department of Pharmaceutical
Chemistry, Czech Republic
Key words: β3-Adrenoreceptor agonist/Arylpropanolamine/Lipolysis
BACKROUND: β3-Adrenoreceptor agonists can stimulate lipolysis in white adipose tissue and thermogenesis in
brown adipose tissue. These activities could be useful in
treatment of obesity and associated metabolic syndrome.
The key success in this area is the discovery of selective
β3-adrenergic receptor agonists that lack cardiovascular
or other effect mediated by β1- or β2-adrenergic stimulation1.
METHODS: The effect of newly synthesized substances A482 and B496, was tested using isolated sliced adiO
O
HO
O
O
R
1
NH
O
H
O
R2
N+
H
Substance
R1
R2
A
-CH2CH3
-COOH
B
-CH3
-COOH
Fig. 1. Structure of newly synthesized substances.
pose tissue as previously mentioned2 and was compared
with Isoprenaline and BRL37344. Briefly, male Wistar
rats (body weight 250-300 g) were fasted for 18 hours.
Rats were decapitated and the epididymal fat pads were
removed and cut into small pieces and samples (50 mg)
were incubated for 90 min in 1 mL Krebs-Ringer buffer
(pH=7.4) containing 5% bovine serum albumin. Drugs
were added directly to the incubation medium in concentration of 1x10–5M and 1x10–7M and were present in
the medium for the whole incubation time. The rate of
lipolysis was determined by glycerol concentration in the
incubation medium using free glycerol reagent (Sigma).
RESULTS: Addition of all drugs in both concentration
resulted in significant increase in glycerol concentration in
incubation medium. The substance A stimulated glycerol
release to 2.2-fold (1x10–5 M) and 2.1-fold (1x10–7 M); substance B stimulated glycerol release to 5.0-fold (1x10–5M)
and 4.5-fold (1x10–7M); (--) Isoprenalin 5.0-fold (1x10–5M)
and 2.1-fold (1x10–7M) and BRL37344 4.5-fold (1x10–5M)
and 4.3-fold (1x10–7M).
CONCLUSION: Isoprenalin is a mixed β-adrenergic
agonist while BRL37344 stimulate lipolysis mainly via
β3-adrenergic receptor in rat adipocytes. Isoprenalin can
induce maximum lipolysis in 1x10–5M concentration.
BRL37344 is more potent and its effect on lipolysis reaches a peak in 1x10–7M. Newly synthesized substance B is
able to produce full lipolysis in 1x10–7M concentration
and its effect on rat epididymal adipose tissue is similar
to specific β3-adrenergic agonist BRL37344.
46
ACKNOWLEDGEMENT
Financial support by the IGA VFU grant no. 29/2006/
FaF is gratefully acknowledged.
2. Lincová D, Misekova D, Kmonickova E, Canova N, Farghali H.
Effect of oxide donors on isoprenaline-induced lipolysis in rat
adipose tissue: Studies in isolated adipose tissue and immobilized
perfused adipocyte. Physiol Res 2002; 51:387–94.
REFERENCES
1. Kordik CP, Reitz AB. Pharmacological treatment of obesity.
Therapeutic strategies. J Med Chem 1999; 42:186–7.
RISK PERCEPTION IN RELATION TO SAFE USE OF ANALGESICS
Milan Kriska, Roman Hudec, Jan Rajec, Andrea Raganova, Monika Lassanova,
Andrea Gazova
Department of Pharmacology, Medical Faculty, Comenius University, Bratislava, 827 13 Slovak Republic
Key words: Drug risk perception/Analgesics/Drug usage
BACKGROUND: Drug risk perception expresses the
subjective emotional feeling of the patient, doctor and
other people in medical community inside the chain of
health services. This could substantially influence the decision making in prescription, in buying an OTC drug as
well as the compliance during the treatment schedule; and,
then, the final results of pharmacotherapy. In the modern,
Western type of medicine there exists a great pressure to
improve the safety of the pharmacotherapy not only by
evaluation of drug risk potential but also by improving the
communication on the drug risk, by increased information of patients. The withdrawal of high-risk medicines as
rofecoxib and cerivastatin is the major case of the pharmacovigilance’s effort. Higher number of the OTC drugs
is followed due to increased possibilities of interactions.
A typical example is ibuprofen, where the self treatment
can diminish protective effect of aspirin in preventive
cardiology; or, the self-treatment with non steroidal antiinflammatory drugs (NSAID) as pain-killers in elderly
people which may cause a fatal bleeding1. Adequate risk
perception in relation to risky factors of patients can be
vital for the safe symptomatic pain treatment. Estimation
of the risk perception serves as one tool for an effective
prevention of harmful drug effects together with careful
selection of an appropriate drug to a high risk patient2.
METHODS: Risk perception evaluation consists
of a battery of indirect tests. The widely used method
consists of structured questionnaire with targeted questions. A part of the questionnaire was the VAS (visual
analogues scale) where a questioned person should sign
his/her own feeling in evaluation of the expected risk. One
selected group of the OTC drugs used as a model were the
NSAIDs. The study group consisted of doctors – general
practitioners (260), rheumatologists (n = 41) and orthopaedists. Comparator groups were students of medicine
(43) and pharmacy (n = 90). The statistical analyses of
the obtained data were done using Statgraphics Plus 5.1
and Microsoft Excel 2000; Student’s t-test was routinely
used here.
RESULTS: The results from the questionnaire showed
relatively similar opinions of responders in expression of
perceived risk of analgesics on the VAS scale. Compared
with the expression of pain intensity with group of responders encountering various situation, this was a different evaluation (Fig. 1). Orthopaedists are dealing
probably with the highest intensity of pain in their patients, but they perceived risk of drug at the lower level
comparing with the group of rheumatologists. More than
a quarter of doctors considered the non-steroidal analgesics as the safest group of analgesics, tramadol was considered as the second most safe, and ibuprofen from the
group of NSAIDs was at the third position. In the group
of students of medicine, ibuprofen dominates with 72%
preference, whereas paracetamol with 9 % was on the
second position. Pharmacy students dealing with greater
variability of opinions considered also ibuprofen as the
safest analgesics with preference of 46 % of subjects, followed by paracetamol (9 %), coxibs (5 %), nimesulid and
diclofenac (both 5 %). Concerning the real analgesic consumption in Slovakia, dominating group is represented
by ibuprofen, nimesulid, diclofenac; in nordic countries,
paracetamol is more preferred, nimesulid is completely
absent in prescription3. Generally, the risk perception
which was studied in selected group of doctors and students as control group expressed similar level of perceived
drug risk. This is probably given by the common cultural
background and as a consequence of educational pregradual and postgradual systems and of other social factors forming a personality. This study confirmed previous
preliminary results published by our group earlier4; similar
signals were published by French authors5.
CONCLUSIONS: Perception of analgesic risk measured by VAS is generally high, but concerning the group
of NSAIDs it seems to be insufficient. Analgesic consumption with domination of NSAIDs and with low paracetamol consumption indirectly support this idea. Low level
of the adverse drug reaction reporting in Slovakia from
the group of analgesics and probably not appropriate an-
47
10
9
Pain intensity
Drug risk
8
7
6
5
4
3
2
1
0
GP
Reum
Ort
LFUK
Farm
FaFUK
Fig. 1. Perception of pain intensity and drug risk perceptions measured by VAS (Visual analogues scale 10, minimal
0 resp. maximal 10 value). Columns: GP – general practitioners, Rheu – rheumatologists, Ort – orthopaedists,
LFUK – students of Medical faculty, Farm - FaFUK students of pharmacy 3rd or 4th study year.
algesics risk perception lead toward an increased misuse
of analgesic with higher probability of interactions. The
clear example is the interactions represented by ibuprofen
with possible subsequent failure of the aspirin prevention
in cardiology.
ACKNOWLEDGEMENT
Financial support was provided by the grant VEGA
2292/05.
REFERENCES
1. Rajec J, Raganova A, Kriska M, Dukat A. Riziko zlyhania aspirínovej prevencie: aktuálny klinický problém. Cardiol 2006;
15:248–56.
2. Kriška M a kol. Hodnotenie rizika liekov vo vztahu k jeho percepcii. Klin Farmakol Farm 2003; 17:12–6.
3. Finnish Statistics on Medicines SLT FSM 2005. National agency
for medicines, Helsinki 2006.
4. Tisonova J, Hudec R, Kriska M. a kol. Problémy vnímania liekového
rizika v skupine monokomponentných analgetických prípravkov vo
vzťahu k ich spotrebe v SR. Klin Farmakol Farm 2005; 19:8–12.
5. Bongard V, Menard-Tache S, Bagherri H et al. Perception of the risk
of adverse reactions: differences between health professionals and
non health professionals. Br J Clin Pharmacol 2002; 54:433–436.
THE RAT SMALL INTESTINE IN SITU PERFUSION TECHNIQUE AS A TOOL
FOR BIOEQUIVALENCE ESTIMATION OF SUSPENSION DRUG FORMS
Martin Kunes, Jaroslav Kvetina, Pavel Kubant, Milan Nobilis, Ilona Smidova,
Vladimir Herout, Zbynek Svoboda
Institute of Experimental Biopharmaceutics, Joint Research Centre of PRO.MED.CS Praha a.s. and the Czech Academy of
Science, Prague, Czech Republic
Key words: Rat small intestine/In situ perfusion/Suspension drug forms/Bioequivalence/Intestinal absorption
BACKGROUND: The bioequivalence (BE) definition is based on the empirical concentration time profile
parameters. Two medical products are considered to be
bioequivalent when their concentrations vs. time pro-
files, from the same molar dose, are so similar that they
are unlikely to produce clinically relevant differences in
therapeutic and/or adverse effects1. The BE estimation
of drug forms is provided by comparison of the active
48
Table 1. Basic pharmacokinetic parameters: test (T1, T2, T3, T4) and reference (R) batches.
R
T1
T2
AUC 0–90
[μg.min/mL]
1848.8 ± 536.2
1251.6 ± 192.3
761.6 ± 100.4
1858.3 ± 370.0
AUC 1–4
[μg.h/mL]
14.7 ± 8.4
8.9 ± 2.9
8.5 ± 1.0
14.6 ± 1.6
11.4 ± 2.3
i.d. application
in vivo
30.4 ± 5.0
22.3 ± 3.9
13.8 ± 3.2
47.9 ± 14.1
33.6 ± 4.4
perfusion
in situ
6.8 ± 2.8
4.0 ± 0.7
3.8 ± 0.7
5.3 ± 0.7
4.5 ± 1.9
i.d. application
in vivo
[min]
16.8 ± 6.7
12.5 ± 6.1
13.3 ± 5.2
12.0 ± 4.5
9.2 ± 2.0
perfusion
in situ
[h]
2.3 ± 0.5
2.0 ± 0.0
2.3 ± 0.5
2.5 ± 0.5
2.5 ± 1.0
i.d. application
in vivo
T3
T4
1431.2 ± 168.5 perfusion in situ
Cmax
[μg/mL]
Tmax
drug bioavailability (BA). Although, the BA is defined
as the rate and extent at which an active drug moiety
becomes available at its desired sites of action2, the BA
includes two aspects: systemic availability and absorption. Systemic availability, which represents the amount
of drug that arrives in the systemic circulation and its
test is based on Cmax and AUC. Absorption, on the other
hand, represents permeation into the intestinal mucosa of
the GI tract3, 4. The recent guidelines concerning a waiver
of bioavailability/bioequvalence studies are based on the
Biopharmaceutics Classification System (BCS) which was
developed over the last few years. The BCS classifes drug
substances in four groups according to their solubility
and permeability properties3. The aim of this study was to
characterize and to estimate the similarity of the pharmacokinetic profiles of the model drug after the suspension
drug form administration. Two experimental models were
used in this study (rat small intestine in situ perfusion and
intraduodenal application in vivo).
METHODS: Suspension drug formulation containing
model drug for oral administration involved four test and
one reference batches. Male Wistar Han II rats from conventional breed Konárovice (average weight 316 ± 40 g)
were employed in the study. The rats were abstained from
solid food for 24 hours before study. The whole experiment has been doing under the general anesthesia (intraperitoneal dose of urethane, 1.5 g/kg body weight)6. The
experiment was approved by the Ethical committee of the
Czech Academy of Sciences.
The unilateral small intestine in situ perfusion was
effected. The mesenterial system is only perfused in this
preparation. The perfusion aparature and the surgical
procedure were used as described previously7. The small
intestine was ligated near the ileocecal junction and close
700
600
Experimental
Control
R
2
T3
R = 0.8803
12
T4
200
100
T2
500
1000
1500
2000
AUC 0-90min (perfusion in situ)
Fig. 1. The correlation of the diferences among pharmacokinetic profiles of particular batches (T1, T2,
T3, T4, R) founded in both experimental models
(R2 = the reliability value).
duodenum
t
we e
n
villi
s
cryp
vil lo
u
villi
cryp
t
we e
n
an c
e
b et
b et
jejunum
d ist
0
d ist
0
d ist
an c
e
2
an c
e
b et
we e
n
s
vil lo
u
4
vil lo
us
0
T1
6
t
8
300
villi
10
400
cryp
14
length [μm]
AUC 1-4h (i.d. application in vivo)
500
16
ileum
Fig. 2. The histological examination of the intestinal
wall. The results are expressed as mean ±SE.
under the bile duct issue for the closed loop creation. The
suspension (6 mL) containing model drug was intraluminaly applicated into the luminal part of small intestine.
The perfusion samples from the portal vein were taken at
2, 5, 10, 20, 30, 40, 60, 80, 90 min after the suspension
administration. Six animals were used for testing each
batch. After the laparatomy, the single dose of suspension (6 mL) was intraluminally administred. The small
intestine was ligated under the puncture and under the
bile duct issue simultaneously. Subsequently, the incisions
were closed. The blood serum was withdrawn for the model drug concentration analysis from aorta abdominalis.
Six animals were used for each withdrawal time.
The high-performance liquid chromatographic method was used for the detection of model drug in biological
samples. The absorbed amounts of model drug into the
portal vein were compared among groups. Results were
expressed as the mean ± standard deviation. F-test and
Student’s t-test were used for statistical evaluations. The
basic pharmacokinetic parameters were calculated using
TableCurve2D software. The histological examination was
effected using light microscopy. The vitality of intestine
mucosa under the perfusion conditions was evaluated using standard histological method described earlier8.
RESULTS: In the first step we examinated the similarity of the pharmacokinetic curves of the model drug after
the suspension administration in two test (T1 and T2)
and one reference batches (R). The significant differences
among particular batches were found in all withdrawal intervals using perfusion method. The differences, although
non-significant, were also found after the i.d. application.
This non-bioequivalence was instigation for further pharmaceutical development and for other batches testing (T3,
T4 vs. R). Although the pharmacokinetic profiles were
still not equivalent, they were more similar than in case of
batches T1, T2 vs R. There were found the same drifts of
the pharmacokinetic profiles (T3, T4) towards the reference batch after the i.d. application. The results are expressed in the form of basic pharmacokinetic parameters
(Table 1). The Fig. 1 expresses the high correlation of
the differences among particular batches founded using
both experimental methods. The histological examination
of the small intestinall wall specified that the intestinal
mucosa is still in the physiological relations under the
perfusion conditions (Fig. 2).
CONCLUSIONS: Within the rat in situ models, the
single pass perfusion model is frequently used for the definition of absorption or the evaluation of drug permeability
changes9–12. The technique allows for single-pass or recirculating experiments involving systemic or luminal drug
administration, including luminal administration in closed
loops or segments13–15. In this preparation, the native architecture of the small intestine is maintained with respect
to the circulation such that the extents of metabolism, absorption, and secretion can be studied simultaneously16, 17.
In our previous experiments we also documented that the
perfusion in situ is a suitable method for study of carrier
mediated transporting substances and for the definition of
the potential competition mechanisms during absorption
49
processes of xenobiotics7, 8. We have verified the usability
of the perfusion technique by comparison with the single dose intraperitoneal suspension application in vivo.
Although the differences are not such expressive among
single batches under the condition of i.d. administration,
the high correlation (0.88) of the pharmacokinetic curves
drifts was found in comparison with drifts under the perfusion technique. At the same time, the smaller differences founded after the i.d. administration can be explain
due to the liver metabolic processes. The liver clearence
can supress the differences founded in the process of intestinal transport using perfusion method.
Thus we refer that the rat in situ perfusion experimental model seems to be a suitable for the bioequivalence
estimation of the suspension drug formulations.
REFERENCES
1. Blume HH, McGilveray IJ, Midha KK. BIO-international 94, conference on bioavailability, bioequivalence and pharmacokinetic
studies: pre-conference satellite on in vivo/in vitro correlation. Eur
J Pharm Sci 1995; 3:113–24.
2. Cao X, Gibbs ST, Fang L, Miller HA, Landowski CP, Shin H-C,
Lennernäs H, Zhong Y, Amidon GL, Yu LX, Sun D. Why is it challenging to predict intestinal drug absorption and oral bioavailability
in human using rat model. Pharm Res 2006; 23:1675–86.
3. Amidon GL, Lennernäs H, Shah VP, Crison A. A theoretical basis
for a biopharmaceutic drug classification: the correlation of in vitro
drug product dissolution and in vivo bioavailability. Pharm Res
1995; 12: 413–20.
4. Lennernäs H. Human jejunal effective permeability and its correlation with preclinical drug absorption models. J Pharm Pharmacol
1997; 49:627–38.
5. Blume HH and Schug BS. The biopharmaceutics classification systém (BCS): Class III drugs – better candidates for BA/BE waiver?
Eur J Pharm Sci 1999; 9:117–21.
6. Goon D, Klaassen CD. Intestinal biotransformation of harmol and
1-naphthol in the rat. Further evidence of dose-dependent phase-II
conjugation in situ. Drug Metab Dospod 1991; 19:340–7.
7. Kunes M, Svoboda Z, Kvetina J, Herout V, Herink J, Bajgar J.
Intestinal single-pass in situ perfusion technique: the influence of
L-carnitine on absorption of 7-methoxytacrine. Biomed Pap Med
Fac Univ Palacky Olomouc Czech Republic 2005;149:433–5.
8. Kunes M, Kvetina J, Svoboda Z, Herout V. Study of the mechanisms of intestinal absorption of xenobiotics using in situ perfusion
of rat intestine. Biologia 2005; 17(60Suppl):89–92.
9. Lane ME, Levi KA, Corrigan OI. Effect of intestinal fluid flux
on ibuprofen absorption in the rat intestine. Int J Pharm 2006;
309:60–6.
10. Lane ME, Levis K, McDonald GSA, Corrigan OI. Comparative
assessment of two indices of drug induced permeability changes
in the perfused rat intestine. Int J Pharm 2006; 312:196–9.
11. Ohta K, Inoue K, Hayashii Y, Yuasa H. Carrier-mediated transport
of glycerol in the perfused rat small intestine. Biol Pharm Bull
2006; 29:785–9.
12. Song NN, Li QS, Liu CHX. Intestinal permeability of metformin
using single-pass intestinal perfusion in rats. World J Gastroenterol
2006; 12:4064–70.
13. Cong D, Fong AKY, Lee R, Pang KS. Absorption of benzoic acid
(BA) by segmental regions of the in situ perfused rat intestine.
Drug Metab Dispos 2001; 29:1539–47.
14. Masaoka Y, Tanaka Y, Kataoka M, Sakuma S, Yamashita S. Site
of drug absorption after oral administration: Assessment of membrane permeability and luminal concentration of drugs in each segment of gastrointestinal tract. Eur J Pharm Sci 2006; 29:240–50.
15. Kamio Y, Saito Y, Utoguchi N, Kondoh M, Koizumi N, Fujii M,
Watanabe Y. Epinephrine is enhancer of rat intestinal absorption.
J Control Release 2005; 102:563–8.
50
16. Pang KS, Cherry WF, Ulm EH.Disposition of enalapril in the perfused rat intestine-liver preparation: absorption, metabolism and
first-pass effect. J Pharmacol Exp Ther 1985; 233:788–95.
17. Xu X, Hirayama H, Pang KS. First pass metabolism of salicylamide.
Studies in the once through vascularly perfused rat intestine-liver
preparation. Drug Metab Dispos 1989; 17:556–63.
METHODS AND RESULTS OF BEHAVIOURAL RESEARCH AT THE DEPARTMENT
OF PHARMACOLOGY, MASARYK UNIVERSITY, BRNO, CZECH REPUBLIC
Leos Landa
Department of Pharmacology, Faculty of Medicine, Masaryk University, Brno, Czech Republic
Key words: Research report/Behavioural pharmacology/Dpt. Pharmacology, Masaryk Univ. Brno
BACKGROUND: Behavioural research at the
Department of Pharmacology, Faculty of Medicine,
Masaryk University in Brno is focused on neurobiological
mechanisms of CNS functions for a 30 year long period.
There were a number of subsequently conducted, and successfully defended 10 grant supported research projects.
The main themes of the mentioned projects were: a) the
interaction of psychotropic and immunomodulating factors; b) the interactions of drugs with neurobiological
mechanisms involved in drug addiction. Various international conferences were organised by the department
for professional discussion, and the acquisition of an
international reputation, e.g.: „3rd Regional Symposium
of Central Europe/USA: Drug Addiction and Aids“
(1997); „Workshop Psychopharmacology of Cannabis and
Ecstasy“(2002), supported by IBRO (International Brain
Research Organisation), and by BAP (British Association
for Psychopharmacology); „Workshop: Addiction and
Eating Disorders – Neurobiology and Comorbidities“
(2007), supported by CINP (Collegium Internationale
Neuropsychopharmacologicum). In the last 10 years,
in terms of the research done in this area, there have
been successful defenses of 1 habilitation, 1 professorship, and 6 Ph.D. Theses (two of which was awarded
the Masaryk University Rector’s Prize, and one Czech
Minister of Education Prize). The members of the pharmacological team were awarded the British Association
for Psychopharmacology Prize for the best pre-clinical
paper published in the „Journal of Psychopharmacology“
in the year 2002 and the 1st Prize in the Competition of
Pharmaceutical Sciences organised by French Embassy
in Prague1, 2.
RESULTS: In the last 3 years the behavioural studies
were focused on: a) phenomenon of behavioural sensitization to methamphetamine, ecstasy and cannabinoid receptor ligands (rodent open field test; agonistic behaviour of
aggressive and timid singly-housed male mice on paired
interactions with non-aggressive group-housed partners;
rat I.V. drug self-administration); b) prediction of novel
potential antidepressants (rodent models of repeated
social defeat and olfactory bulbectomy combined with
immune measurement of leukocyte phagocytic activity).
Behavioural sensitization to methamphetamine and ecstasy was confirmed after their repeated administration
but also after repeated pretreatment with CB1 receptor
agonist while blockade of this receptor system the development of sensitization inhibited3, 4. In collaboration
with neuroanatomists and pathophysiologists it has been
shown that repeated intake of methamphetamine is associated with CB1 receptor down-regulation in the brain ventral tegmental area (immunofluorescent detection), and
decrease in CB1 mRNA expression (real-time PCR).
CONCLUSIONS: Behavioural, neuroendocrinological
(collaboration with the Endocrinological Institute of the
Slovak Academy of Science) and immunological results
suggest potential antidepressant like effects resembling
those of citalopram, carbamazepine and valproate in antiepileptics of the 3rd generation felbamate and tiagabine2.
REFERENCES
1. Vinklerova J, Novakova J, Sulcova A. Inhibition of methamphetamine self-administration in rats by cannabinoid receptor antagonist
AM 251. J Psychopharmacol 2002;16:139–43.
2. Pistovcakova J, Makatsori A, Sulcova A, Jezova D. Felbamate reduces hormone release and locomotor hypoactivity induced by
repeated stress of social defeat in mice. Eur Neuropsychopharm.
2005; 15:153–8.
3. Landa L, Sais K, Sulcova A. Involvement of cannabinoid CB1 and
CB2 receptor activity in the development of behavioural sensitization to methamphetamine effects in mice. Neuroendocrinol Lett
2006a; 27:63–9.
4. Landa L, Slais K, Sulcova A. Impact of cannabinoid receptor
ligands on behavioural sensitization to antiaggressive methamphetamine effects in the model of mouse agonistic behaviour.
Neuroendocrinol Lett 2006b; 27:703–10.
51
ON THE POSSIBLE INTERACTION OF PLATINUM CHEMOTHERAPEUTICS
WITH HUMAN LIVER MICROSOMAL CYTOCHROMES P450
Vlastimil Maseka, Marketa Machovaa, Eva Anzenbacherovab, Viktor Brabecc,
Pavel Anzenbachera
a
Department of Medical Chemistry and Biochemistry, Faculty of Medicine and Dentistry, Palacky University, Hnevotinska 3,
775 15 Olomouc, Czech Republic
c
Institute of Biophysics, v.v.i., Academy of Sciences of the Czech Republic, Kralovopolska 135, 612 65 Brno, Czech
Republic
b
Key words: Cisplatin/Transplatin/Cytochrome P450/CYP
BACKGROUND: For almost forty years now, platinum
complexes are known to possess the antineoplastic activity1. The most prominent antineoplastic drug of this class
is the cisplatin (cis-diamminedichloroplatinum (II)) used
to treat many types of cancer either in monotherapy or in
combination with other drugs or with radiotherapy.
The proper positioning of the ligands of the complex
appeared to be crucial for the therapeutic effect of this
drug as the trans – complex of the same chemical composition having the identical ligands of platinum (i.e. two
amino groups or two chlorides at the opposite side of
the square-like planar platinum complex) does not exhibit
the desired antitumor activity2. Although the platinumbased chemotherapeutic drugs are in use for long time,
little is known about their interactions with liver microsomal cytochrome P450 enzymes which are known to
play a crucial role in metabolism of the majority of drugs
and other xenobiotics3. There is in the literature only one
study performed with human liver microsomal preparations dealing with cisplatin; there was no effect of this
compound on the activity of CYP3A4 observed4. On the
other hand, platinum cytostatics are responsible for an
upregulation of CYP2E1 and CYP2A1 genes in the rat
liver and kidney; inhibition of CYP2E1 has been shown
to protect against hepatotoxicity and nephrotoxicity due
to the effect of cisplatin5–7. This work presents the results
of a pilot study on the inhibition of various human liver
microsomal CYP activities by cis- and transplatin.
METHODS: Cisplatin and transplatin were supplied
by Sigma Aldrich CZ (Prague); chlorzoxazone, 6-hydroxychlorzoxazone, diclofenac, 4-hydroxydiclofenac, bufuralol, 6-hydroxybufuralol and 6β-hydroxytestosterone were
supplied by Ultrafine Chemicals (Salford, UK). P450-Glo®
substrate for determination of CYP2C9 activity by luminescence spectrometry was product of Promega (Madison,
WI, USA) obtained through East Port (Prague, Czech
Republic). Ethoxyresorufin (for estimation of CYP1A2
activity) was supplied by Fluka (Buchs, Switzerland).
All other chemicals were purchased from Sigma Aldrich
(Prague, Czech Republic). Pooled human liver microsomes were purchased from Advancell (Barcelona, Spain).
Microsomes were obtained in accordance with ethical
regulations of the country of origin (Spain).
Individual CYP activities were measured according
to standard protocols8 and were described elsewhere9.
The following microsomal CYP activities were tested:
Fig. 1. Inhibition of CYP2C9 by cisplatin (circles) and
transplatin (triangles).
Fig. 2. Inhibition of CYP2E1 by cisplatin (circles) and
transplatin (triangles).
52
CYP3A4, testosterone 6β-hydroxylation; CYP2C9, P450Glo® substrate luciferin-ME biotransformation (Promega
Technical Bulletin, http://www.promega.com); CYP2E1,
chlorzoxazone 6-hydroxylation; CYP1A2, 7-ethoxyresorufin O-deethylation; CYP2D6, bufuralol 1’-hydroxylation;
CYP2A6, coumarin 7-hydroxylation; For determination
of metabolites formed from specific substrates, HPLC
system Shimadzu Class VP (Kyoto, Japan) with UV (6βhydroxytestosterone, 6-hydroxychlorzoxazone) or with
fluorescence detection (1’-hydroxybufuralol) was used.
A TECAN Infinity absorbance/ fluorescence/ luminescence reader (Tecan, Vienna, Austria) was used for detection
of other metabolites (resorufin, 7-hydroxycoumarin, luciferin derivatives).
Inhibition of CYP enzymes in microsomal fractions:
For each enzyme assay, the Km and Vmax values were determined to get the substrate concentration suitable for
the inhibition experiments. The Km values corresponded
well to known literature data10. Substrate concentrations
were used near the Km. Inhibition experiments were performed with six levels of inhibitor concentration ranking
from 10 to 400 μM; the stock solutions were 1.6 mM
(cisplatin) and 0.2 mM (transplatin), both in deionized
water. Experimental conditions were the same as for determination of individual CYP activities; preincubation
of reaction mixtures with inhibitor for 30 min was kept
in all determinations. Inhibition of individual CYP activities was in all cases calculated as a mean from two
experiments (differing less than 10 %) and expressed as
the fraction of remaining activity after addition of the
inhibitor.
RESULTS: Cisplatin was found to influence the
CYP2C9 and CYP2E1 only slightly (the remaining activity of the respective CYP form was at about 80 % at
concentrations of cisplatin reaching 100 μM). Activities
of CYP3A4, 1A2, 2A6 and 2D6 were not affected by the
presence of cisplatin in the medium. On the other hand,
effect of transplatin to CYP2C9 and CYP2E1 activities
was much more pronounced as only about 50 % of the
original activity was remaining at inhibitor concentrations
already at about 50 μM (Figs. 1 and 2). Activity of the
CYP1A2 form was again, as it was with the cisplatin, low-
ered to 80%; no significant changes in the enzymatic activity of the CYP3A4, CYP2D6, and CYP2A6 was found.
CONCLUSIONS: The results of experiments on inhibition of activities of the human liver microsomal cytochromes P450 indicate that (i) No prominent inhibition of
CYP activities by cisplatin takes place; (ii) The transplatin
behaves in a different manner inhibiting the CYP2C9 and
CYP2E1 activities already at lower concentrations. On
the other hand, as the transplatin is not taken as a drug
because of lack of its efficacy, the results do not constitute
a molecular basis for possible drug-drug interaction.
ACKNOWLEDGEMENT
Financial support from the KAN200200651 project of the
Grant Agency of the Czech Academy of Sciences is gratefully
acknowledged.
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385–6.
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KT, Zaigler M, Barthold D, Fuhr U. Screening for inhibitory effects
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Cancer Res 1992; 52:540–7.
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cisplatin-induced cytotoxicity to renal proximal tubular epithelial
cells. Anticancer Res 2002; 22:863–8.
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89:515–23.
8. Cytochrome P450 Protocols (Phillips IR, Shephard EA, ed).
Totowa, NJ: Humana; 1998.
9. Nekvindova J, Masek V, Veinlichova A, Anzenbacherova E,
Anzenbacher P, Zidek Z, Holy A. Inhibition of human liver
microsomal cytochrome P450 activities by adefovir and tenofovir.
Xenobiotica 2006; 36:1165–77.
10. Walsky RL, Obach RS. Validated assays for human cytochrome
P450 activities. Drug Metab Dispos 2004; 32:647–60.
53
INTERACTION OF NABUMETONE WITH CYTOCHROMES P450 IN VITRO:
COMPARISON OF THE MAN AND PIG
Jaroslav Matala, Eva Anzenbacherovab, Alena Veinlichovaa, Milan Nobilisc,
Pavel Anzenbachera
a
Department of Medical Chemistry and Biochemistry, Faculty of Medicine and Dentistry, Palacky University, Hnevotinská 3,
775 15 Olomouc, Czech Republic
c
Institute of Experimental Biopharmaceutics, Joint Research Center of PRO.MED.CS Praha a.s. and Academy of Sciences
of the Czech Republic, Hradec Kralove, Czech Republic
b
Key words: Nabumetone/6-MNA/Cytochrome P450/ CYP/Pig
BACKGROUND: Nabumetone (4-(6-methoxy-2naphtyl)-butan-2-one) is a nonsteroidal anti-inflammatory prodrug, clinically used mainly for treatment of
osteoarthritis or rheumatoid arthritis to reduce pain and
inflammation. It undergoes rapid and extensive first-pass
metabolism in liver to form the main circulating active metabolite 6-methoxy-2-naphthylacetic acid (6-MNA), which
is a potent inhibitor of cyclooxygenase (COX-2). Three
main metabolic pathways of nabumetone were described:
O-demethylation, reduction of ketone to an alcohol, and
an oxidative cleavage of the side-chain1. Possible interac-
tions of nabumetone with liver microsomal cytochromes
P450 (CYPs, which are often responsible not only for
drug metabolism, but also for unwanted effects such an
enzyme inhibition or induction2) are studied here at the
level of human and pig liver microsomes. To prove the
suitability of pig as an experimental model for following of
metabolism and pharmacokinetics of drugs we compared
the inhibition of activities of CYP forms presented in human and pig liver microsomal fraction.
METHODS: Nabumetone, 4-(6-methoxy-2-naphtyl)butan-2-one was obtained from PRO.MED.CS Praha a. s.,
Human
120
activity (%)
100
80
60
40
20
0
1A2
2A6
2B6
2C8
2C9
2D6
2E1
3A4
Pig
120
activity (%)
100
80
60
40
20
0
1A2
2A6
2B6
2C8
2C9
2D6
2E1
3A4
Fig. 1. Effect of nabumetone on the liver microsomal CYP activities in
human and pig. Effect expressed as fraction of remaining activity
after addition of nabumetone.
54
Czech Republic, chlorzoxazone, 6-hydroxychlorzoxazone,
diclofenac, 4-hydroxydiclofenac, bufuralol, 6-hydroxybufuralol and 6β-hydroxytestosterone were supplied by
Ultrafine Chemicals (Salford, UK). P450-Glo® substrate
for determination of CYP2C8, CYP2C9 activities by luminescence spectrometry was product of Promega (Madison,
WI, USA) obtained through East Port (Prague, Czech
Republic). 7-Ethoxy-4-(trifluoromethyl)coumarin was
supplied by Fluka (Buchs, Switzerland). All other chemicals were purchased from Sigma Aldrich (Prague, Czech
Republic). Pooled human liver microsomes were purchased from Advancell (Barcelona, Spain). Microsomes
were obtained in accordance with ethical regulations of
the country of origin (Spain). Pooled pig liver microsomes
were prepared according to established methods3 from experimental material obtained in a local slaughterhouse.
Individual CYP activities were measured according
to standard protocols3 and were described elsewhere4.
The following microsomal CYP activities were tested:
CYP3A4, testosterone 6β-hydroxylation; CYP2C9,
diclofenac 4’-hydroxylation or P450-Glo® substrate luciferin-ME biotransformation; CYP2E1, chlorzoxazone
6-hydroxylation; CYP1A2, 7-ethoxyresorufin O-deethylation; CYP2D6, bufuralol 1’-hydroxylation; CYP2A6,
coumarin 7-hydroxylation; CYP 2B6, 7-ethoxy-4-(trifluoromethyl)coumarin O –deethylation; CYP2C8, luciferin-ME demethylation (Promega Technical Bulletin
No.325, http://www.promega.com). For determination
of metabolites formed from specific substrates, HPLC
system Shimadzu Class VP (Kyoto, Japan) with UV (6βhydroxytestosterone, 6-hydroxychlorzoxazone, 4’-hydroxydiclofenac, 4’-hydroxymephenytoin) or with fluorescence
detection (1’-hydroxybufuralol) was used. A TECAN
Infinity absorbance/ fluorescence/ luminescence reader
(Tecan, Vienna, Austria) was used for detection of other
metabolites (resorufin, 7-hydroxycoumarin, 4-(trifluoromethyl)coumarin, luciferin derivatives).
Inhibition of CYP enzymes by nabumetone in human
and pig microsomal fractions: For each enzyme assay, the
Km and Vmax values were determined to get the substrate
concentration suitable for the inhibition experiments.
The Km values corresponded well to known literature
data5. Substrate concentrations were used near the Km.
Inhibition experiments were performed with six levels of
nabumetone concentration 10, 50, 100, 150, 200, 400 μM;
the stock solution was 25 mM in 60% (v/v) DMSO, except
for measurement of activity of CYP 2E1 (here the stock
solution contained 17.3 mM nabumetone in acetonitril).
Experimental conditions were the same as for determina-
tion of individual CYP activities; preincubation of reaction mixtures with inhibitor (nabumetone) for 30 min
was kept in all determinations. Inhibition of individual
CYP activities was in all cases calculated as a mean from
two experiments (differing less than 10 %) and expressed
as the fraction of remaining activity after addition of
nabumetone.
RESULTS: Fig. 1 documents that the majority of CYP
activities is not markedly influenced either in human or
pig microsomes with 100 μM nabumetone. However,
prominent changes were observed with pig CYP1A2 activity (decreased to 33 %, contrary to the human sample with
CYP1A2 activity unchanged) and CYP2E1 (decrease to
36 % in human and 74 % in the pig sample). Changes
in CYP activities not exceeding 20 % were found with
activities of CYP2A6, CYP2B6, CYP2C8, CYP2C9, and
CYP2D6. Interestingly, activity of CYP3A4 was in both
samples (human and pig) influenced to approximately the
same extent (71 % and 62 %, respectively).
CONCLUSIONS: The results, as expressed in the
Figure, show that the pig microsomes are giving (with an
exception of CYP1A2) good information on the influence
of the compound studied on the respective CYP activities
(as it is in the cases of CYP2B6, CYP2C9, CYP2D6, and
CYP3A4). With respect to possible in vitro interactions of
nabumetone with human liver microsomal CYP enzymes,
it can be expected that this compound can influence the
metabolism of compounds metabolized by CYP2E1 and
probably also the CYP3A4 enzymes.
ACKNOWLEDGEMENT
Financial support from the COST 861 project (MSM
1P05OC050) and from the Internal grant of the Palacky
University, Faculty of Medicine (J.M.) 91110061 is gratefully
acknowledged.
REFERENCES
1. Hedner T, Samuelsson O, Wahrborg P, Wadenvik H, Ung KA,
Ekbom A. Nabumetone: therapeutic use and safety profile in the
management of osteoarthritis and rheumatoid arthritis. Drugs
2004; 64:2315–43.
2. Anzenbacher P, Anzenbacherová E. Cytochromes P450 and metabolism of xenobiotics. Cell Mol Life Sci 2001; 58:737–47.
3. Cytochrome P450 Protocols (Phillips IR, Shephard EA, ed).
Totowa, NJ: Humana; 1998.
4. Nekvindová J, Masek V, Veinlichová A, Anzenbacherová E,
Anzenbacher P, Zídek Z, Holý A. Inhibition of human liver microsomal cytochrome P450 activities by adefovir and tenofovir.
Xenobiotica 2006; 36:1165–77.
5. Walsky RL, Obach RS. Validated assays for human cytochrome
P450 activities. Drug Metab Dispos 2004; 32:647–60.
55
THE AFFINITY OF RECEPTOR SPECIFIC PEPTIDES 111IN-DOTA-NOC AND
111
IN-DOTA-TATE TO SOMATOSTATIN RECEPTORS IN VIVO AND IN VITRO
Ludmila Melicharovaa, Milan Lazniceka, Alice Laznickovab, Milos Petrikb
a
Department of Pharmacology and Toxicology, and
Department of Physical Chemistry, Faculty of Pharmacy, Charles University, 500 05 Hradec Kralove, Czech Republic
b
Key words: Somatostatin receptors/Internalization/Somatostatin analogs
BACKGROUND: The uptake of radiolabelled somatostatin analogs by tumour cells through receptor-mediated
internalization is a critical process for the in vivo targeting
of tumoural somatostatin receptors1. Five human somatostatin receptor subtypes (sst1 – sst5) are known to be
overexpressed to some degree on various tumours, sst2
being the most important one. Clinically used somatostatin based radiopeptides target exclusively sst2. Earlier
studies have shown that modification of the octapeptide
octreotide in positions 3 and 8 may result in compounds
with increased somatostatin receptor affinity that display improved uptake in somatostatin receptor-positive
tumours2. In this study we analyzed an internalization of
111
In-DOTA-Tyr3-octreotate (111In-DOTA-TATE) and 111InDOTA-1-Na13-octreotide (111In-DOTA-NOC) to rat pancreatic tumour cells in vitro. The results were compared
with uptakes of the peptides under study to somatostatin
receptor-rich rat organs (namely the adrenals and pancreas) in vivo. Whereas radiolabelled DOTA-TATE with
the affinity only to sst2 is clinically used at present, radiolabelled DOTA-NOC represents a new receptor-specific
peptide with an extended spectrum of affinity also to sst3
and sst5.
METHODS: AR42J (from ECACC) cells were grown
in RPMI-1640 (suplemented with 2 mM glutamine and
10% fetal calf serum) in air containing 5 % CO2 at 37 °C.
Subculturing was performed employing a trypsin/EDTA
solution. On the day of the experiment, the cells were
treated with trypsin/EDTA solution and concentrated to
1×106 cells per 1 mL of internalization medium (RPMI–
1640 supplemented with 2 mM glutamine and 1% fetal
calf serum) per epppendorf tube. Incubation was started
by addition 10 ng of radiolabelled peptide per tube. Cells
were incubated at 37 °C in triplicates for the indicated
time periods. Cellular uptake was stopped by removal of
the medium and washing of the cells with ice-cold PBS
two times. Thereafter the cells were incubated twice at
ambient temperature in acid wash buffer (50 mM glycine
buffer pH 2.8, 0.1 M NaCl) for 5 min. The supernatant
was collected (receptor-bound radioligand fraction). Cells
were lysed by treatment in 1M NaOH and cell radioactivity collected (internalised radioligand fraction).
Internalization of 111In-DOTA-NOC and
111
In-DOTA-TATE
% of D
10
111
9
In-DOTA-NOC
8
7
111
In-DOTA-TATE
6
5
4
3
2
1
0
0
60
120
180
time (min)
240
Fig. 1. Comparison of internalization rate of peptides into AR42J cellls. Values are expressed as specific internalization (percentage of added dose).
56
Table 1. Radioactivity concentrations in selected organs and tissues after intravenous administration of peptides
under study to rats (% dose per 1% of body weight).
111
111
In-DOTA-NOC
In-DOTA-TATE
1 hour
24 hrs
1 hour
24 hrs
Blood
0.632 ± 0.116
0.008 ± 0.001
0.329 ± 0.048
0.005 ± 0.001
Liver
0.858 ± 0.124
0.528 ± 0.047
0.252 ± 0.030
0.138 ± 0.007
Muscle
0.128 ± 0.018
0.012 ± 0.001
0.066 ± 0.004
0.006 ± 0.001
Pancreas
26.89 ± 4.65
12.59 ± 2.59
17.33 ± 1.31
8.03 ± 0.63
Adrenals
71.40 ± 27.00
38.32 ± 9.65
39.25 ± 7.60
28.12 ± 6.59
Animal experiments were performed in compliance with
generally accepted guidelines governing such work. Male
Wistar rats (180-260 g) were used in the experiments.
Rats were injected intravenously under ether anesthesia
with 0.2 μg of the peptide per animal. At selected time
points, rats were sacrificed under ether anesthesia. Organs
and blood were collected and the radioactivity was determined using a gamma-counter.
RESULTS: Both radiopeptides are internalized in
the same range as seen on Fig.1. Values are expressed
as specific internalization (percentage of dose added to
1 million cells at 1.5 nM concentration) and are result of
three independent experiments with triplicates in each
experiment. Biodistribution studies in male rats showed
rapid radioactivity clearance from the blood and most tissues. High and long-term uptake of radioactivity in somatostatin receptor-positive organs (the pancreas, adrenals,
and some others) was determined. The radioactivity accumulations in the pancreas and adrenals and in selected
sst-negative tissues are shown in Table 1. Due to higher
lipophilicity of 111In-DOTA-NOC its elimination rate by
glomerular filtration is lower (due to its higher plasma
protein binding) and blood radioactivity concentrations
higher in comparison with that of 111In-DOTA-TATE. On
the other hand, radioactivity accumulations in organs with
high density of somatostatin receptors are higher after
In-DOTA-NOC when compare with 111In-DOTA-TATE.
The reason of this difference is probably in both slower
blood radioactivity-time decrease and a partial binding of
111
In-DOTA-NOC to other somatostatin receptor subtypes
than sst2 in comparison with 111In-DOTA-TATE.
CONCLUSIONS: The AR42J rat pancreatic tumour
cell line is known to express sst2 receptors in vivo and in
vitro. It may be the reason why the both radiopeptides under study are internalized to these cells in the same range.
In in vivo experiments a slower blood radioactivity-time
decrease and higher radioactivity uptake in somatostatinexpressed organs of rats for 111In-DOTA-NOC were found
in comparison with 111In-DOTA-TATE.
111
ACKNOWLEDGEMENT
The study was supported by the grant n. 305/07/0535 of
the Grant Agency of Czech Republic.
REFERENCES
1. Cescato R et al. Internalization of sst2, sst3, and sst5 Receptors:
Effects of Somatostatin Agonists and Antagonists. J Nucl Med
2006; 47:502–11.
2. Wild D et al. DOTA-NOC, a high-affinity ligand of somatostatin
receptor subtypes 2, 3 and 5 for labelling with various radiometals.
Eur J Nucl Med Mol Imaging. 2003; 30:1338–47.
57
CHANGES IN CARDIAC TROPONIN T DURING ACUTE AND CHRONIC
CHOLESTASIS IN RATS
Stanislav Micudaa, Michaela Adamcovab, Eva Brcakovaa, Leos Fuksaa, Jolana Cermanovaa,
Jitka Hajkovaa, Gabriela Kolouchovaa, Jirina Martinkovaa, Frantisek Staudc
a
Department of Pharmacology, Faculty of Medicine, Charles University in Prague, Simkova 870, 500 38 Hradec Kralove,
Czech Republic
b
Department of Physiology, Faculty of Medicine, Charles University in Prague, Simkova 870, 500 38 Hradec Kralove,
Czech Republic
c
Department of Pharmacology and Toxicology, Faculty of Pharmacy, Charles University in Prague, Heyrovskeho 1203,
500 05 Hradec Kralove, Czech Republic
Key words: Cholestasis/Troponin T/Myocardial injury
BACKGROUND: Cardiac troponin T (cTnT), a subunit of a thin-filament-associated complex regulating the
formation of actin-myosin crossbridges during the contraction of cardiomyocytes, has unique amino acid sequences
that differentiate it from skeletal muscle isoforms1. Serum
cTnT has been shown to be highly sensitive and specific
marker of myocardial cell injury2. Importantly, increase
in cTnT serum concentrations closely correlates with
the extent of myocardial damage3. In addition, decreased
cTnT content in the myocardium might also indicate
heart impairment4. Cholestatic liver disease is commonly associated with many cardiovascular events such as
bradycardia, hypotension, QT prolongation, resistance
against ischemia/reperfusion-induced arrhythmia, and
hyporesponsiveness of cardiovascular system to adrenergic stimulation5. In addition to electrophysiologic disturbances, contractility of myocardium is also significantly
decreased in long term cholestasis-induced liver impairment6. On the contrary, little is known about potential
myocardial damage after short-term cholestasis. In addition, lack of such studies is available to describe influence
of cholestasis on cTnT. The purpose of the present study
was to evaluate damage of cardiomyocytes in rats with
short-term cholestasis induced by 1- or 7-day bile duct ligation. Measurement of cTnT concentrations in the serum
was used as the marker for myocardial injury together with
direct measurement of myocardial cTnT content.
METHODS: Male Wistar rats (n = 5 in each group)
weighing 280–320 g were subjected to bile duct ligation
(BDL) or sham-operation (controls) under general anesthesia (pentobarbital sodium 50 mg/kg body wt i.p.).
Blood samples were taken one (Sham-1 and BDL-1) or
seven (Sham-7 and BDL-7) days after surgical procedure
with consequent removal of heart, which was snap frozen
in liquid nitrogen and stored at –80 °C until analysis. The
concentrations of cTnT were measured in serum using
Elecsys Troponin T STAT Immunoassay (Roche, Basel,
Schwitzerland) on the Elecsys 2010 immunoassay analyzer (Roche) with the detection limit of 0.010 ng.mL–1. The
concentrations of total and conjugated bilirubin in serum
and urine were measured on Cobas Integra ® 800 (Roche
Diagnostics, Mannheim, Germany) according to manufacturer‘s instructions. The cTnT content in cardiomyocytes
was determined by Western blot analysis. Whole cell homogenate (WCH) was prepared from rat myocardium of
sham operated and BDL animals by lysis for 2 hours on
ice in RIPA buffer (Sigma) with consequent centrifugation
for 15 min at 15,000 × g at 4 °C. Hundred micrograms of
supernatant protein were separated on a 10% polyacrylamide gel. After the proteins were transferred to a nitrocellulose membrane (Bio-Rad), it was blocked for 1 h at room
temperature with 5 % nonfat dry milk in Tris-buffered
saline containing 0.05 % Tween 20 (TBST). The membrane was then incubated with anti-cTnT antibody (clone
JLT-12, Sigma, 1 : 500) for 1 h, washed, and incubated
for 1 h with a peroxidase-conjugated goat anti-mouse IgG
antibody (1 : 2,000). Chemiluminescence development
was performed using ECL reagents (GE Healthcare). The
immunoreactive bands on the autoradiography films were
scanned with GS-800 Calibrated Densitometer (Bio-Rad
Laboratories, Hercules, CA, USA) and semiquantified using the QuantityOne software v. 4.2.2 (Bio-Rad). Data are
presented as means and SEMs. Differences between means
were evaluated by ANOVA (Graphpad Instat 3.0).
Fig. 1. Representative Western blot analysis of myocardial cTnT from Sham and BDL animals. JLT-12 antibody
identified one band in a position corresponding to ~40 kDa.
58
ular weight position corresponding to ~40 kDa. Fig. 2
shows the changes in cTnT determined by densitometry of
obtained cTnT bands. Myocardial content of cTnT significantly decreased after one day of cholestasis to 65 ± 2.7 %
(P < 0.01) of Sham-1 animals. Seven days after BDL, the
amount of myocardial cTnT remained decreased to 77.1
± 2.8 % (P < 0.05) of Sham-1 animals.
CONCLUSIONS: The present study is the first report using cardiac troponin T for description of serious
myocardial injury shortly after induction of extrahepatic
cholestasis. We observed increased serum cTnT concentrations after one day of cholestasis together with decrease
in its myocardial content. This observation raised the
question about the possibility of myocardial damage in
patients with acute extrahepatic cholestasis, the frequent
pathophysiological condition in clinical practice.
Fig. 2. cTnT alterations determined by Western blot
analysis in ventricles of Sham and BDL rats.
* P < 0.05; ** P < 0.01 compared with controls.
ACKNOWLEDGEMENT
Financial support by the grant GAUK 116807/C/2007 is
REFERENCES
RESULTS: To verify efficacy of the model of cholestasis used, we evaluated endogenous bilirubin serum concentrations in sham-operated and BDL rats. Extrahepatic
cholestasis was associated with an increase in the serum
total (conjugated) bilirubin concentration from 1.2 ±
0.2 μmol.L–1 (below detection limit < 0.1 μmol.L–1)
in sham operated rats to 64.1 ± 8.7 μmol.L–1 (53.2 ±
8.6 μmol.L–1, P < 0.05) and 173.4 ± 28.2 μmol.L–1 (127.4 ±
19.5 μmol.L–1, P < 0.05) in BDL-1 and BDL-7 rats, respectively. Most of elevated serum bilirubin was in conjugated
form. Serum cTnT concentrations were below detection
limit in sham-operated rats while a significant increase to
0.17 ± 0.06 (P < 0.01) was observed in BDL-1 animals.
Seven-day cholestasis produced an increase in serum cTnT
only in two animals thus the difference from controls was
insignificant. Fig. 1 shows a representative Western blot
analysis of myocardial cTnT in the sham-operated, BDL-1,
and BDL-7 groups. One cTnT band migrated to a molec-
1. Chen Y, Serfass RC, key-Bojack SM et al. Cardiac troponin T alterations in myocardium and serum of rats after stressful, prolonged
intense exercise. J Appl Physiol 2000; 88:1749–55.
2. Mair J, Puschendorf B, Michel G. Clinical significance of cardiac
contractile proteins for the diagnosis of myocardial injury. Adv Clin
Chem 1994; 31:63–98.
3. Metzler B, Hammerer-Lercher A, Jehle J et al. Plasma cardiac troponin T closely correlates with infarct size in a mouse model of
acute myocardial infarction. Clin Chim Acta 2002; 325:87–90.
4. Ricchiuti V, Sharkey SW, Murakami MM, Voss EM, Apple FS.
Cardiac troponin I and T alterations in dog hearts with myocardial
infarction: correlation with infarct size. Am J Clin Pathol 1998;
110:241–7.
5. Hajrasouliha AR, Tavakoli S, Jabehdar-Maralani P et al. Cholestatic
liver disease modulates susceptibility to ischemia/reperfusion-induced arrhythmia, but not necrosis and hemodynamic instability:
the role of endogenous opioid peptides. J Hepatol 2005; 43:491–
8.
6. Ebrahimi F, Tavakoli S, Hajrasouliha AR et al. Contribution of
endogenous opioids and nitric oxide to papillary muscle contractile
impairment in cholestatic rats. Eur J Pharmacol 2005; 523:93–
100.
GADOLINIUM-CONTAINING MRI CONTRAST AGENTS AND NEPHROGENIC
SYSTEMIC FIBROSIS – CURRENT PHARMACOVIGILANCE ISSUE
Jana Mlada, Jiri Lamka, Radek Bela
State Institute for Drug Control (SUKL), Pharmacovigilance Unit, Prague, Czech Republic
Key words: Nephrogenic systemic fibrosis/Gadolinium-containing contrast agents
BACKGROUND: In January 2006, gadolinium-containing MRI contrast agents were believed to contribute
to the development of a rare but sometimes fatal disorder called nephrogenic systemic fibrosis (NSF). NSF was
first recognised in the USA in 1997 as an idiopathic skin
condition characterised by thickening and hardening of
the skin of the extremities and sometimes of the trunk
and increase in the number of dermal fibroblast-like cells
associated with collagen remodelling and mucin deposition. The exact disease mechanism has to be elucidated;
59
Table 1. Currently marketed gadolinium contrast agents.
Brand
name
Generic name
Acronym
Chemical
structure
Charge
Elimination
pathway
Protein
binding
NSF
Yes/No
Omniscan
gadodiamide
Gd-DTPA-BMA
Linear
Nonionic
Kidney
None
Yes
Linear
Nonionic
Kidney
None
Yes
OptiMARK gadoversetamide Gd-DTPA-BMEA
Magnevist
gadopentetate
dimeglumine
Gd-DTPA
Linear
Ionic
Kidney
None
Yes
MultiHance
gadobenate
dimeglumine
Gd-BOPTA
Linear
Ionic
97 % Kidney
3 % Bile
<5%
Yes
Primovist
gadoxetic acid
disodium salt
Gd-EOB-DTPA
Linear
Ionic
50 % Kidney
50 % Bile
< 15 %
No
Vasovist
gadofosveset
trisodium
Gd-DTPA
Linear
Ionic
91 % Kidney
9 % Bile
> 85 %
No
ProHance
gadoteridol
Gd-HP-DO3A
Cyclic
Nonionic
Kidney
None
No
Gadovist
gadobutrol
Gd-BT-DO3A
Cyclic
Nonionic
Kidney
None
No
Dotarem
gadoterate
meglumine
Gd-DOTA
Cyclic
Ionic
Kidney
None
No
physicochemical properties of gadolinium-containing
agents and corresponding pharmacokinetics of released
gadolinium probably belong among the important factors
trigerring NSF.
METHODS: Initially, twenty cases of NSF from
Denmark, and further five cases from Austria were identified in patients with renal impairment receiving gadolinium containing contrast agent gadodiamide before
development of the disorder. During 2006 the worldwide data collection and assessment were performed
by the Pharmacovigilance Working Party (PhVWP) of
the Committee for Medicinal Products for Human Use
(CHMP) (scientific board of European Agency for the
Evaluation of Medicinal Products (EMEA)) in collaboration with external experts.
RESULTS: To date, there have been no reports of
NSF in patients with normal kidney function. The patients
at risk are those with severely impaired renal function.
Several researchers have suggested that liver transplant
patients with renal dysfunction are also prone to NSF.
A review of the available data does not suggest that the
risk of NSF in patients with advanced renal impairment is
the same for all gadolinium-based contrast agents. Distinct
physicochemical properties affect their stabilities and thus
the release of free gadolinium ions and pharmacokinetic
properties influence how long the contrast agent remains
in the body. The non-ionic linear chelates (see Table 1)
are associated with the highest risk of NSF, they are more
likely to release Gd3+ from the chelate complex. By contrast, the cyclic chelates are considered the most stable
and likely to have the lowest risk of NSF. New data suggest that the ionic linear chelate (Magnevist) is associated
with an increased risk of NSF in patients with severe renal
impairment, but not as great a risk as that with linear nonionic structure. The risk of NSF associated with the other
ionic chelates remains under investigation.
CONCLUSIONS: More than 250 cases of NSF worldwide have been associated with gadolinium-containing
contrast agents in patients with severe renal impairment.
The use of Omniscan and Magnevist has been contraindicated in patients with severe renal impairment.
REFERENCES
1. Increased risk of nephrogenic fibrosing dermopathy/nephrogenic
systemic fibrosis and gadolinium-containing MRI contrast agents
– Public Assessment Report, The Medicines and Healthcare products Regulatory Agency (MHRA).
60
ADVERSE REACTION REPORTING – STATE INSTITUTE FOR DRUG CONTROL
Jana Mlada, Jiri Lamka, Radek Bela
State Institute for Drug Control (SUKL), Pharmacovigilance Unit, Prague, Czech Republic
Key words: Drug monitoring/Adverse drug reaction reporting/Risk evaluation/Adverse effects
BACKGROUND: A new medicine must pass three
hurdles before it gets approval by the national drug regulatory authority. Sufficient evidence is required to show
the new drug is of 1) good quality, 2) efficacious, and 3)
safe for the purpose or purposes for which it is proposed.
While the first two criteria must be met before approval
procedure, the issue of the safety is less certain. Safety is
not absolute, and it can be judged only in relation to efficacy, requiring assessment on the part of the regulators in
deciding on acceptable limits of safety. Adverse drug reactions (ADRs) occur in 1.75–8% patients in hospital care
and 3–6% hospitalisations are being caused by adverse
drug reactions1–4. In 2006 there were 2 144 936 patients
hospitalized in the Czech Republic, which according to
very rough estimate represents 100 000 to 300 000 hospitalised patients who experienced adverse drug reactions.
METHODS: Collection of the spontaneous reports of
adverse drug reactions. The adverse drug reaction reporting is mandatory for all healthcare professionals in the
Czech Republic. In order to achieve maximum facilitation
in reporting State Institute for Drug Control (SUKL) provides on its website an on-line reporting form.
RESULTS: In 2006 1354 suspected adverse drug reac-
tions were sent to SUKL. Two-thirds of them represented
adverse drug reactions following BCG vaccination. The
last third constituted 360 physicians, approximately 1 %
of prescribing physicians in the Czech Republic.
CONCLUSIONS: Adverse drug reactions are associated with almost any pharmacotherapy with various rate
of seriousness. It is impossible to recognise all adverse
drug reactions in pre-authorisation clinical trials and for
this reason the importance of post-authorisation surveillance is very high. In 2006, the outcome of reporting in
the Czech Republic was so low that it makes detection of
possible risks in pharmacotherapy almost impossible.
REFERENCES
1. Lazarou J. Incidence of Adverse Drug Reaction in Hospitalized
Patients. JAMA, 1998; 279:1200–5.
2. Bond CA. Adverse Drug Reaction in United States Hospital.
Pharmacotherapy 2006; 26:601–8.
3. Haramburu F. Incidence and prevalence of adverse drug reaction.
Presse Med 2000; 29:111–4.
4. Pouyanne P. Admission to hospital caused by adverse drug reactions: cross sectional incidence study. BMJ 2000; 320:1036
5. Aktuální informace Ústavu zdravotnických informací a statistiky
České republiky č.4, Nemocnice v České republice v roce 2006,
12.3.2007
Adverse Reaction Reporting
Is ADR SERIOUS ?
Death ?
Life threatening ?
Hospitalization or its
prolongation ?
Serious health damage ?
Permanent health
damage ?
YES
NO
Is ADR EXPECTED ?
(~Compliance with
SPC (ref.5) ?
NO
REPORTING TO SÚKL
Suspicion of QUALITY DEFECT
Drug abuse ?
Overdose ?
Off label use ?
Lack of efficacy ?
Drug interaction ?
Matter with serious
impact on patient
YES
61
PROAPOPTOTIC EFFECT OF 1-METHOXYBRASSININ
Jan Mojzisa, Martina Pilatovaa, Peter Kutschyb, Marek Sarisskya,
Gabriela Mojzisovac, Ladislav Mirossaya
a
Department of Pharmacology, Faculty of Medicine, P.J. Safarik University, Kosice, Slovak Republic
Department of Organic Chemistry, Faculty of Natural Science, P.J. Safarik University, Kosice, Slovak Republic
c
Department of Experimental Medicine, Faculty of Medicine, P.J. Safarik University, Kosice, Slovak Republic
b
Key words: Cruciferous phytoalexins/Antiproliferative effects/Apoptosis/Leukemia
BACKROUND: A group of vegetables with considerable anticancer properties are the plants from
Cruciferous family. Epidemiological studies have shown
that a diet rich in cruciferous vegetables such as broccoli,
Brussels sprouts, cabbage and cauliflower, can lower the
risk of various cancers1–5. The major active compounds in
cruciferous vegetables, indole-3-carbinol and sulforaphane exhibit promising cancer protective properties in vitro
and in vivo6–9. Another indole-based group of compounds
naturally occurring in cruciferous vegetables are phytoalexins. These secondary metabolites, which are synthesized
de novo in response to diverse forms of stress, including
fungal infection, are part of the plants‘ chemical and biochemical defense mechanisms10. Moreover, there are few
indices that phytoalexins may also act as chemopreventive
or antiproliferative agents. The present study was conducted to examine the proapoptotic effect of 1-methoxybrassinin in the human leukemic Jurkat cell line.
METHODS: 1-Methoxybrassinin was synthetised
by Kutchy and co-workers11. Cycle TESTTM PLUS DNA
Reagent Kit, annexin V-FITC and propidium iodide
were purchased from Becton Dickinson, USA. Jurkat
cells (human acute T-lymphoblastic leukemia cells) were
kindly provided by Dr. M. Hajdúch (Olomouc, Czech
Republic). Cells were maintained in RPMI 1640 medium with Glutamax-I supplemented with 10% foetal calf
serum, penicillin (100 IU.mL–1) and streptomycin (100
μg.mL–1) (all from Invitrogen, UK), in the atmosphere 5%
CO2 in humidified air at 37 oC. Cell viability, estimated by
trypan blue exclusion, was greater than 95 % before each
experiment. Cytotoxic effect of the tested compounds was
studied by using colorimetric microculture assay with the
MTT end-point12. Cell cycle distribution in cells treated
with the tested agent was analyzed by propidium iodide
DNA staining using Cycle TESTTM PLUS DNA Reagent
Kit (Becton Dickinson, USA). The data were analyzed
using Win MDI software. Percentages of cells corresponding to G0/G1, S and G2/M phases of the cell cycle were
calculated. Sub-G0/G1 fraction of cells was identified as
an apoptotic population. Instrument sensitivity and performance were checked prior to each acquisition by using
DNA QC Particle Kit (Becton Dickinson, USA). Annexin
V/PI staining was performed as described previously13.
DNA fragmentation Assay: treated (1-methoxybrassinin
for 24, 48 and 72 hours) and untreated cells (1x10–6) were
washed twice with 1 x PBS calcium and magnesium free.
Table 1. Flow cytometric analysis of cell cycle distribution and apoptosis in Jurkat cells treated with 100 μmol.L–1
of 1-methoxybrassinin (in %).
Cell cycle
Time (hr)
sub-G0/G1
G0/G1
S
G2/M
Control
2.0
71.0
17.0
10.0
24
65.0
25.0
6.0
4.0
48
87.0
9.0
2.0
2.0
72
92.0
6.0
1.5
0.5
Apoptosis
An-/PI-
An+/PI-
An+/PI+
Control
83.0
8.0
5.0
72
4.0
48.0
47.0
An-/PI- – live cells; An+/PI- – early apoptic cells ; An+/PI+ – late apoptotic/necrotic cells
62
Lysation of cells was provided in lysis buffer contains
(10 mmol.L–1 TRIS, 10 mmol.L–1 EDTA, 0.5% Triton X100). Proteinase K (1 mg/mL) was added and cells were
incubated at 37 °C 1 hour. Then were heated at 70 °C
10 minutes and after adding RNAase (200 μg/mL) cells
were incubated at 37 °C 1 hour, again. Samples were
transfered to 2 % agarose gel and run with 40V at 3 hours.
DNA fragments were visualized by UV illuminator.
R E S U LT S: At the highest concentration
(100 μmol.L–1), 1-methoxybrassinin decreased cell survival to 38.2 % of the control value. However, significant
decrease in cell survival was also observed at concentration 1 μmol.L–1. Cells exposed to tested compound exhibited a time-dependent increase in the sub-G0/G1 fraction
with the onset already after 24 h. After 72 hours of incubation, more than 90 % of cells exposed to 1-methoxybrassinin were found as having sub-G0/G1 DNA content. Very
few cells were found in the S and G2/M phases throughout
the time-points examined (Table 1). These results indicate
that 1-methoxybrassinin causes the appearence of the fraction of cells with sub-G0/G1 DNA content wich is suggestive of apoptosis. In control cells, less than 8 % of the early
apoptotic cells (Annexin V+ /PI-) were detected. Exposure
of cells to 1-methoxybrassinin resulted in an evident increase in the proportion of these early apoptotic cells (to
48.1 %). Moreover, increase in the number of apoptotic
cells was accompanied by more than 10-fold increase in
the annexin V+/PI+ subpopulations of the cells (4.5 vs.
47.7 %) (Table 1). In order to ascertain the induction of
cell death by 1-methoxybrassinin, DNA fragmentation, reflecting the endonuclease activity characteristic of apoptosis, was analyzed. Ttreatment with 1-methoxybrassinin at
a concentration of 100 μmol.L–1 for 24, 48 and 72 hours
resulted in the formation of definite fragments that could
be seen via electrophoretic examination as a characteristic
ladder pattern.
CONCLUSIONS: In conclusion, we found that 1methoxybrassinin exerted the significant cytotoxic and
apoptosis-inducing activity in Jurkat cells. However,
further studies are required to investigate the underlying
mechanism of the action 1-methoxybrassinin.
ACKNOWLEDGEMENT
This work was supported by the Slovak Research and
Development Agency under the contract No. APVV-0514-06
and by VEGA grants 1/4236/07 and 1/3365/06.
REFERENCES
1. Yuan JM, Gago-Dominguez M, Castelao JE, Hankin JH, Ross RK,
Yu MC. Cruciferous vegetables in relation to renal cell carcinoma.
Int J Cancer 1998; 77:211–6.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
Jain MG, Hislop GT, Howe GR, Ghadirian P. Plant foods, antioxidants,
and prostate cancer risk: findings from case-control studies in Canada.
Nutr Cancer 1999; 34:73–84.
Cohen JH, Kristal AR, Stanford JL. Fruit and vegetable intakes and
prostate cancer risk. J Natl Cancer Inst 2000; 92:61–8.
Graham S, Dayal H, Swanson M, Mittelman A, Wilkinson G. Diet in
the epidemiology of cancer of the colon and rectum. J Natl Cancer Inst
1978; 61:709–14.
Agudo A, Esteve MG, Pallares C, Martinez-Ballarin I, Fabregat X,
Malats N, Machengs I, Badia A, Gonzalez CA. Vegetable and fruit
intake and the risk of lung cancer in women in Barcelona, Spain. Eur
J Cancer 1997; 331256–61.
Wattenberg LW, Loub WD. Inhibition of polycyclic aromatic hydrocarbon-induced neoplasia by naturally occurring indoles. Cancer Res
1978; 38:1410–3.
Cover CM, Hsieh SJ, Tran SH, Hallden G, Kim GS, Bjeldanes LF,
Firestone GL. Indole-3-carbinol inhibits the expression of cyclin-dependent kinase-6 and induces a G1 cell cycle arrest of human breast
cancer cells independent of estrogen receptor signaling. J Biol Chem
1998; 273:3838–47.
Frydoonfar HR, McGrath DR, Spigelman AD. The effect of indole-3carbinol and sulforaphane on a prostate cancer cell line. ANZ J Surg
2003; 73:54–6.
Jin L, Qi M, Chen DZ, Anderson A, Yang GY, Arbeit JM, Auborn KJ.
Indole-3-carbinol prevents cervical cancer in human papilloma virus
type 16 (HPV16) transgenic mice. Cancer Res 1999; 59:3991–7.
Pedras MS, Nycholat CM, Montaut S, Xu Y, Khan AQ. Chemical
defenses of crucifers: elicitation and metabolism of phytoalexins and
indole-3-acetonitrile in brown mustard and turnip. Phytochemistry
2002; 59:611–25.
Kutschy P, Suchy M, Monde K, Harada N, Maruskova R, Curillova
Z, Dzurilla M, Miklosova M, Mezencev R, Mojzis J. Spirocyclization
strategy toward indole phytoalexins. The first synthesis of (+/-)-1-methoxyspirobrassinin, (+/-)-1-methoxyspirobrassinol, and (+/-)-1-methoxyspirobrassinol methyl ether. Tetrahedron Lett 2002; 43:9489–92.
Mosmann T. Rapid colorimetric assay for cellular growth and survival:
application to proliferation and cytotoxicity assays. J Immunol Methods
1983; 65:55–63.
Kravtsov VD, Greer JP, Whitlock JA, Koury MJ. Use of the microculture kinetic assay of apoptosis to determine chemosensitivities of
leukemias. Blood 1998; 92:968–80.
63
REACTIVITY OF URINARY BLADDER SMOOTH MUSCLE
Juraj Mokry, Gabriela Nosalova
Key words: Urinary bladder/Smooth muscle reactivity/In vitro/Pharmacology
BACKGROUND: Urinary bladder smooth muscle
(UBSM) with its properties assumes unsubstitutable position in morphological as well as functional state of organism and is responsible for appropriate bladder function.
Impaired activity of urinary bladder leads to disturbed
voiding an affects the quality of life in these patients.
Clinically, incidence of unstable (overactive) bladder is
relatively frequent and increases with age1. This led us
to evaluation of the underlying mechanisms and several
possibilities of pharmacological modulation.
The aims of presented study were as follows: 1) to use
modified in vitro method of organ baths for UBSM strips,
2) to evaluate and compare the reactivity of UBSM to
contractile agents (acetylcholine, carbachol), 3) to follow
the interspecies variability in UBSM reactivity, 4) to elucidate the role of acetylcholinesterase inhibition in UBSM
reactivity, 5) to evaluate the age dependence of UBSM
reactivity, and, 6) to modify the reactivity of UBSM with
various drugs.
METHODS: The in vitro method of organ baths was
used in this study, previously described in several papers
about airway smooth muscle reactivity testing2,3. All experiments were approved by Local Ethical Committee
at Jessenius Faculty of Medicine, Comenius University
in Martin. Adult male guinea pigs (n=56, 250–450 g),
adult rabbits (n = 16, 1500–2500 g) and newborn rabbits (n = 8, 30–40 g) were sacrified and urinary bladder
was removed. Furthermore, UBSM samples from male
patients (n = 16, 55–80 years) after open prostatectomy
due to benign prostatic hyperplasia were evaluated. Thin
UBSM strips (approx. 2x2x15 mm) were inserted into organ chambers (30 ml) containing pre-heated (37 ± 0.5 °C)
and pneumoxide-saturated Krebs-Henseleit‘s solution and
connected to force transducer, amplifier, and recorder.
After one hour adaptation period, cumulative doses of
acetylcholine or carbachol were added into the chambers (both subst. Sigma Aldrich). After several scours
with fresh Krebs-Henseleit‘s solution (every 10 minutes),
tested substance was administered to reach desired
concentration: neostigmine 10–4 mol.L–1, oxybutynine
10–6–10–4 mol.L–1, dicyclomine 10–6–10–3 mol.L–1, propantheline 10–6–10–3 mol.L–1, aminophylline 10–4–10–3 mol.L–1, caffeine 10–5–10–3 mol.L–1, and theophylline 10–5–10–3 mol.L–1;
(all subst. Sigma Aldrich). After 15 min incubation period, contractile agents were applied again in cumulative
manner.
The contraction force is expressed in grams recalculated to 100 mg of tissue. ANOVA test was used for statistical analysis; p < 0.05 was considered as statistically
significant.
RESULTS: Cumulative adding acetylcholine and
carbachol into organ chambers resulted in continual increase of contractile responses in all experiments. The
UBSM reactivity to carbachol was significantly higher
comparing to acetylcholine (p < 0.01; in concentrations
of 10–7–10–5 mol.L–1). Similar diference was observed in
human strips (p < 0.01; 10–6–10–5 mol.L–1).
Incubation with neostigmine (acetylcholinesterase inhibitor) led to significant increase of contractile responses
to acetylcholine and not carbachol4. Evaluating the interspecies differences, reactivity to both acetylcholine and
carbachol was significantly higher in guinea pigs comparing to human as well as rabbit UBSM strips. The UBSM
reactivity to both contractile agents was significantly
higher in adult rabbits, comparing to newborns (p < 0.01;
10–8–10–5 mol.L–1). The administration of oxybutynine, dicyclomine as well as propantheline caused significant decrease of guinea pig UBSM reactivity to acetylcholine; the
most pronounced effect was observed with oxybutynine5–7.
By evaluation of several xanthine derivatives, significant
decrease of UBSM reactivity was observed in adult rabbits after incubation with aminophylline (10–3 mol.L–1)8,
and in guinea pigs after incubation with caffeine, theobromine and theophylline in concentrations of 10–4 and
10–3 mol.L–1.
CONCLUSIONS: We suppose that higher contractile
potency of carbachol comparing to acetylcholine results
from higher affinity of carbachol to muscarine receptors,
as well as from 90-fold more effective stimulation of phospholipase C (ref.9). Furthermore, increased UBSM reactivity to acetylcholine after incubation with neostigmine
(inhibitor of acetylcholinesterase) supports a participation of various pharmacokinetic properties in different
reactivity to these contractile agents10.Our results are in
accord with results of Yoshida et al.11, who demonstrated
higher UBSM reactivity and increased resistance to antimuscarinic therapy in older patients. We suppose that
the lower reactivity of UBSM in newborn period could
be based on immaturity of contractile mechanism and
nervous system.
Several literature sources described the mechanism of
action of muscarinic antagonists12, 13, showing that antagonism of muscarinic receptors (especially M3 subtype) led
to suppression of second messengers creation and calcium
release to cytoplasm as well as to decreased Rho kinase
activation followed by smooth muscle relaxation14. Our
results supported this fact, as administration of oxybutynine, propantheline and dicyclomine led to decreased
reactivity and thus point out their possible effectiveness in
treatment of overactive bladder. Decreased UBSM reactiv-
64
ity in rabbits, guinea pigs as well as in humans caused by
incubation with xanthine derivatives could be explained
by their ability to inhibit the enzyme phosphodiesterase,
which is responsible for intracellular degradation of cyclic
AMP (ref.15). However, a significant relaxing effect was
observed only after applying higher concentrations.
Based on our results we can conclude that: 1) UBSM
reactivity can be evaluated in vitro in various species as
well as under different conditions, 2) UBSM reactivity to
acetylcholine and carbachol was significantly higher in
guinea pigs comparing to rabbits and human, 3) inhibition
of acetylcholinesterase led to increase of UBSM reactivity
to acetylcholine and not to carbachol, 4) UBSM reactivity was significantly higher in adult rabbits comparing to
newborn rabbits, 5) anticholinergics caused significant decrease of UBSM reactivity in guinea pigs with the highest
potency of oxybutynine, and 6) methylxanthines administration resulted in significantly lower contractile responses, however, only in relatively high concentrations.
ACKNOWLEDGEMENT
Financial support by the grant APVT- 20-013102 is gratefully acknowledged.
REFERENCES
1. Svihra J, Baska T, Martin M, et al. Prevalence of female urinary
incontinence in Slovak Republic. Urologia 2001; 7: 29–34.
2. Franova S. The influence of inhaled furosemide on adverse effects of ACE-inhibitors in airways. Bratisl Lek Listy 2001; 102:
309–13.
3. Mokry J, Svihra J, Nosalova G, Kliment J. Mechanisms of reactivity of urinary bladder smooth muscle. Bratisl Lek Listy 2002; 103:
279–83.
4. Mokry J, Jakubesova M, Svihra J, Urdzik J, Hudec M, Nosalova G,
Kliment J. Reactivity of urinary bladder smooth muscle in guinea
pigs to acetylcholine and carbachol: participation of acetylcholinesterase. Physiol Res 2005; 54:453–8.
5. Urdzik J, Jakubesova M, Hudec M, Mokry J, Svihra J. In vitro reactivity of urinary bladder smooth muscle influenced by oxybutynin,
propranolol, and indomethacin in guinea pigs Acta Med Martin
2003; 3:23–9.
6. Jakubesova M, Rusnakova H, Pasztoova K, Mokry J, Svihra J.
Influence of dicyclomine on in vitro reactivity of urinary bladder
smooth muscle in guinea pigs. Acta Med Martin 2004; 4:12–7.
7. Mokry J, Nosalova G, Jakubesova M. Propantheline and in vitro
reactivity of urinary bladder smooth muscle in guinea pigs. Bratisl
Lek Listy 2005; 106:151–4.
8. Hudec M, Jakubesova M, Urdzik J, Mokry J, Svihra J. The influence of aminophylline on the contractility of urinary bladder
smooth muscle in rabbits. Acta Med Martin 2003; 3:9–15.
9. Jankovic SM, Kouvelas D, Mirtsou-Fidani V. Time course of isolated rat fundus response to muscarinic agonists: a measurement
of intrinsic efficacy. Physiol Res 1998; 47:463–70.
10. Nakahara T, Kubota Y, Sakamoto K, Ishii K. The role of cholinesterases in rat urinary bladder contractility. Urol Res 2003; 31:223–
6.
11. Yoshida M, Homma Y, Inadome A. Age-related changes in cholinergic and purinergic neurotransmission in human isolated bladder
smooth muscles. Exp Gerontol 2001; 36:99.
12. Schneider T, Fetscher C, Krege S, Michel MC. Signal transduction
underlying carbachol-induced contraction of human urinary bladder. J Pharmacol Exp Ther 2004; 309: 1148–53.
13. Andersson KE, Chapple C, Wein A. The basis for the drug treatment of the overactive bladder. World J Urol 2001; 19:294–8.
14. Peters SLM, Schmidt M, Michel MC. Rho kinase: a target for treating urinary bladder dysfunction? Trends Pharmacol Sci 2006; 27:
492–7.
15. Nosalova G. New opinions on the mechanism of action of xanthine
derivatives. Slovakofarma Revue 2000; 10: 51–5.
CARDIOPROTECTIVE EFFECTS OF NEFIRACETAM – A NEWLY SYNTHESIZED
POTENTIAL NOOTROPIC ANALOG OF PIRACETAM
Jiri Necasa, Lenka Bartosikovaa, Oldrich Farsab, Tomas Bartosikc, Galina Kuzminaa, Jiri Luzaa
a
Department of Physiology, Faculty of Medicine and Dentistry, Palacky University, Hnevotinska 3, 775 15 Olomouc, Czech
Republic
b
Department of Chemical Drugs, Faculty of Pharmacy, Veterinary and Pharmaceutical University, Palackeho 1–3,
612 42 Brno, Czech Republic
c
Department of Anaesthesiology and Intensive Care, St. Anne’s University Hospital Brno, Pekarska 53, 656 91 Brno, Czech
Republic
Key words: Nefiracetam/Heart ischemia-reperfusion/Free oxygen radicals
BACKGROUND: Racetames are the group of drugs
characterized by the presence of an ω-lactame moiety in
their structure, in most it uses to be a fragment of pyrrolidin-2-one, but substances with piperidin-2-one and azepan2-one have also been appearing. Typical representatives of
such structures are piracetam, etiracetam, levetiracetam
and a newly synthesized nefiracetam. Nefiracetam, N(2,6-dimethylphenyl)-2-(2-oxopyrrolidin-1-yl) acetamide,
patented by Betzing et al. in 1982 (ref.1), is a representa-
tive of substances with improved lipophilicity and thereby
ability of crossing the blood-brain barrier (BBB) to the
brain. Effects of nefiracetam to many tissues and organs
both in vitro and in vivo were evaluated, but there is lack
of information about its in vitro effect to the heart, in part
due to its poor solubility in water. The aim of the study
was to monitor potential cardio-effect of nefiracetam in
pre-clinical experiment in vitro.
METHODS: The study was performed on 30 male
Wistar SPF (AnLab, Germany) laboratory rats of identical age (6 month) and comparable weight (254 ± 17 g).
After a recovery period, the animals were divided randomly into 2 groups (n = 10) – treated and placebo. All
rats were anesthetized with an intraperitoneal injection of
(2% Rometar 0.5 ml + 1% Narkamon 10 ml, dose 0.5 mL
solution/100 g body weight) and after the intraperitoneal
administration of heparine injection of 500 IU dose, the
heart were excised and perfused. In all experiments, modified Langendorf’s method and the universal apparatus
Hugo Sachs Electronic, UP 100, (Germany HSE) were
used. Working schedule: stabilization/ischaemia/reperfusion were proceeded in intervals 10/30/20 min. The tested
substance was added to perfusate in the dose of 50 mg/L
all along time of experiment (treated hearts). Placebo hearts underwent ischemia-reperfusion only. Biomechanical
parameters from isolated heart – left ventricle pressure,
end-diastolic pressure, contractility (LVP, LVEDP, dP/
dtmax) were measured using a ball filled with liquid (8–
12 mmHg), inserted through the left atrium in the left
ventricle connected to the analog convertor (Isotec HSE,
DIF modul HSE)2.
65
RESULTS: In placebo hearts left ventricular pressure
(LVP) recovered until 67 ± 5 % of preischemic values at
the end of the reperfusion. The treated hearts showed
improved postischemic recovery, reaching LVP values of
53 ± 5 % at the end of the reperfusion. In placebo hearts left ventricular end-diastolic pressure (LVEDP) rise
from 10.3 ± 0.2 mmHg to 33.0 ± 3.0 mmHg after 20 min
of reperfusion. In the treated hearts LVEDP was 41 ±
3 mmHg at the end of reperfusion. The nefiracetam added
to perfusate of the treated hearts improved myocardial
contractility (+dP/dtmax) recovery during reperfusion to 68
± 5 %. These values were significantly greater than those
obtained from the placebo hearts (80 ± 5 %).
CONCLUSIONS: The results demonstrate that newly
synthesized nefiracetam showed some effect on defined
biomechanical parameters. The effect of this substance is
probably employment by supporting myocardial anaerobic
metabolism during ischemic period.
REFERENCES
1. Betzing H, Biedermann J, Materne C; Nauser V. GE 2924011, April
8th, 1982.
2. Kozlovski VI, Vdovichenko VP, Chlopicki S, Malchit SS, Praliyev
KD, Zcilkibayev OT. Anti-arrhytmic profile and endothelial action
of novel decanhydroquinoline derivatives. Polish J Pharmacol 2004;
56:767–74.
REDUCED EXPRESSION OF ACE2 IN THE KIDNEYS OF SPONTANEOUSLY
HYPERTENSIVE RATS: THE EFFECT OF PERINATAL ADMINISTRATION
OF LOSARTAN
Peter Ochodnickya, Michael Olvedya, Peter Kreneka, Katarina Mackovicovaa, Jan Klimasa,
Frantisek Kristekb, Sona Cacanyiovab, Jan Kyselovica
a
Institute of Normal and Pathological Physiology, Slovak Academy of Sciences, Bratislava, Slovak Republic
b
Key words: ACE2/Hypertension/Losartan/Real-time PCR/Kidney
BACKGROUND: Recently discovered homologue
of angiotensin-converting enzyme (ACE) termed ACE2
degrades vasoconstrictory angiotensin II to angiotensin
(1–7) with vasodilatory and antiproliferative properties1.
Therefore, ACE2 might serve as an important regulator
of renin-angiotensin system (RAS) counteracting the detrimental effects of angiotensin I(ref.2). Moreover, ACE2
epxression in the kidney has been implicated in the development of hypertension3, though its precise physiological
role remains to be established. Spontaneously hypertensive rats (SHR) develop hypertension at early age and
display overactivation of intrarenal RAS (ref.4). Therefore,
an early intervention in RAS might retard the development of hypertension. In the present study we examined
the expression of ACE2 in the kidneys of young SHRs.
Additionally, we studied the effect of perinatal administration of angiotensin II AT1 blocker losartan on the
expression of ACE2 and its relations to the changes in
blood pressure.
METHODS: Blood pressure was measured weekly by
non-invasive tail-cuff method in Wistar rats (n=5), SHR
(n = 5) and SHR treated with losartan (20 mg/kg/day,
p.o. twice daily) either perinatally up to 9 weeks of age
(SHR+LOS 0–9, n = 5, dams during gestation/lactation
period and pups from weaning at 4 weeks) or perinatally,
but withdrawn after weaning at 4 weeks of age (SHR+LOS
0–4, n = 5). The animals were sacrificed at the age of
9 weeks, kidneys were weighed and processed for the
histological assessment of renal damage. Following isolation of RNA from renal cortex, ACE2 mRNA levels were
determined by real-time PCR using relative comparison
method with GAPDH as a housekeeping gene.
RESULTS: Perinatal losartan treatment prevented the
rise of blood pressure in the SHRs. This effect however
66
Table 1. Body weight (BW), kidney weight (KW), systolic blood pressure (SBP) in Wistar, SHR, and SHR rats perinatally treated with losartan up to 9 weeks (SHR+LOS 0-9), and SHR rats perinatally treated with losartan, which
was withdrawn after weaning at 4 weeks (SHR+LOS 0-4).
WISTAR
BW (g)
KW(mg/g BW)
SBP (mmHg)
SHR
SHR+LOS 0-9
SHR+LOS 0-4
209 ± 3
195 ± 5
196 ± 5
206 ± 5
3.8 ± 0.2
3.5 ± 0.1
3.4 ± 0.1
3.6 ± 0.2
109 ± 2
149 ± 2*
105 ± 3#
145 ± 2*
n = 5 in each group, average ± SEM, * p < 0.05 vs. Wistar, # p < 0.05 vs. SHR
ACKNOWLEDGEMENT
Financial support by the grant of Comenius University
REFERENCES
1. Santos RA, Ferreira AJ. Angiotensin-(1-7) and the renin-angiotensin system. Curr Opin Nephrol Hypertens 2007; 16:122–8.
2. Danilczyk U, Penninger JM. Angiotensin-converting enzyme II in
the heart and the kidney. Circ Res 2006; 98:463–71.
1,5
Relativ e ex pression of ACE2
did not sustain after losartan withdrawal (Table 1). Renal
expression of ACE2 was reduced by 60 % ±12 % in SHR
when compared to Wistar rats (Fig. 1). Perinatal losartan
treatment failed to show any significant effect on ACE2
mRNA levels in the kidney, despite of the tendency for
recovery.
CONCLUSIONS: Renal ACE2 mRNA levels are reduced in young SHR rats, potentially contributing to local
RAS activation in these animals. Perinatal intervention in
RAS results only in temporary decrease of blood pressure
and fails to alter renal ACE2 expression. Drugs regulating
renal ACE2 expression might provide novel therapeutical
strategy in the treatment of essential hypertension.
1
*
0,5
0
WISTAR
SHR
SHR+LOS 0-9 SHR+LOS 0-4
Fig. 1. Relative expression of ACE2 in renal cortex of
four experimental groups (average ± SEM, n = 5
per group, *p < 0.05 vs. Wistar).
3. Krum H, Gilbert RE. Novel therapies blocking the renin-angiotensin-aldosterone system in the management of hypertension
and related disorders. J Hypertens 2007; 25:25–35.
4. Igase M, Strawn WB, Gallagher PE, Geary RL, Ferrario CM.
Angiotensin II AT1 receptors regulate ACE2 and angiotensin-(17) expression in the aorta of spontaneously hypertensive rats. Am
J Physiol Heart Circ Physiol 2005; 289:H1013–19.
INTOXICATIONS BY DRUGS WITH ANALGESIC EFFECT IN CHILDREN
HOSPITALIZED IN CHILDREN’S FACULTY HOSPITAL IN BRATISLAVA
IN YEARS 1996–2005
Elena Ondriasovaa, Anna Harcarovaa, Iveta Chovancovaa, Silvia Plackovab,
Marta Benedekovac, Laszlo Kovacsd, Rudolf Riedele, Magdalena Kuzelovaa
a
National Toxicological Information Centre, Faculty of Medicine, Comenius University, Bratislava, Slovak Republic
c
I. Children’s Clinic and
d
II. Children’s Clinic, Faculty of Medicine, Comenius University, Bratislava, Slovak Republic
e
Children’s Clinic of Anesthesiology and Intensive Medicine, Bratislava, Slovak Republic
b
Key words: Drug intoxications/Analgesics/Children/Adolescents
BACKGROUND: Drug intoxications in children and
adolescents have still increasing tendency. Generally,
childhood poisoning can be attributed to insufficient
parental supervision and accessibility of products with
poisoning potential. Analgesic acting drugs are one of
the most frequent causes of intoxications in children and
adolescents.
METHODS: Medical records of patients intoxicated with analgesic acting drugs and hospitalized at the
I. and II. Children’s Clinics and Children’s Clinic of
67
60
number of intoxications
A
50
40
PSS 0
PSS 1
30
PSS 2
PSS 3
20
10
0
1996-2000
2001-2005
B
number of intoxications
60
50
40
accidental
30
intentional
20
10
0
boys
girls
1996-2000
boys
girls
2001-2005
Fig. 1. A. Intoxications according to the poisoning severity score (PSS).
B. Accidental and intentional intoxications according to patient’s gender.
Anesthesiology and Intensive Medicine of Children’s
Faculty Hospital in Bratislava during 10 years period
1996–2005 were analyzed. The analyzed group included
181 patients up to 18 years of age, which formed 11.5 %
of children and adolescents who were hospitalized with
the diagnosis “intoxication” during the observed period.
The highest number of patients hospitalized was in the
15–18-age category (99 i.e. 54.7 %). The second most frequent age group were children at the age of 0–5 years (49
i.e. 27.1 %).
RESULTS: The significantly (p < 0.05) higher number
of girls (130) suffered from intoxication than boys (51)
(Fig. 1). Suicidal intoxications (117) predominated over
accidental intoxications (61). The majority of the hospitalized children had minor symptoms of intoxication
(poisoning severity score – PSS – 1)1 or they were without
symptoms (PSS – 0). There was no fatal intoxication (PSS
– 4). Comparing the two five year periods (1996–2000
and 2001–2005) in the second period (2001–2005) increased the number of intoxicated patients by 5.7 % and
there were more cases with PSS – 2/moderate intoxication
and with PSS – 3/severe intoxication (Fig. 1). Paracetamol
(acetaminophen) was the most frequently ingested drug
from the group of analgesics-antipyretics, followed by ibu-
profen from the group of nonsteroidal antiinflammatory
drugs (Table 1). From the group of opioid analgesics tramadol was the most frequent cause (7 intoxications) of
children intoxications.
CONCLUSIONS: This study defines the characteristic
pattern of pediatric intoxications with analgesic acting
drugs with respect to different age groups and gender.
Most adolescent intoxications occurred in girls and were
Table 1. Number of intoxicated patients
Drug
1996-2000 2001-2005 1996-2005
paracetamol
42
35
77
ibuprofen
31
40
71
ASA, salicylates
19
9
28
diclofenac
8
13
21
methamizole
8
9
17
propyphenazone
4
6
10
68
intentional. Parental education and intensified child supervision are indicated measures of prevention for unintentional poisoning.
REFERENCES
1. Persson HE, Sjoberg GK, Haines JA, Pronczuk de Garbino J.
Poisoning severity score. Grading of acute poisoning. J Toxicol
Clin Toxicol 1998; 36:205–13.
CAMALEXIN AND ITS DERIVATIVE BENZOCAMALEXIN WITH VARIOUS
SUBSTITUENTS AND THEIR ANTIPROLIFERATIVE EFFECT ON DIFFERENT
CANCER CELL LINES
Martina Pilatovaa, Peter Kutschyb, Jan Mojzisa, Zuzana Curillovab, Maria Repovskab,
Marek Sarisskya, Andrej Mirossaya , Roman Mezencevc, Ladislav Mirossaya
a
Department of Pharmacology, Faculty of Medicine, P. J. Safarik University, 040 11 Kosice, Slovak Republic
Department of Organic Chemistry, Institute of Chemical Sciences, Faculty of Science, P. J. Safarik University,
040 11 Kosice, Slovak Republic
c
Ovarian Cancer Institute, School of Biology, Georgia Institute of Technology, Atlanta, USA
b
Key words: Camalexin/Benzocamalexine/Indole phytoalexins/Antiproliferative
BACKGROUND: Camalexin (3-thiazol-2’-yl-indole)
is an indole phytoalexin produced by important plants
of the family Crucicerae. Phytoalexins are low molecular weight compounds produced by plants in response
to physical, biological or chemical exposure. These so
called phytoalexins show great structural diversity and
are often restricted to a limited number of plant species1. With respect to the epidemiologically proven cancer chemopreventive properties of brassica vegetables,
Fig. 1. Camalexin
Table 1. Effects of tested compounds on viability of cancor cell lines cells after 72 h
incubation detected by MTT test (in %).
37
44
K22
K23
K24
K45
Concentration
mol.L–1
10–4
5x10–5
10–5
10–4
5x10–5
10–5
10–4
5x10–5
10–5
10–4
5x10–5
10–5
10–4
5x10–5
10–5
10–4
5x10–5
10–5
Jurkat
A-549
MCF
MDA
HeLa
25
43
83
0
0
65
55
65
80
40
45
70
60
65
75
70
80
90
70
90
100
0
0
100
70
90
90
50
60
100
60
70
80
70
75
85
85
100
100
0
0
100
60
90
100
75
75
100
50
80
100
65
75
85
36
67
85
0
0
61
50
50
90
75
75
75
30
45
75
50
60
75
24
50
88
0
0
75
40
50
85
25
65
85
30
50
75
60
70
85
antiproliferative and anticarcinogenic activities of indole
phytoalexins have been studied. Several indole phytoalexins (i.e. brassinin, spirobrassinin, brassilexin, camalexin,
1-methoxyspirobrassinin, 1-methoxyspirobrassinol and
methoxyspirobrassinol methyl ether) have been found to
possess significant antiproliferative activity against various
cancer cells2. Based on this study we tested camalexin and
its derivative benzocamalexin with various substituents to
discover possible relationship between structure, character of substituent and antiproliferative effects on different
human cancer cell lines.
METHODS: We examined: camalexin (37) (Fig. 1),
benzocamalexin (44), 1-methylbenzocamalexin (K22),
1-methyl-6’-methoxybenzocamalexin (K23), 1-methyl-6’fluorobenzocamalexin (K24), 1-methyl-6’-cyanobenzocamalexin (K45) on different human cancer cell lines:
Jurkat-acute T- lymphoblastic leukemia, MDA-MB-231,
MCF-7 – mammary gland adenocarcinoma, A-549 lung
carcinoma, HeLa – cervical carcinoma.
Cytotoxic effect of the tested compounds was studied
using colorimetric microculture assay with the MTT endpoint. The amount of MTT reduced to formazan is proportional to the number of viable cells. Briefly, 5x104 cells
were plated per well in 96-well polystyrene microplates
(Sarstedt, Germany) in the culture medium containing the
tested chemicals at final concentrations 10–4, 5x10–5, and
10–5 mol.L–1. After 72 hours incubation, 10 μL of MTT
(5 mg.mL–1) were added in each well. After additional 4 h,
during which insoluble formazan was produced, 100 μL
of 10% sodium dodecylsulphate were added in each well
and another 12 h were allowed the formazan to be dissolved. The absorbance was measured at 540 nm using
the automated MRX microplate reader (Dynatech labo-
69
ratories UK). Absorbance of control wells was taken as
100%, and the results were expressed as a percent of control (Table1).
RESULTS: Our data has shown that camalexin possesses antiproliferative activity. The highest effectivity
was observed in inhibition of proliferation Jurkat and
HeLa cells at concentration 10–4 mol.L–1 with efficiency
75 % and at concentration 5x10–5 mol.L–1 with efficiency
50–57 %. The binding of benzene on camalexin structure
significantly enhanced its cytotoxic effect and complete
inhibition of cell proliferation was achieved at concentration 10–4 mol.L–1 and 5x10–5 mol.L–1 in all used cell lines.
Methylation of benzocamalexin resulted in decreased antiproliferative activity and neither addition of methoxy,
fluoro or cyano group increased it.
CONCLUSIONS: These results suggest the valid interest in structure and antiproliferative activitity relationship in searching for anticancer agent. Further studies
are necesary to investigate the mechanism of action and
to find out the relationship between structure, character
and position of substituents and their antiproliferative
activity.
ACKNOWLEDGEMENT
This work was supported by the Slovak Research and
Development Agency under the contract No. APVV-0514-06.
REFERENCES
1. Glawischnig E. Camalexin. Phytochemistry 2007; 401–6.
2. Mezencev R, Mojzis J, Pilatova M, Kutschy P. Antiproliferative and
cancer chemopreventive activity of phytoalexins: focus on indole
phytoalexins from crucifers. Neoplasma 2003; 239–45.
ANTHRACYCLINE CARDIOTOXICITY AND THE POSSIBILITY
OF THE PHARMACOLOGICAL CARDIOPROTECTION
Olga Popelovaa, Martin Sterbaa, Tomas Simunekb, Yvona Mazurovac, Michaela Adamcovad,
Ivana Guncovac, Vladimír Gersla
a
Department of Pharmacology, Faculty of Medicine in Hradec Kralove, Charles University in Prague, 500 38 Hradec
Kralove, Czech Republic
b
Department of Biochemical Sciences, Faculty of Pharmacy in Hradec Kralove, Charles University in Prague, 500 38
Hradec Kralove, Czech Republic
c
Department of Histology and Embryology, and
d
Department of Physiology, Faculty of Medicine in Hradec Kralove, Charles University in Prague, 500 38 Hradec Kralove,
Czech Republic
Key words: Anthracycline cardiotoxicity/Cardioprotection/Iron chelators
BACKGROUND: Anthracyclines (e.g., doxorubicin
and daunorubicin) belong to the most widely used anticancer drugs in today clinical practice. However, their
clinical impact is considerably limited by the occurrence
of the cardiac toxicity. Mainly the chronic and delayed
types of cardiotoxicity are considered to be a serious
healthcare problem, since both may result to severe and
largely irreversible myocardial injury manifesting itself
as the congestive heart failure1. Despite the more than
40 years of intensive research, the precise pathogenesis of
70
anthracycline cardiotoxicity remains still elusive, though
several hypothesis were proposed. Nevertheless, so called
“iron and free radicals” hypothesis is currently still among
the most widely accepted. In part, this is due to the fact,
that the only well evidenced and available so far approach
to pharmacological cardioprotection is the administration of dexrazoxane – the pro-drug of the iron-chelating
compound ADR-925. Although this drug has been clearly
shown to be clinically effective, it was assumed that in
Europe it is used only in 6–7 % of patients treated with
anthracyclines. This is likely due to the relative high costs
of therapy and the risk of myelotoxicity. Hence, the urgent
need for novel cardioprotectant remains.
The aim of the present study was to investigate the
potential cardioprotective properties of novel lipophilic
aroylhydrazone chelators (pyridoxal 2-chlorobenzoyl
hydrazone – o-108, salicylaldehyde isonicotinoyl hydrazone – SIH) as well as dexrazoxane (DEX, model cardioprotectant) and deferiprone (novel clinically used iron
chelator) on the rabbit model of chronic anthracycline
cardiotoxicity.
METHODS: In the experiment 1, potential cardioprotective properties of two aroylhydrazone iron chelators
(o-108 and SIH) were studied. The anthracycline cardiotoxicity was induced in Chinchilla male rabbits (body
weight at the start of the study = 3.53 ± 0.11 kg) with
daunorubicin (DAU, 3 mg/kg, i.v., once weekly, 10 weeks,
n = 15). The control group (n=11) received saline in the
same schedule. In the o-108 group (n=8) the chelator
was administered in 10% Cremophor EL in the dose of
10 mg/kg 30 min before each daunorubicin administration (once weekly, 10 weeks as in the DAU group). In the
SIH group (n = 9), the chelator SIH was administered i.v.
(in the vehicle composed of PEG 300/ethanol/ saline) in
the dose of 1.0 mg/kg 10 min before each DAU administration. The general toxicity parameters (mortality, body
weight etc.) were followed throughout the study. At the
end of experiment heart function was examined through
the left ventricular (LV) catheterization using polyethylen
catheter (under pentobarbital anesthesia, 30 mg/kg, i.v.).
The pressures were recorded and derived using appropriate pressure transducers using ADI Powerlab 8SP and the
Chart software v. 5.4.2. (ADInstruments, Australia) and
the index of the LV contractility (dP/dtmax) was obtained.
In the experiment 2, the cardioprotective properties of
DEX and novel clinically used iron chelating compound
were studied in the pilot study on the same rabbit model.
DEX (n = 6) was administered in the dose of 60 mg/kg
i.p. 30 min before each DAU administration, while deferiprone (kindly provided by ApoPharma Inc, in 0.5%
carboxymethylcellulose, n = 6) was administered orally
in the dose of 10 mg/kg 45 min before daunorubicin. The
same parameters as in experiment 1 were evaluated. The
LV contractility was examined using the same approach
as stated above, with exception of employment of MicroTip Pressure Catheter (2.3F Nylon, ADInstruments,
Australia). The results were compared with the new control (n = 8) and DAU groups (n = 11) evaluated using the
same equipment.
RESULTS: In the experiment 1, the premature mortality (33 %) was observed only in the DAU group. The
necropsy performed immediately after exitus revealed
massive hydrothorax and ascites as the signs of blood
congestion. All animals co-treated with o-108 as well as
SIH survived until the end of the experiment without apparent signs of toxicity. Unlike the control animals and
o-108 co-treated animals, the animals in the DAU and
SIH co-treated group tend to reduce their food and water
intake. Therefore the significant body weight gain (beginning vs. end of the study) was determined only in the
control (3.45 ± 0.11 vs 4.18 ± 0.12 kg, p < 0.05) and o108 co-treated (3.54 ± 0.06 vs 3.94 ± 0.08 kg) animals,
while the body weights in the DAU (3.34 ± 0.11 vs 3.66 ±
0.14 kg) and SIH co-treated (3.82 ± 0.12 vs 3.83 ± 0.14 kg)
groups remained unchanged. At the end of experiment,
the index of the LV contractility (dP/dtmax) was dramatically lower in the DAU group in comparison with the
results of the control group (Fig. 1A). Importantly, the
co-treatment with both aroylhydrazone chelators under
study was able to completely overcome not only overall
Fig. 1. Index of the left ventricular contractility – A) experiment 1, B) experiment 2. Statistical significance (ANOVA,
p<0.05): c (control), o (o-108), s (SIH), d (DEX). L1- deferiprone.
mortality induced by DAU, but also significantly preserve
the LV systolic function. These results were very promising and they further supported the involvement of iron
in the pathogenesis of the anthracycline cardiotoxicity.
Moreover, with exception of DEX, in this study it was for
the first time shown that iron chelation can be a successful
approach for the cardioprotection. However, in further
experiments it was shown that increasing the dose of both
chelators did not lead to the improvement of the cardioprotective effects. Instead the cardioprotective effects
virtually disappeared, though the chelators themselves
were shown non-toxic and very well tolerated. Although
we can not explain this uncommon dose-response relationship observed in this study, we can speculate on the
principle which is behind this phenomenon. It is plausible
that short biological half-life determined recently for both
of these aroylhydrazones2 could be one of their limitations. It can not be ruled out that the increasing of the
dose of aroylhydrazone iron chelators can result in high
peak concentrations of these strong chelators within the
cardiomyocytes which can “overshoot” desirable degree of
chelation and partially contribute to the changes in iron
homeostasis induced by anthracyclines.
In the experiment 2, it was clearly shown that DEX is
capable to completely overcome both DAU-induced mortality and LV dysfunction (Fig. 1B). During the autopsy of
these animals no macroscopic abnormalities were found.
This is in line with the results of clinical trials performed
so far1. Furthermore, this experiment also supported the
clinical relevance of our model of chronic anthracycline
cardiotoxicity. In second part of this pilot experiment
which was focused on the novel iron chelator deferiprone
we did not find any sign of cardioprotective efficacy of
this compound. Two animals in the group co-treated with
deferiprone died prematurely; the excessive pleural as well
as abdominal effusion was noted in addition to pulmo-
71
nary edema and hepatomegalia. Also the results of the
examination of the LV function shown dramatic drop of
contractility which was comparable with the DAU alone
and significantly lower than in both the control and DEX
group (Fig. 1B). In both groups co-treated with iron chelators the mild, however, insignificant body weight gains
(beginning vs. end) were observed (DEX: 3.52 ± 0.09
vs 3.96 ± 0.09 kg, deferiprone: 3.48 ± 0.04 vs 4.00 ±
0.16 kg).
CONCLUSIONS: In this study it is shown that tridentate cell-permeable aroylhydrazone iron chelators
(o-108, SIH) are able to overcome DAU-induced mortality and markedly diminished LV systolic dysfunction.
Furthermore, the model drug DEX, was shown to afford
complete protection regarding both overall mortality a left
ventricular function. In contrast, novel clinically used
bidentate iron chelator deferiprone was shown to have
no cardioprotective effects in this pilot study. However,
further study including the dose escalation is needed to
fully assess the possible cardioprotectantve potential of
deferiprone against anthracycline cardiotoxicity.
ACKNOWLEDGEMENT
The financial support by the grant of Charles University
Grant Agency No. 89/2006/C and the Research project
MSM0021620820 is gratefully acknowledged. Deferiprone
was kindly provided by Dr. John Connelly, ApoPharma Inc.,
Canada.
REFERENCES
1. Jones RL, Swanton C, Ewer MS. Anthracycline cardiotoxicity.
Expert Opin Drug Saf 2006; 5:791–809.
2. Kovarikova P, Klimes J, Sterba M, Popelova O, Gersl V, Ponka
P. HPLC determination of a novel aroylhydrazone iron chelator
(o-108) in rabbit plasma and its application to a pilot pharmacokinetic study. J Chromatogr B Analyt Technol Biomed Life Sci 2006;
838:107–12.
USE OF DESLORATADIN IN THE THERAPY OF ALLERGIC RHINITIS IN PEDIATRY
Juraj Prochaska, Tatiana Foltanova, Ingrid Tumova, Magdalena Kuzelova, Pavel Svec
Slovak Republic
Key words: Desloratadin/Allergic rhinitis/Pediatry/Eeffective reduction of symptoms
BACKGROUND: Allergic rhinitis is defined as an
abnormal inflammation of the membrane lining the
nose. It is characterized by nasal congestion, rhinorhea,
sneezing, itching of the nose and/or postnasal drainage1.
Although the onset of allergic rhinitis may occur at any
age, it is most common in children and at adolescence2.
Desloratadin is a no sedating antihistamine, with satisfied
evidence of improving the nasal airflow, total symptoms,
and total nasal symptoms. It is recommended also for the
therapy of AR in children, older than one year3.
METHODS: In the time period from september 2005
to september 2007, we perform an analysis of the use
of desloratadin in pediatric patients with the diagnose
allergic rhinitis (AR). The monitoring is performed in
pediatric ambulance in Levice. Dates of visits are not
established in advance. Patients come because of their
symptoms, acute infection or to prescribe medication. In
this work we analyze first three visits, which include one
pollen season (september 2005 – december 2006). In total 71 patients were included. Main inclusion criterion
72
Table 1. Characteristics of the studied population.
Nb of patients
71
girls/boys (%)
33.8/66.2 (24/47)
year of birth – modus
2000 (1987–2006)
Visit 1 most frequent month of visit
september (9–3)
march (1–12)
jun (1–12)
New diagnosed patients (%)
23.9 (17 pts)
AR (intermittent/persistent) (%)
60.6/23.9 (43/17 pts)
Alergic complications (%)
15.5 (11 pts)
Obstructive bronchitis (%)
29.58 (21 pts)
Allergic cought syndrom (%)
22.54 (16 pts)
Bronchial asthma (%)
08.45 (6 pts)
Average symptom score visit. 1
7.69 SD4.36 (0–19)
3.62 SD 2.71 (0–11)
1.77 SD 2.04 (0–7)
Skin test (%)
63.38 (45 pts) (1–6)
Decrease of the global symptom score
value of the global symptom score
14
12
10
8
6
4
2
0
-2
1
2
3
visit
Fig. 1. Decrease of the global symptom score.
was diagnosis of AR. The doctor files the information into
the in advance prepared questionnaire. The questionnaire
is oriented in character and intensity of the symptoms.
Results of skin testing were noted. New variable “skin
test” was computed. The value of skin test was the sum
of all positive results of skin testing (range 0–6). Patient
with skin test zero, was the one, in whom the skin test
wasn’t performed. The effect of desloratadin was evaluated as decrease of the intensity of frequent symptoms
as well as decrease of the global symptom score of AR.
Global symptom score was computed as sum of the in-
tensity of all symptoms (range 0–45). Statistical analyses
were performed using SPSS 10.0 for Windows statistical
program. The data were analyzed using nonparametric
tests – Wilcoxon test, cross tabulations 2 × 2 and chisquare tests. The significance of the results is presented
as P-values.
RESULTS: Two times more boys than girls were included. Skin testing was performed in 63.38 % children
(45 patients). The most frequent allergen was pollen
(59.15 %, 42 children). Majority of children was allergic
to more than one allergen. The most patients (18 chil-
dren) were allergic to four different allergens (range 1–5)
(Table 1). The higher was the value of skin test, the more
intensive were the symptoms (p < 0.001), on third visit
this result was not significant. Although frequency of visits
and intensity of symptoms increased during the pollen
season, the differences were not significant. The most frequent symptoms were rhinorrhea (83.10 %, 59 children),
cought (80.28 %, 57 children) and smell disturbances
(64.79 % 46 children). Desloratadin significantly (p <
0.001) reduced the intensity of all frequent symptoms,
except increased temperature. The global score of the
symptoms of AR was significantly reduced only on the
second visit (p < 0.001) (Fig. 1). No adverse events were
observed.
73
CONCLUSION: Desloratadin approved its position
as effective and safe antihistaminic drug in pediatric
praxis.
ACKNOWLEDGEMENT
Financial support by the grants FaF UK/33/2007 is
REFERENCES
1. Bosquet J. Allergic rhinitis and its impact on asthma (ARIA). Clin
Exp All Rev 2003; 3:43–5.
2. Bachert C. Decongestant efficacy of desloratadine in patients with
seasonal allergic rhinitis. Allergy 2001; 56 (suppl 65):14–20.
3. Hakansson K, Thomsen SF, Ulrik CS, Porsbjerg C, Backer V.
Increase in prevalence of rhinitis among Danish children from
1986 to 2001. Pediatr Allergy Immunol 2007; 18:154–9.
EXPRESSION AND PRODUCTION OF SPINAL CYCLOOXYGENASE 1 AND 2
IN THE MODEL OF EXPERIMENTAL OSTEOARTHRITIS
Michaela Prochazkovaa, Peter Zanvitb, Tomas Dolezala, Ludmila Prokesovab, Miloslav Krsiaka
a
Department of Pharmacology, 3rd Faculty of Medicine, Charles University, Prague, Czech Republic
Institute of Immunology and Microbiology, 1st Faculty of Medicine, Charles University, Prague, Czech Republic
b
Key words: Osteoarthritis/Cyclooxygenase/Spinal cord/Pain
BACKGROUND: Osteoarthritis (OA) is a degenerative
joint disease. The joints are characterized by a progressive
degeneration of articular cartilage leading to inflammation and pain. In an attempt to study the pathophysiology
of OA, numerous experimental models that mimic human OA have been developed. Chemical models of OA
involve intraarticular injection of chemical compounds
that can have a number of effects on joint physiology.
Surgical models of OA induce joint instability by partial
menisectomy. One of the most frequently used model is
the model of monoiodoacetate (MIA) induced OA. Local
injection of monoiodoacetate, an inhibitor of glycolysis,
disrupts chondrocyte metabolism and produces cartilage
degeneration. In mice, injection of monoiodoacetate led
to strong inhibition of proteoglycan synthesis in the patella and in the medial part of the tibial plateau1. The
loss of locomotor activity and the severity of cartilage
lesions in patella were observed in rats injected with MIA
(ref.2). It was previously described, the important role
of synovial cyclooxygenase-1 in patient with primary osteoarthritis3. The role of spinal cyclooxygenase (COX)
isoenzymes in the osteoarthritis is not known. The aim of
the present study was to ascertain the role of spinal COX1 and COX-2 in the model of monoiodoacetate induced
osteoarthritis.
METHODS: Adult male Wistar albino rats (weight
200–220 g) obtained from VÚFB Konárovice (Czech
Republic) were used in all experiments. The animals
were housed under standard laboratory conditions (in
a temperature-controlled (21±1°C) room with a normal
12-h light/dark cycle). Osteoarthritis was induced by
a single injection of monoiodoacetate (2 mg) into the
right knee joint of anaesthetised rats in a total volume of
25 μl. Control animals were injected 25 μL of vehicle
into the right knee joint under the same conditions.
Thermal hyperalgesia was assessed by the plantar test
(Ugo Basile) following method of Hargreaves4 before
and in defined times during 31 days after injection of
monoiodoacetate. A mobile radiant heat source was located under the plantar surface of the hind paw and paw
withdrawal latencies were recorded. Both hind paws were
tested independently for three times per side with a 10
minutes waiting period between trials. Tactile allodynia
was measured with von Frey hairs (Ugo Basile). Animals
were placed into wire mesh bottom cages and tactile allodynia was tested by touching the plantar surface of the
animal‘s hind paw with von Frey hairs in ascending order of force until a paw-withdrawal response was elicited.
Each von Frey hair was applied to the paw for 5 seconds
or until a response occurred. Once a withdrawal response
was established, the paw was retested. The lowest amount
of force required to elicit a response was recorded as
withdrawal threshold in grams. RNA isolation was performed after disruption and homogenization of samples
from the lumbar section of spinal cord stabilized with
the RNAlater (Qiagen) using Ultra-Turrax (Ika). Total
RNA was isolated with the RNeasy lipid tissue isolation
kit (Qiagen) according to the manufacturer’s instruction.
Real-time PCR was run on an ABI PRISM 7300 SDS
analytical cycler (Applied Biosystems).The amounts of
relative amount of mRNA
74
***
6
COX-1
COX-2
***
5
4
3
***
***
**
* *
2
control
0
1
5
14
31
days after induction of OA
Fig. 1. Relative expression of COX-1 and COX-2
mRNA in lumbar section of spinal cord 1, 5,
14 and 31 day after induction of osteoarthritis
as determined by real-time PCR. Each column
represents measurements from 8 rats, data are
displayed as means±S.E.M. Asterisks indicate
significant difference between MIA injected
and control animals at respective time-points
(* P < 0,05; ** P < 0,01 and *** P < 0,001).
COX-1 and COX-2 protein were measured on Multiscan
RC reader by 450 nm.
RESULTS. Intraarticular injection of monoiodoacetate into the right knee joint induced marked hyperalgesia measured by von Frey thresholds and paw
withdrawal latencies. Decreased paw withdrawal latencies and paw withdrawal thresholds were evident from
the first day following injection of monoiodoacetate, with
the maximum on the 5th day (statistically significant at
all observed times compared to baseline and to control
animals). Expression of COX isoenzymes was measured
in 4 intervals. First day after MIA injection spinal levels
of COX-1 mRNA and COX-2 mRNA were moderately
increased (2.3 and 2.6 times, respectively; P < 0.05). The
expression of spinal COX-2 mRNA was much higher on
the day 5 (5.2 times; P < 0.001) and remained increased
at this level until the day 31 (4.9 times; P < 0.001). On
the other hand, expression of spinal COX-1 mRNA increased gradually during the whole testing period reaching maximum on the day 31 (4.5 times; P < 0.001) when
the relative expression of both genes was almost equal
(Fig.1). The production of spinal COX proteins was parallel to the expression results. The production of COX-1 and
COX-2 protein was almost similar at the beginning of the
experiment. The highest production of COX-2 protein was
observed on day 5 after induction of osteoarthritis (increased 3.9 times). The levels of COX-1 protein increased
gradually with maximum on day 31 (3.4 times). All results
are expressed in comparison with control animals.
CONCLUSIONS: The present findings indicate that
not only expression and production of COX-2 but also
that of COX-1 is significantly increased in the spinal cord
in osteoarthritis pain. In contrast to other pain models
– e.g. inflammatory pain, the increased expression and
production of spinal COX-1 might play an important role
in osteoarthritis pain.
ACKNOWLEDGEMENT
Supported by the grants VZ MSM0021620816 and IGA
NR/9072-3.
REFERENCES
1. van Osch GJ, van der Kraan PM, van den Berg WB. Site-specific
cartilage changes in murine degenerative knee joint disease induced
by iodoacetate and collagenase. J Orthop Res 1994; 12:168–75.
2. Guingamp C, Gergout-Pottie P, Philippe L, Terlain B, Netter P,
Gillet P. Mono-iodoacetate-induced experimental osteoarthritis:
a dose-response study of loss of mobility, morphology, and biochemistry. Arthritis Rheum 1997; 40:1670–7.
3. Knorth H, Dorfmüller P, Lebert R, Schmidt WE, Wittenberg RH,
Heukamp M, Wiese M, Willburger R. Participation of cyclooxygenase-1 in prostaglandin E2 release from synovitis tissue in primary
osteoarthritis in vitro. Osteoarthritis and Cartilage 2004; 12:658–
66.
4. Hargreaves K, Dubner R, Brown F, Flores C, Joris J. A new and
sensitive method for measuring thermal nociception in cutaneous
hyperalgesia. Pain 1988; 32:77–88.
EFFECTS OF A NEWLY SYNTHESIZED FLUOROPHENYLPIPERAZINE
DERIVATIVE ON BASIC CARDIOVASCULAR FUNCTIONS
Eva Racanskaa, Matej Balaza, Ivan Malikb
a
Department of Pharmacology and Toxicology, Faculty of Pharmacy, Bratislava
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Bratislava
b
Key words: Substituted fluorophenylcarbamate/Isolated aorta/Isolated atria/Antihypertensive activity
BACKGROUND: Series of substituted N-phenylpiperazine derivatives of phenylcarbamates as potential ß-blockers with combined vasodilator activity were prepared1.
Incorporation of the carbamoyl group (-NHCOO-) into
connecting chain of the original aryloxyaminopropanol
moiety is the principal intervention into basic skeleton of
the compounds. On the basis of our previous studies mainly 4’-fluorophenylpiperazine substitution positively influenced majority of the evaluated cardiovascular functions2,3.
The present studies were performed to investigate in in
vitro experiments antiisoprenaline (potential ß-adrenolytic)
and vasodilator activities as well as in vivo antihypertensive
OH
H
N
H5C2O
O
N
N
F
O
Fig. 1. Chemical structure of the evaluated compound
IIIb.
efficiency of one choosen representative compound from
numerous series of the original structures.
METHODS: To the strip of isolated rat thoratic aorta
in Krebs-Henseleit solution noradrenaline (NA) at conc.
10–9–10–5 mol.L–1 was cumulatively added. After washing
and re-equilibration the evaluated compound was given
30 min prior subsequent NA application. The effect of
drug was evaluated according its ability to inhibit maximal NA-induced contraction of aorta. The ability of the
compound to antagonise positive chronotropic effect of
isoprenaline (IPN) after its cumulative application was
measured isometricaly on right atria of guinea pigs. The
evaluated compound was given to the bath with atria
20 min before application of isoprenaline. The affinity
for IPN without and with the compound was expressed
as EC50 values and the antiisoprenaline potency of the
compound was calculated from the shift in CRC of IPN
and expressed as dissociation constant (pA2 value) according to the modified method of Van Rossum4. Male
normotensive Wistar rats and spontaneously hypertensive rats (SHR) were used to evaluate effect on blood
presure. Systolic and diastolic blood pressure (BP) were
measured by the indirect tail cuff method. The measurements were performed just before and 30 min, 1, 3, 5 and
24 h after single i.p. administration of the compound at
dose 10 mg.kg–1. The magnitudes of the effects elicited
after injections were expressed relatively and compared
to saline group values.
RESULTS: The evaluated compound IIIb at conc.
10–5 mol.L–1 significantly depressed (by 44.9%) the maximal response of NA-induced contractions of the aorta.
The calculated pA2 = 7.88 ± 0.19 confirmed that the studied compound had ability to specificly and competitively
75
inhibit effect of isoprenaline on the heart chronotropy
similary as other potent β-blockers of aryloxyaminopropanol type. In both evaluated rat groups the compound
decreased BP. In normotensive rats onset of effect on
systolic and diastolic BP developed during followed 30
min and its maximum reached in 3rd h after the compound
administration (–10.5 and/or –9.2 %, respectively). In the
SHR group the antihypertensive effect had maximally developed in 5th h after the compound application (–12.3
and/or –13.8 %, respectively). However, in all cases the
values of blood pressure have returned back to the initial
values till 24 h after the compound application.
CONCLUSIONS: It seems that incorporation of
the carbamoyl group between phenyl ring and aminopropanol chain did not influence significantly potential
β-adrenolytic activity in comparison with the original
aryloxyaminopropanols. Moreover, additive property to
inhibit contractibility of aortal strips in vitro and decrease
blood pressure in normotensive as well as hypertensive
rats enable to consider the evaluated compound as proper
representative of potential antihypertensives. However,
the mechanisms of its vasodilator effect need other experimental approaches.
ACKNOWLEDGEMENT
Financial support by the Grant Agency VEGA (Bratislava)
grant No. 1/3415/06 is gratefully acknowledged.
REFERENCES
1. Malik I., Sedlarova E., Csollei J., Racanska E., Cizmarik J., Kurfurst
P. Synthesis, physico-chemical properties and biological activity of
1-(fluorophenyl)-4-[3-(2-,3- and 4-alkoxyphenylcarbamoyloxy)-2-hydroxypropyl]piperaziniumchlorides. Sci Pharm 2004; 72:283–91.
2. Mlynarová R., Celkova H., Racanska E. Preparation and pharmacological characteristic of aryloxyaminopropanol derivatives with
assumed cardiovascular effects. (in Slovak) Ces slov Farm 1996;
45:260–4.
3. Racanska E., Tumova I., Foltanova T. 2-, and 4-fluorophenylpiperazine derivatives of alkoxyphenylcarbamic acid and their potential
antidysrhythmic effects. (in Slovak) Farm Obzor 2005; 74:247–
50.
4. Van Rossum JM. Cumulative dose-response curves II. Technique
for the making of dose response curves in isolated organs and the
evaluation of drug parameters. Arch Int Pharmacol Ther 1963; 143:
299–330.
THE MODEL OF PREMENOPAUSAL BREAST CANCER: EFFECTS OF LETROZOLE
Vladimira Sadlonovaa, Peter Kubatkaa, Karol Kajob, Gabriela Nosalovaa
a
Department of Pathological Anatomy, Jessenius Faculty of Medicine, Comenius University, Martin
b
Key words: Mammary carcinogenesis/Chemoprevention/Rat/Aromatase inhibitors/Letrozole
BACKGROUND: Estrogens and their metabolites
play an important role in both the initiation and the progression of breast cancer. In postmenopausal women,
aromatase inhibitors block biotransformation of adrenal
androgens to estrogens in peripheral tissues, resulting in
undetectable levels of plasma estrogens. Recently potent
and specific third-generation aromatase inhibitors have
been introduced: two non – steroidal anastrozole and
letrozole, and the steroidal exemestane. Based on results
of clinical trials1–3, letrozole, anastrozole and exemestane
were approved for the treatment of advanced breast cancer in postmenopausal women. Recent adjuvant trials
76
Table 1. Chemopreventive effects of letrozole in NMU-induced mammary carcinogenesis in female Sprague-Dawley
rats at the end of experiment.
Group
tumour incidence (%)
tumour frequency per group*
tumour latency* (days)
cumulative tumour volume** (cm3)
CONT
LETRO 1
LETRO 10
75.0
5.2 a
0b
1.60 ± 0.29
0.05 ± 0.05 a
0b
98.00 ± 2.82
112.00
-
14.14
4.15
-
*Data are expressed as means ± SEM, **data are expressed as a sum of volumes.
Significantly different, a P < 0.00002 vs CONT, b P < 0.000005 vs CONT
have established the value of above mentioned drugs4–6
in early-stage breast cancer. Single-agent therapy with
aromatase inhibitors has no established role in premenopausal women with breast cancer. Several experimental
methods have been used to determine the biological importance of breast in situ estrogen production versus uptake of estradiol from plasma by breast tissue7, 8. Results
of above cited experiments suggested the importance of
in situ estrogen production in the breast and led to the
hypothesis that an important determinant of tissue estradiol levels is local production in the mammary gland.
The efficacy and toxicity of aromatase inhibitors in the
treatment of premenopausal breast cancer patients are
discussed among oncologists9–11. Our model of mammary
carcinogenesis in intact female rats mimics situation in
high-risk premenopausal women. The aim of this experiment was the evaluation of antineoplastic activity of letrozole in the chemoprevention of experimental mammary
carcinogenesis.
METHODS: Female rats of Sprague-Dawley strain
aged 33–37 days were used in the experiment. Letrozole
(Femara) was administered in the chow in two concentrations – 1 mg/kg (0.0001 %), and 10 mg/kg (0.001 %).
Mammary carcinogenesis was induced by repeated administration of N-methyl-N-nitrosourea on postnatal days
44 and 51. Chemoprevention with letrozole began a week
before carcinogen administration and lasted until the end
of the experiment – 17 weeks after carcinogen application.
Animals were randomly assigned to one of three experimental groups: (1) control group without chemoprevention; (2) chemoprevention with letrozole administered in
chow in concentration of 1 mg/kg; (3) chemoprevention
with letrozole administered in chow in concentration of 10
mg/kg. Each group consisted of 20 animals. The animals
were weekly weighed and palpated in order to register
the presence, number, location and size of each palpable
tumour. In the end of the experiment, the animals were
quickly decapitated. The mammary tumours, the uterus
as well as the vagina of each animal were excised and
the tumour size was recorded. The effect of letrozole on
uterine and vaginal weights was observed. Specimens of
mammary tumours, uteri and vaginas were fixed in 10%
buffered formalin and later prepared for histological examination. Blood was collected, at sacrifice, from each an-
imal. In the serum, the concentrations of triacylglycerols,
total cholesterol, and cholesterol values of the low-density
lipoprotein (LDL), and high-density lipoprotein (HDL)
fractions were measured. The tumours were classified according to the criteria for the classification of rat mammary tumours12. The following parameters of mammary
carcinogenesis were evaluated in each group: tumour incidence, tumour frequency, and cumulative tumour volume
and latency period determined by the appearance of first
tumour in an animal. The individual body weight gain and
food intake of animals were recorded. Tumour incidence
was evaluated by Mann-Whitney U-test, other parameters
by one-way analysis of variance (ANOVA) or KruskalWallis test. All procedures were carried out according to
EU directives and reviewed by Ethics Committee of the
Comenius University.
RESULTS: This study revealed high tumour suppressive effects of letrozole in experimental mammary carcinogenesis (Table 1). In the groups with letrozole, uterine and
vaginal atrophy and decrease in the weight of both organs
(P<0.0001) were found at the end of the experiment. In
letrozole – treated animals in comparison with control
animals, increased plasmatic triacylglycerol concentrations (P < 0.0001) were observed. Total cholesterol and
cholesterol of low-density and high-density lipoprotein
fractions were not significantly changed. A statistically
significant increase in body weight gain and food intake
was found in both letrozole groups in comparison with
untreated rats (P < 0.0001).
CONCLUSIONS: This study is the first report on
tumour suppressive effects of letrozole in the model of
premenopausal mammary carcinogenesis in female rats.
Oncostatic effects of letrozole were recorded in all evaluated parameters of mammary carcinogenesis. This experiment provided a rationale for clinical trials of letrozole
in premenopausal women with breast cancer. The role of
aromatase inhibitors in premenopausal breast cancer women is area of next exploration. Administration of these
drugs in premenopausal women will be limited by their
adverse effects and risk/benefit ratio will be considered.
Further studies with longer follow – up are required to
clarify the effects of aromatase inhibitors on lipid metabolism and cardiovascular health. Also the monitoring
and management of bone loss associated with their ap-
plication are essential and are being addressed in clinical
trials. The results from ongoing prevention trials with
aromatase inhibitors will determine their suitability for
use as chemopreventives in healthy women with risk of
breast cancer developing.
ACKNOWLEDGEMENT
Financial support by the grant UK/66/2007 and
UK/67/2007.
REFERENCES
1. Thürlimann B, Hess D, Koberle D, et al. Anastrozole (‘Arimidex’)
versus tamoxifen as first-line therapy in postmenopausal women
with advanced breast cancer: Results of the double-blind crossover SAKK trial 21/95 – a sub-study of the TARGET (Tamoxifen
or ‘Arimidex’ Randomized Group Efficacy and Tolerability) trial.
Breast Cancer Res Treat 2004; 85:247–54.
2. Mouridsen H, Gershanovich M, Sun Y, et al. Phase III study of
letrozole versus tamoxifen as first-line therapy of advanced breast
cancer in postmenopausal women: analysis of survival and updates
of efficacy from the International Letrozole Breast Cancer Group.
J Clin Oncol 2003; 21:2101–119.
3. Paridaens R, Therasse P, Dirix L, et al. First-line hormonal treatment (HT) for metastatic breast cancer with exemestane or
tamoxifen in postmenopausal patients – A randomized phase III
trial of the EORTC Breast Group. Proc Am Soc Clin Oncol 2004;
22:A 515.
77
4. Thürlimann BJ, Keshaviah A, Coates AS, et al. A comparison of
letrozole and tamoxifen in postmenopausal women with early breast cancer, N. Engl. J Med 2005; 353:2747–57.
5. Baum M, Buzdar A, Cuzick J, et al. Anastrozole alone or in combination with tamoxifen versus tamoxifen alone for adjuvant treatment
of postmenopausal women with early-stage breast cancer: results of
the ATAC (Arimidex, Tamoxifen Alone or in Combination) trial
efficacy and safety update analysis. Cancer 2003; 98:1802–10.
6. Coombes RC, Hall E, Gibson LJ, et al. A randomized trial of exemestane after two to three years of tamoxifen therapy in postmenopausal women with primary breast cancer. N Engl J Med
2004; 350:1081–92.
7. Santen RJ, Martel J, Hoagland F, et al. Stromal spindle cells
contain aromatase in human breast tumors, J Clin Endocrinol
Metab 1994; 79:627–32.
8. Bernstein LM, Larionov AA, Kyshtoobaeva AS, Pozharinsski KM,
Semiglazov VF, Ivanova OA. Aromatase in breast cancer tissue
localization and relationship with reproductive status of patients.
J Cancer Res Clin Oncol 1996; 122:495–8.
9. Santen RJ, Yue W, Naftolin F, Mor G, Berstein L. The potential
of aromatase inhibitors in breast cancer prevention. Endocr Rel
Cancer 1999; 6:235–43.
10. Goss PE, Strasser K. Aromatase inhibitors in the treatment and
prevention of breast cancer. J Clin Oncol 2001; 19:2767.
11. Freedman OC, Verma S, Clemons MJ. Pre-menopusal breast cancer
and aromatase inhibitors: treating a new generation of women.
Breast Cancer Res Treat 2006; 99:241–7.
12. Russo J, Russo IH, Rogers AE, van Zwieten MJ, Gusterson B.
Pathology of tumours in laboratory animals. Tumours of the rat.
Tumours of the mammary gland. IARC Sci Publ 1990; 99:47–78.
NEUROPROTECTION IN TWO DIFFERENT TISSUE CULTURE MODELS OF BRAIN
ISCHEMIA: INFLUENCE OF CEREBROLYSIN
Evelyn Schauera, Robert Wronskia, Jitka Patockovab, Herbert Moesslerc, Edith Dopplerc,
Birgit Hutter-Paiera, Manfred Windischa
a
JSW – Research, Institute of Experimental Pharmacology, Graz, Austria
Department of Pharmacology, Charles University, 3rd Faculty of Medicine, Prague, Czech Republic
c
EBEWE Pharma, Unterach, Austria
b
Key words: Ischemia/Neuron/Apoptosis/Necrosis/Cerebrolysin
BACKGROUND: All attempts to reduce neuronal
damage after acute brain ischemia by the use of neuroprotective compounds have failed to prove efficacy in clinical trials so far. In spite of the promising pre-clinical data
none of the compounds could show significant therapeutic
effects1,2. In this study two different tissue culture models
of ischemia, excitotoxic lesion by the use of glutamate and
oxygen-glucose deprivation, were used to investigate the effect of delayed application of Cerebrolysin (Cere) on neuronal survival. Cere is a drug consisting of low molecular
weight peptides and amino acids. Different publications
indicate that this drug has growth factor like activities
and is promoting neuronal survival and sprouting similar to the naturally occurring nerve growth factor3,4. The
published data are derived both from pre-treatment5,6 and
post-treatment7 application of Cere.
METHODS: Primary neuronal cultures from 8 days
old Lohman brown chicken embryo telencephalons
were prepared as described previously8, the cells were
maintained in Dulbecco’s minimum essential medium
(DMEM) supplemented with Nu serum, glutamine and
gentamycine. Glutamate lesion was performed on the
third day by adding a glutamate solution to each well.
The toxic substance remained with the cells for 24 hours.
Cere was added to the samples on the day five in vitro
(24 hours time window) or on the day eight (96 hours
time window). Measurement of viability was measured on
the day 7, 9, 11, 14 (24 hours time window) or on the day
9, 11, 14, 17 (96 hours time window). Oxygen glucose deprivation (OGD) was done by the replacement of DMEM
on the third day by glucose-free DMEM. The plates were
placed in OGD-chamber. The entire lesion period was
24 hours. After the lesion the media were replaced by
medium with glucose (1 g.L–1), 2% fetal calf serum, 0.01 %
gentamycin-sulfate and 2mM L-glutamine. The plates were
kept until the end of experiment in an incubator at 37 °C,
78
a1
24 h drug addition
MTT 6d after lesion
b1
96 h drug addition
MTT 6d after lesion
0,10
0,30
0,08
0,25
0,20
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0,10
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2.5μl
5μl
10μl
20μl
40μl
80μl
160μl
0μl
1.25μl
2.5μl
b2
24 h drug addition
MTT 8d after lesion
a2
0,00
5μl
10μl
20μl
40μl
80μl
160μl
40μl
80μl
160μl
40μl
80μl
160μl
96 h drug addition
MTT 8d after lesion
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0,30
0,08
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a3
2.5μl
5μl
10μl
20μl
40μl
80μl
160μl
0,00
0μl
b3
24 h drug addition
MTT 11d after lesion
2.5μl
5μl
10μl
20μl
96 h drug addition
0,10
0,30
1.25μl
0,08
0,25
0,06
0,20
0,15
0,04
0,10
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5μl
10μl
20μl
40μl
80μl
160μl
0,00
0μl
1.25μl
2.5μl
5μl
10μl
20μl
Fig. 1. Neuroprotective effects of Cere application to chick embryonic cortical neurons in culture 24h (a) and 96 h
(b) after 24 hours exposure to L-glutamate. (1) Measurement of viability on day 6 after lesion, (2) day 8 after
lesion (3) day 11 after lesion. Values show mean +/- SEM. Number of experiments n=6. Grey, hatched, black
and angular chequered columns represent samples significantly different from controls.
95% humidity and 5% CO2. Firstly OGD was performed
on the third day in vitro. MTT assay was performed 24
hours after Cere addition. Cere was added on the days
3,4,5,6 in vitro. Secondly Cere was added directly after
the lesion (day 4 in vitro). The evaluations were done on
the days 8, 10, 12, 15 in vitro. Statistica 99’, Statsoft Inc.,
USA was used for data processing.
RESULTS: Glutamate exposure for 24 hours kills
approximately 60 % of the neurons within 4 days. The
application of Cere with a delay of 24 hours after the
lesion showed a significant, dose dependent increase in
the number of viable neurons (p < 0.05) on the 4th day
(Data not shown), almost displaying double the number
of viable cells in case of 80 μL of Cere per mL of tissue culture medium (p < 0.001). 6 days after the lesion
there is a further significant reduction in living neurons
(p < 0.001) in the control wells (Fig 1. a1), but also at that
time point Cere still exhibits a significant neuroprotection
(p < 0.05) with a dosage of 10 μL per mL, showing again
a peak effect at 80 μL per mL (p < 0.0001), practically
preventing further cell death with this dosage. This significant protective effect can be demonstrated up to day 11
after lesion (Fig 1. a3), but the range of effective dosages
moves to the higher concentrations of 40, 80 and 160 μL
per mL displaying an almost 3 fold compared to controls
with a delay of Cere for 96 hours after the lesion (Fig 1.
b1). The first assessment of cell viability was performed
6 days after the lesion, 2 days after the treatment with
the drug. 10 μL per mL of tissue culture medium produced a significant (p < 0.05) increase in viability, and the
protected effect was again dose dependent with highest
rescue in 80 and 160 μL per ml (Fig.1. a3). Evaluation
A1
B1
Cere addition in parallel to the lesion (0h)
0,10
0,20
0,08
0,15
0,05
0,10
0,03
0,05
0,00
79
Cere addition after 24h
MTT 6d after lesion
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0μl
1.25μl
2.5μl
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10μl
20μl
40μl
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2.5μl
A2
5μl
10μl
20μl
40μl
80μl
160μl
40μl
80μl
160μl
40μl
80μl
160μl
Cerebrolysin/ml medium
Cer ebr olysin/ ml medium
MTT 8d after lesion
B2
0,10
0,20
0,08
0,15
0,05
0,10
0,03
0,05
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B3
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Cerebrolysin/ ml medium
0μl
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2.5μl
5μl
10μl
20μl
Fig. 2. Dose and time dependent effects of Cerebrolysin in cultures of cortical neurons after oxygen glucose deprivation. (A ) Cerebrolysin was applied together with onset of the lesion (A1), or with a delay of 24 hours (A2),
or 48 hours (A3) after lesion respectively. Viability of cortical neurons was assessed with the MTT-assay 24
hours after addition of the drug. (B) The drug was added to the cultures immediately after end of the 24 hours
lesion period and the assessment of viability was performed using MTT assay 6 days (B1), 8 days (B2) or 11
days (B3) after lesion respectively. Values shown mean +/- SEM. Number of experiments n=6. Grey, hatched,
black and angular chequered columns represent samples significantly different from controls.
of the living neurons 6 days after lesion shows that there
is still a significant continuing cell death (p < 0.01), and
the Cere effects are completely maintained as shown at
the earlier assessment time point (Fig. 1. b1). Further
evaluations of cell viability were performed 8 and 11 days
after lesion, showing that there is still a small, significant
decline in the number of living neurons, but at both time
points Cere shows a clear (p < 0.05 to p <0.001) neuroprotection (Fig. 1. b2 and 1.b3). In both of the highest
dosages (80 μl and 160 μl) the cell counts do almost not
change over the whole experimental period. It seems that
the effective dosages are moving over time to higher Cere
concentrations, indicating that most likely active factors
are consumed.
In a 2nd series of experiments the oxygen glucose
deprivation model was used to simulate brain ischemia,
significantly reducing the number of viable neurons under
control conditions. There is no further ongoing cell death
between the assessment 24 hours after end of the lesion
and 48 hours time point (Fig. 2. A1 to A3). Treatment
immediately at the time point of re-oxygenation resulted
in a significantly increased (p < 0.05) number of neurons
already at the Cere dosage of 20 μL per mL, reaching
a maximum at 40 μL per mL(p < 0.001) (Fig. 2. A1).
80
A 24 h delay in treatment showed even a higher degree
of neuroprotection, (Fig. 2. A2). Further delay of drug
application for 48 (Fig. 2. A3) and 72 h (Data not shown)
resulted in lower efficacy and significant rescue could
be achieved only with higher dosage (p < 0.05). To get
more information about the time course of Cere activity after lesion again OGD was performed and all wells
were treated with the drug immediately after end of the
24 hours hypoxic period (Fig 2. B1-B3), and the assessment of viability was performed 4 (Data not shown), 6, 8
and 11 days after the lesion, again displaying a similar correlation between time point of assessment and neuronal
viability as in the glutamate experiments. At 4 days post
lesion a significant protection was shown at 5 and 10 μL
per mL but higher dosages showed inconclusive data and
160 μL even significantly decreased the number of living
neurons (p < 0.001). 6 days after the lesion the significant
neuroprotection by Cere was still maintained between 5
and 40 μL per mL (p < 0.05 to p < 0001, Fig. 2. B1). Even
8 and 11 days after lesion a significant neuroprotection
by Cere in the dose range between 5 and 160 μl per ml
(p < 0.05 to p < 0.001, Fig. 2. B2) and of 40 to 160 μl per
ml (p < 0.05 to p < 001, Fig. 2. B3) respectively. In summary all the data indicate a clear neuroprotective potency
of Cere even if treatment is initiated with a delay of up
to 96 hours after onset of the lesion. The long lasting
progressive cell death after ischemic events advocates for
an early initiation of neuroprotective treatment to rescue
a maximum of cells.
CONLUSIONS: All the data demonstrate that Cere
displays a neuroprotective potency even if treatment is
initiated with a delay up to 96 hours after the onset of the
lesion. Nevertheless the ongoing cell death after ischemic
event advocates for an early initiation of neuroprotective
treatment to rescue a maximum of cells. The data are indicating that Cere is active during the apoptotic phase of
delayed neuronal cell death. It also seems that some of the
active compounds are consumed over time, what would
explain the shift of efficacious dosage from low to high.
ACKNOWLEDGEMENT
Financial support by EBEWE Pharma and the grant
MSM0021620816 is gratefully acknowledged.
REFERENCES
1. Calabresi P, Centonze D, Cupini LM, Costa C, Pisani F, Bernardi
G. Ionotropic glutamate receptors: still a target for neuroprotection
in brain ischemia? Insights from in vitro studies. Neurobiol Dis
2003;12:82–8.
2. Liebeskind DS, Kasner SE. Neuroprotection for ischaemic stroke
– an unattainable goal? CNS Drugs 2001; 15:165–74.
3. Satou T, Itoh T, Tamai Y, Ohde H, Anderson AJ, Hashimoto S.
Neurotrophic effects of FPF-1070 (Cerebrolysin) on cultured neurons from chicken embryo dorsal root ganglia, ciliary ganglia, and
sympathetic trunks. J Neural Transm 2000;107:1253–62
4. Windisch M, Albrecht E, Eggenreich U, Paier B. Neurotrophic effects of the nootropic drug Cerebrolysin – a summary. Adv Biosci
1993; 87:355–56.
5. Gutmann B, Hutter-Paier B, Skofitsch G, Windisch M, Gmeinbauer
R. In vitro models of brain ischemia: the peptidergic drug cerebrolysin protects cultured chick cortical neurons from cell death.
Neurotox Res 2002; 4:59–65.
6. Hutter-Paier B, Grygar E, Windisch M. Death of cultured telencephalon neurons induced by glutamate is reduced by the peptide
dervative Cerebrolysin. J Neural Transm 1996; 47:267–73.
7. Patockova J, Krsiak M, Marhol P, Tumova E. Cerebrolysin Inhibits
Lipid Peroxidation Induced by Insulin Hypoglycemia in the Brain
and Heart of Mice. Physiol Res 2003; 52:455–60.
8. Pettmann B, Louis J, Sensenbrenner M. Morphological and biochemical maturation of neurones cultured in the absence of glial
cells. Nature 1979; 281:378–80.
THE IMPACT OF RECOMBINANT LACTOBACILLUS PLANTARUM ON POLLEN
ALLERGY DEVELOPMENT
Martin Schwarzera, Andreas Repab,c, Ursula Wiedermannb, Tomas Hrncira,
Catherine Danield, Bruno Potd, Renata Stepankovaa, Tomas Hudcovica,
Helena Tlaskalova-Hogenovaa, Martina Souckovaa, Hana Kozakovaa
a
Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic
Department of Specific Prophylaxis and Tropical Medicine, Medical University of Vienna, Austria
c
Department of Neonatology, University Children’s Hospital Vienna, Austria
d
Institute Pasteur, Lille, France
b
Key words: Pollen allergy/Germ-free mice/Recombinant Lactobacillus plantarum
BACKROUND: Allergy is caused by an immune reaction that is out of all proportion to the antigenic stimuli.
The birch pollen allergy belongs to the type I allergy,
which is characterized by an imbalance of T helper (Th)1- and Th-2-like immune responses with exaggerated production of interleukin (IL)-4, IL-5 and IL-13 leading to
production of immunoglobulin (Ig)-E towards otherwise
innocuous birch pollen. In the last few years the role of
specific lactic acid bacteria strains in the prevention of
allergic diseases has become more evident, in particular
the relation between the composition of the intestinal
flora and the prevalence of allergic diseases. It has been
ARBITRARY ELISA UNITS
400
350
300
250
200
150
*
100
50
0
L. plantarum
Germ-free
Fig. 1. The level of Bet v 1 specific IgE in sera after intraperitoneal priming with Bet v 1/Al(OH) 3.
Mice precolonized by recombinant L. plantarum
(white bar) and germ-free controls (black bar).
*P ≤ 0.01
also shown that intranasal application of recombinant
Lactobacillus (L.) plantarum producing major birch pollen
allergen (Bet v 1) induce shift towards non-allergic Th-1
responses after subsequent sensitization1.
METHODS: A mouse model of type I allergy to birch
pollen was used in order to evaluate the role of the microflora for the prevention of allergy by mucosal tolerance
induction. Germ-free (GF) BALB/c mice were monocolonized by recombinant L. plantarum NCIMB8826,
harboring plasmid pMEC181 with Bet v 1 gene under lactatedehydrogenase promotor. Mice were sensitized twenty
days after colonization with three intraperitoneal injections of 1 μg Bet v 1/Alum in ten-day intervals. Seven days
81
after the last injection sera, spleens and small intestines
were collected. The specific Bet v 1 antibody responses
were measured in sera and in small intestine homogenates
by ELISA. Splenocytes were cultivated with/without Bet
v 1. The levels of IL-5 and INF-gamma were measured in
supernatants by ELISA after 48 hours of cultivation.
RESULTS: Recombinant L. plantarum remain genetically stable during whole experiment. The level of Bet
v 1 specific IgA in the small intestine was significantly
increased as compared with GF controls. In L. plantarumcolonized mice Bet v 1 specific IgE in sera (Fig. 1) was
significantly lower than in sensitized GF mice. The level
of INF-gamma was increased in precolonized mice.
CONCLUSION: Monocolonization with recombinant
L. plantarum reduced the level of specific Bet v 1 specific
IgE antibody and stimulated production of INF-gamma
in spleen cells supernatants. We conclude that monocolonization by recombinant L. plantarum shifts the immune
response towards Th-1 direction and that it is a promising
vaccine candidate against type I allergy.
ACKNOWLEDGEMENT
This study was supported by grants 303/05/2249 of the
Czech Foundation of the Czech Republic and IAA500200710
from the Grant Agency of the Academy of Sciences of the
Czech Republic and by Institutional Research Concept
AVOZ50200510.
REFERENCES
1. Daniel C, Repa A, Wild C, Pollak A, Pot B, Breiteneder H,
Wiedermann U, Mercenier A. Modulation of allergic immune responses by mucosal application of recombinant lactic acid bacteria
producing the major birch pollen allergen Bet v 1. Allergy 2006;
61:812–9.
OPTIMIZATION OF BACTERICIDAL EFFECT OF TIME-DEPENDENT ANTIBIOTICS
(EXAMPLES OF THERAPEUTIC DRUG MONITORING)
Josef Sedivya, Anna Jedlickovaa, Olga Cernab
a
Institute of Clinical Biochemistry and Laboratory Diagnostics, and
Department of Paediatrics and Adolescent Medicine, General University Hospital and 1st Faculty of Medicine, Charles
University, 128 08 Prague, Czech Republic
b
Key words: Therapeutic drug monitoring/Growth/Killing rates/Amoxicillin/Clavulanate/ Vancomycin
BACKGROUND: Therapeutic drug monitoring (TDM)
is a method used for individual next therapy optimization.
Rate of bactericidal effect of two time-dependent antibiotics (ATB), vancomycin (VAN) and amoxicillin/clavulanate, was assessed according not only to individual drug
concentration/time fluctuations but also to growth/killing
microbial parameters in vitro. Regardless of all immunologic factors, and based on assumptions of both short
post-antibiotic effect and correlation between dynamic
parameters in vitro and in vivo, examples of optimization
of the individual dosage protocol are presented in order to
hasten the therapy and to decrease the hazard of growing
microbial resistance.
METHODS: Antibiotic use was indicated i.a. according to minimal inhibition concentration (MIC) of etiologic
agent(s), examined by haemoculture or deep wound swab
of patients using microdilution technique1. Serum vancomycine concentration was analyzed using fluorescence
82
polarization immunoassay method; serum amoxicillin concentrations were analyzed using validated high-performance liquid chromatography method2. Pharmacokinetic
analysis and simulation of individual concentration/time
oscillations was performed using MW/PHARM (Kinfit
3.30, MediWare, The Netherlands)3. Evaluation of microbial growth/killing parameters of inoculated bacteria
was performed in vitro in Mueller-Hinton broth without or
with stable concentrations of ATB at a rigorous sampling
schedule, respectively. The inoculum size was verified and
the number of colony-forming units counted using standard spread-plate technique. The maximal killing rate was
calculated as the maximal slope of log-linear part of the
net killing value/time curve. The non-linear sigmoid Emax
model by Zhi4 was applied to describe the relationship
between antibiotic concentration and predicted time to
decrease the relative number of viable bacteria to 10–6,
adopted as the mildest surrogate criterion of the bactericidal effect5. The rate of change in the net log number of
viable bacteria over time was calculated using the equation
dN/dt =[λ – Emax . Cγ/(EC γ50+Cγ)] . N0,
where λ= the maximal growth rate, Emax= the maximal
slope of net killing data, EC50= the antibiotic concentration at which 50% of the maximal effect was obtained,
γ = Hill′s exponent, C= concentration of ATB, N0= starting number of bacteria. Moreover, a microbial static concentration (at which bacterial growth is in equilibrium
with bacterial killing) was described by the equation4–6):
SC = EC50 . [λ/(Emax–λ)](1/γ).
After logarithmic data transformation, the growth/killing parameters were solved by numerical integration using
EDSIM 2.06 as a part of the MW/PHARM software3.
Finally, the time necessary for the required decrease in
the number of microbes could be individuallly assessed
and the drug dosage subsequently optimized.
RESULTS: Example No. 1: Newborn baby (HU,
1 month old, 2.2 kg, 45 cm), admitted to the Paediatric
ICU, suffered from staphylococcal sepsis with good susceptibility to VAN (MIC= 1 mg.L–1). The administration
of VAN (daily dose of 9.5 mg/kg, divided in three one-hour
intermittent IV infusions every 8 hours) turned to low
detected serum concentration on the next day (C6=1.84
mg/L). Changing the VAN dose to 20 mg/kg/day, divided
in four infusions every 6 hours, restored the effective drug
concentrations (C2 = 6.24 mg.L–1). Based on calculated
dynamic parameters of isolated Staphylococcus aureus in
vitro (see Table 1), the decreased relative number of viable
bacteria to 10–6 was predicted in 69 hours (at the start of
therapy) and 54 hours (after the change the dose), respectively. This difference was relatively small.
On the other hand, beta-lactam antibiotics are not being monitored. As published previously2, very low serum
ATB concentrations were detected at the end of usually
recommended dosage interval in some pacients. Example
No. 2: 61 years old men (99 kg, 170 cm) suffered from
diabetic foot infection (detected Staphylococcus aureus,
MIC < 0.5 mg.L–1, its static concentration was assessed as
Fig. 1. Serum concentration/time oscillations of amoxicillin (amoxicillin/clavulanate 875mg/125mg orally every
12 hrs) and the relative number of viable Staphylococcus aureus (MIC<0.5 mg.L–1, SC=0.21 mg.L–1) (as descending line with corresponding right ordinate) in one patient (see Example No.3). The surrogate criterion
of the bactericidal effect was predicted in 89 hours.
83
Table 1. Dynamic parameters of Staphylococcus aureus (see Example No. 1)
in vitro against vancomycine (MIC= 1 mg.L–1).
STAU
Max. growth rate
(logCFU/hr)
Max. killing rate
(logCFU/hr)
EC50
(mg.L–1)
γ
Static concentration
(mg.L–1)
0.38
0.69
0.52
1.19
0.63
0.21 mg.L–1). Amoxicillin/clavulanate was indicated (IV
infusion of 1g/200 mg over 30-min every 8 hrs), but serum
amoxicillin concentration at the end of his dosing interval
was detected as low as 0.04 mg.L–1. Then, the decreased
relative number of viable bacteria to 10–6 was predicted in
65 hours. When the same dose would be given in the first
24 hrs continuously (and then intermittently as above),
the surrogate criterion of the bactericidal effect could be
predicted earlier, in 44 hours.
Example No. 3: Other convalescent patient, 44 years
old men (89 kg, 187 cm), suffered from the same infection
with similar susceptibility. Standard oral doses of amoxicillin/clavulanate (875mg/125mg every 12 hrs) were administered orally. During the PK/PD analysis, two different
phases were visible in the predicted bactericidal effect: at
first, decreasing number of bacteria when ATB concentration was high enough, and secondly, increasing number of
remaining bacteria when ATB concentration was near or
under the microbial static concentration. The lower the
antibiotic concentration, the exponentially longer time
was needed to effectively decrease the number of bacteria. Thus, due to low serum concentrations at the end of
dosing interval, the decreased relative number of viable
bacteria to 10–6 was predicted in 89 hours (see Fig. 1).
When dosing interval would be reduced to 8 hours, the
surrogate criterion of the bactericidal effect could be predicted in 43 hours.
CONCLUSIONS: Based on growth/killing microbial
parameters in vitro and individual drug concentration/
time fluctuations, the rate of bactericidal effect of two
time-dependent antibiotics, vancomycin and amoxicillin/
clavulanate, was assessed to optimize the next therapy.
Predicted time to decrease the number of viable bacteria
to 10–6 (as compared with the start of therapy) was adopted as surrogate criterion of the bactericidal effect. Even
of highly susceptible strains, microbial killing activity of
amoxicillin tends to slow down when its concentration
declines under about 4-times the MIC-level (or about 10times the SC-level). It takes place mainly at the end of too
long intermittent dosage interval. Shortening this interval
(being much more effective than higher dose administered
intermittently) or continuous IV-infusion of standard daily
dose of evaluated antibiotics accelerates their bactericidal
effect in pacients with high degree elimination. Therefore,
the dosage interval for standard oral forms of amoxicillin/clavulanate should not be longer than 8 hours, even
in highly susceptible microbes. The recommendation to
hold Cmin of vancomycine over 5 to 10 mg/L seems to be
rational and satisfactory for individual dosage optimization in cases of susceptible infection. Drug monitoring of
time-dependent antibiotics is supported.
REFERENCES
1. Urbaskova P. Rezistence bakterií k antibiotikům – vybrané metody.
Praha: Trios; 1998.
2. Sedivy J, Petkov V, Jirkovska A, Stambergova A, Ulbrichova Z,
Lupinkova J, Fejfarova V, Bem R. Optimalizace léčby amoxicilín/
klavulanátem na základě PK/PD parametrů u nemocných s infekcí
při syndromu diabetické nohy. Klin mikrobiol inf lék 2004;10:67–
75.
3. Proost JH, Meijer DKF. MW/PHARM, an integrated software
package for drug dosage regimen calculation and therapeutic drug
monitoring. Comput Biol Med 1992; 27:155–63.
4. Zhi J, Nightingale CH, Quintiliani R. A pharmacodynamic model
for the activity of antibiotics against microorganisms under nonsaturable conditions. J Pharm Sci 1986; 75:1063–7.
5. Neef C, van Gils SA, IJzerman WL: Analogy between temperaturedependent and concentration-dependent bacterial killing. Comput
Biol Med 2002; 32:529–49.
6. Mouton JW, Vinks AATMM, Punt NC. Pharmacokinetic-pharmacodynamic modeling of activity of ceftazidime during continuous
and intermittent infusion. Antimicrob Agents Chemother 1997;
41:733–8.
84
THERAPEUTIC DRUG MONITORING (TDM) OF GENTAMICIN IN CRITICALLY
ILL NEONATES
Iva Selke-Krulichovaa, Jirina Martinkovab, Pavla Pokornac, Jiri Zahoraa
a
Department of Biophysics, and
Department of Pharmacology, Charles University of Prague, Faculty of Medicine in Hradec Kralove, Czech Republic
c
Intensive Care Unit, General Teaching Hospital, Departmet of Pediatrics and Adolescent Medicine, Prague, Czech
Republic
b
Key words: Gentamicin/Neonate/Plasam level/Pharmacokinetics
BACKGROUND: Gentamicin (GE) is an aminoglycoside antibiotic frequently used to treat serious infections
and suspected or proven sepsis in neonates due to Gram
negatives and staphylococci. In adults, the risk of nephrotoxicity/ototoxicity or poor efficacy requires monitoring of plasma GE concentration. In neonates, the risk is
further increased due to recognized wide intra- and interpatients variability in the pharmacokinetic parameters
of GE, strongly dependent on the dynamic maturational
process at this extreme of age1,2. In addition, the pharmacokinetic profile in extremely sick septic neonates and in
those with birth asphyxia and renal compromise may be
different than in normal population. Such infants need
individualized GE dosing protocol and close monitoring
of plasma GE concentrations. Therefore, GE dosing in
neonates continues to be a challenge, in view of rapidly
changing and variable pharmacokinetic parameters. In
this prospective study the kinetic profile of GE has been
studied to enable the development of optimized dosage
schedules for neonatal intensive-care units and to stress
the relationship between pharmacokinetic parameters and
several demographic, developmental and clinical factors
which might be associated with changes in GE disposition. A new regimen was developed to produce the assumed target concentrations for GE which were defined
to be 0.5 – 2 mg.L–1 and 6 – 10 mg.L–1 for trough and peak
levels at steady state (Ctroughss, Cpeakss) respectively3.
METHODS: Thirty- two newborn patients of 25 – 42
weeks gestational age, and 0.8 – 4.56 kg birth weight at
their first week of life, prescribed GE for treatment of
suspected or proven sepsis (30/32), and adnate pneumonia (2/32) were admitted at the Neonatal Intensive
Care Unit and included at this prospective study. The
neonates were critically ill, suffering from a different stage
of multiorgane dysfunction. A culture-positive infection
was found in 8 cases. Two empirical GE i.v. loading doses
(given by a 30-min infusion) predicted according to the
body weight and gestational age4 were followed by individually predicted maintenance doses. Peak GE plasma
concentration (0.5 h after dose 1 termination), trough GE
plasma levels (0.5 h just before next dose), and two more
blood samples in between were analyzed. Plasma samples
were collected via a central venous catheter and analysed
by a fluorescence polarization immunoassay technique
(TDx, Abbott Diagnostics) at the Biochemical Laboratory
of University Hospital in Prague. Intra and interday coefficients of variation were < 6 %. The kinetic profile of GE
was estimated using two-compartment (84 %) or one-comparment (16 %) models. Both individual dosage regimens
(maintenace doses), and simulation of plasma concentration fluctuation were developed by MW Pharm 3.15.
RESULTS: Target trough and peak plasma concentrations predicted for a steady state (Ctroughss and Cpeakss)
achieved at dose 4 were compared with those measured
at the same time (Fig 1). Agreement between Ctroughss
and Cpeakss predicted and practically measured was assessed by Bland-Altman analysis. Two groups of infants
were constituted: group 1 free of co-variates accused of
modifying dosage adjustement, and group 2 gathering the
rest of neonates. In group 1 the Bland-Altman analysis
resulted in a mean difference (MD) (predicted minus
monitored concentrations) of 0.22 mg.L–1 and limits of
agreement (LA) of –0.25 to 0.69 mg.L–1 for Ctroughss
whereas for Cpeakss the MD was 0.12 mg.L–1 and LA
–1.47 to 1.71 mg.L–1. In both cases all the values of differences lied within the LA. There was a bias in Ctroughss
predictions. They tended to be higher than monitored
values. Despite of it the LA for Ctroughss were within
a priori defined clinically acceptable targets. The LA for
Cpeakss were wider. To this may have contributed a lower
number of neonates enrolled in group 1. In group 2 the
MD and LA were 0.49 mg.L–1 and – 0.44 to 1.42 mg.L–1,
for Ctroughss, respectively and 1.98 mg.L–1 and – 2.03 to
5.99 mg.L–1 for Cpeakss, respectively. The calculated LA
in this group were clinically inacceptable.
Several co-variates were considered to explain the
pharmacokinetic behaviour of GE: birth weight (BW), gestational age, clearence of creatinine (Clcr), creatininemia,
therapy with furosemide and/or systemic capillary leak
syndrome (Table 1). The relationship determined between
GE pharmacokinetic parameters (estimated after the first
dose of GE) and studied co-variates: BW, clearance of
GE (Clge), Clcr, and creatininemia was assessed by correlation analysis (Pearson’s and Spearman’s correlation).
A value of p < 0.05 was regarded as indicative of significance. The significant correlation was found between BW
and Clge (r = 0.80, p < 0.0001), CLge and CLcr (r = 0.5,
p = 0.002), BW and Clcr (r = 0.4, p = 0.04), and BW and
85
Table 1. Co-variates and pharmacokinetic parameters of GE in neonates.
Group 1
No 13
Group 2
No 19
Stat. significance
Group 1: Group 2
Gestational age
(weeks)
36.00 ± 4.242
35.111 ± 5.063
NS
BW (kg)
2.823 ± 0.894
2.372 ± 0.864
NS
Female sex (%)
44
46
Clearance of creatinine
(mL/min.kg–1)
7.37*
min = 1.20 max = 20.43
4.43*
min = 0.38 max = 19.98
p = 0.31**
Clearance of GE
(mL/min.kg–1)
0.91 ± 0.30
0.54 ± 0.30
p < 0.001
Volume of distribution V1
(L/kg)
0.472 ± 0.144
0.467 ± 0.149
NS
Systemic capillary leak
syndrome between doses 1-4
–
suspected or proven in
12 neonates
Furosemide i.v.
given to 3 neonates
< 1 mg
given to 10 neonates
0.14–12.7 mg
* Values have not normal distribution, so the median, the minimal and maximal values were used; ** Values have not
normal distribution, so the Mann-Whitney test was used.
ACKNOWLEDGEMENT
Supported by the grant IGA MZ 1A 8671-5/05.
REFERENCES
1. de Hook M, van den Anker JN. Aminoglycosides and glycopeptides, In: Yaffe SJ, Aranda JV, editors. Neonatal and pediatric pharmacology. 3rd ed. Philadelphia: Lippincott Williams and Wilkins;
1995. p. 377–401.
2. Semchuk W, Shenchuk Y.M., Sankaran K, Wallace S.M.
Prospective, randomized, controlled evaluation of a gentamicin
loading dose in neonates. Biol.Neonate 1995; 13:13–20.
GE plasma concentrations after 1st and 4th dose
(two-compartment model)
10
GE clearance (mg/L)
creatininemia (r = – 0.1, p = 0.6). This outcome indicates
that BW is a good co-variate for predicting the kinetic profile of GE and dosage individualizing. At the beginning of
study, the only difference between two groups was lower
Clge found in group 2 (Table 1). Hearing evaluation using
auditory evoked potentials to acoustic stimuli was done
on all neonates before discharge from hospital. No sign
of ototoxicity was found.
CONCLUSIONS: First dose plasma concentrations
of GE in neonates during the first week of postnatal age
seems to be predictive for steady state concentrations
in neonates without co-variates significantly modifying
pharmacokinetics of GE. However, the results of BlandAltman analysis indicate a lack of agreement between
predicted and monitored GE concentrations in neonates
administered furosemide, and/or suffering from systemic
capillary leak syndrome. The unexplained variability between observed and predicted values may originate from
an effect of co-administered drugs and/or more serious
clinical status of neonates. The variability requires further
analysis.
8
6
4
2
0
0
1
2
3
4
5
6
7
8
9
duration of treatment (days)
Fig. 1. Prediction of GE plasma concentrations at steady
state (Css) using data obtained after loading
dose 1.
The neonate: BW = 1.39 kg, gestational age = 30
weeks
Comparison of predicted and measured Cpeakss
and C troughss in a neonate from group 1.
After dose 1 plasma concentrations were measured and used to predict Ctrough and Cpeak at
steady state (ss) reached after dose 4. Two loading doses (5.6 mg/48 h) were predicted according
to BW and gestational age of the infant. The individualized maintenance dose was: 4 mg/48 h.
Ctroughss predicted : 0.66 mg/L and measured:
0.6 mg/L
Cpeakss predicted: 6.0 mg/L and measured:
6.1 mg/L
86
3. Rocha MJ, Almeida AM, Afonso E, Martins et al: The kinetic
profile of gentamicin in premature neonates. J Pharm Pharmacol
2000; 52:1091–7.
4. Isemann BT, Kotagal UR, Mashni SM, Luckhaupt EJ, Jophnson
CJ: Optimal gentamicin therapy in preterm neonates includes
loading doses and early monitoring. Ther Drug Monitoring 1996;
18:549–55.
EVALUATION OF IN VITRO PROTECTION BY SALICYLALDEHYDE
ISONICOTINOYL HYDRAZONE (SIH) AGAINST HYDROGEN PEROXIDEAND ANTHRACYCLINE-INDUCED TOXICITY TO CARDIAC CELLS
Tomas Simuneka, Petra Kovarikovab, Martin Sterbac, Olga Popelovac, Helena Kaiserovaa,
Michaela Adamcovad, Premysl Ponkae, Vladimír Gerslc
a
Department of Biochemical Sciences and bDepartment of Pharmaceutical Chemistry and Drug Control, Charles University
in Prague, Faculty of Pharmacy in Hradec Kralove, Czech Republic
c
Department of Pharmacology and dDepartment of Physiology, Charles University in Prague, Faculty of Medicine in Hradec
Kralove, Czech Republic
e
Lady Davis Institute for Medical Research, McGill University, Montreal, Canada
Key words: Salicylaldehyde isonicotinoyl hydrazone/Hydrogen peroxide/Daunorubicin/ Cardiotoxicity/Cardioprotection
BACKGROUND: Anthracycline antibiotics (e.g.
doxorubicin, daunorubicin, epirubicin) rank among the
most effective and frequently used antineoplastic agents.
However, repeated treatment with anthracyclines is accompanied by the risk of cardiomyopathy and heart failure
development. Pivotal pathophysiological role has been attributed to the iron-catalyzed intramyocardial production
of reactive oxygen species (ROS). Displacement of free
or loosely bound iron via its chelation can inhibit Fenton
and Haber-Weiss reactions and thus reduce the ROSinduced cellular injury. We have previously shown that
salicylaldehyde isonicotinoyl hydrazone (SIH) – a strong,
cell permeable iron chelator – is highly protective against
oxidative stress. In H9c2 rat cardiomyoblast cell-line, SIH
was capable to fully prevent hydrogen peroxide (H2O2)-induced alterations in cell morphology, dissipation of mitochondrial inner membrane potential, as well as to abolish
both apoptosis (annexin-V staining, nuclear chromatin
shrinkage, TUNEL positivity) and necrosis (propidium
iodide staining)1. In this study we aimed to examine and
compare the protective effects of SIH against the H2O2and anthracycline-induced toxicity to isolated primary
ventricular cardiomyocytes from neonatal rats.
METHODS AND RESULTS: Cardiomyocytes treated
with 10 μmol.L–1 daunorubicin (DAU) initially increased
beating rate, which was followed by irregular activity,
discontinuation of cellular monolayer and eventually the
arrest of their contractility. DAU exposure resulted in progressive and significant release of lactate dehydrogenase
(LDH) to the culture medium– at 24 h and 48 h its activity
was 3.2 and 5.9 fold higher, respectively, than the control
values. Co-incubation with SIH, tested at concentrations
3 – 100 μmol.L–1, partially but significantly reduced the
DAU-induced LDH release. However, no apparent dosedependency was observed, as the LDH activity reduction
achieved with 100 μmol/l SIH (25 %) was comparable to
that of 3 μmol.L–1 (22 %). The lowest SIH concentration
used (3 μmol.L–1) was the only concentration that did not
induce LDH release when added to cardiomyocytes alone.
Higher SIH concentrations dose-dependently increased
the LDH activity in culture media, probably indicating
unfavorable toxicity due to iron depletion after prolonged
incubation in this in vitro system. Only partial protection
of cardiomyocytes by SIH against DAU was in contrast
Table 1. Protective action of SIH against H2O2 and daunorubicin toxicity in primary rat cardiomyocytes.
Cardiomyocyte treatment
IC50
Maximal achieved degree of cardiomyocyte
protection
H2O2 500 μmol.L–1
(6h exposure)
2.1 μmol.L–1
100 %
(10 μmol.L–1)
Daunorubicin 10 μmol.L–1
(48h exposure)
not reached
25 %
(100 μmol.L–1)
Cytoprotection assessed as lactate dehydrogenase release reduction.
IC50: Calculated concentration of SIH resulting in 50% protection against the toxicity of oxidative stressor.
with our observation that SIH rapidly enters myocytes and
efficiently displaces iron from a fluorescence-quenched
intracellular iron-calcein as well as iron-DAU complexes.
Moreover, SIH completely protected the primary cardiomyocytes against the oxidative toxicity induced by acute
(6 h) exposure to 500 μM H2O2 (Table 1).
The observed discrepancy between high protective efficiency of SIH against acute injury (H2O2) and only partial
reduction of toxicity induced by prolonged DAU exposure
has led us to assess stability of SIH in cell-culture medium
(serum-free DMEM). Previously we have observed rather
short plasma half-life of SIH during a pilot in vivo pharmacokinetics experiment2. Therefore, samples of SIHcontaining media (incubated with H9c2 cells in 37 °C /
5 % CO2) were collected over 24 hours and analyzed by
HPLC with UV detection. HPLC analysis was performed
on C18 column using a mixure of methanol and phosphate
buffer (48:52, v/v) as a mobile phase. As seen in Fig. 1,
an exponential decay of SIH concentration was observed
with t1/2= 210 minutes.
CONCLUSIONS: The present study suggests applicability of iron chelation in cardiovascular diseases where
oxidative stress is involved. However, further investigation
is necessary to detect whether the only moderate protective efficiency of SIH against the DAU-induced cardiomyocyte injury is primarily due to the (i) rapid decay of SIH
concentration, (ii) toxicity of iron depletion by prolonged
SIH exposure, or (iii) the possibility that other, Fentontype ROS-independent, mechanisms play an important
role in the DAU-induced damage of cardiomyocytes.
87
Fig. 1. Time-dependent decrease in SIH concentration
in cell-culture medium. First rate-order kinetics;
t1/2= 210 minutes.
ACKNOWLEDGEMENT
Study supported by the Charles University Grant No.
124307/C/2007.
REFERENCES
1. Simunek T, Boer C, Bouwman RA, Vlasblom R, Versteilen AM,
Sterba M, Gersl V, Hrdina R, Ponka P, de Lange JJ, Paulus WJ,
Musters RJ. SIH--a novel lipophilic iron chelator--protects H9c2
cardiomyoblasts from oxidative stress-induced mitochondrial injury
and cell death. J Mol Cell Cardiol 2005; 39:345–54.
2. Kovarikova P, Klimes J, Sterba M, Popelova O, Mokry M, Gersl
V, Ponka P. Development of high-performance liquid chromatographic determination of salicylaldehyde isonicotinoyl hydrazone
in rabbit plasma and application of this method to an in vivo study.
J Sep Sci 2005; 28:1300–6.
BIOLOGICAL CHARACTERIZATION OF VENOM PEPTIDES
FROM THE NEOTROPICAL SOCIAL WASP POLISTES MAJOR MAJOR
(DOMINICAN REPUBLIC)
Jirina Slaninova, Vladimir Fucik, Lenka Borovickova, Vaclav Cerovsky
Institute of Organic Chemistry and Biochemistry, v.v.i., Academy of Science of the Czech Republic. Flemingovo 2,
166 10 Prague 6, Czech Republic
Key words: Wasp venom/Antimicrobial peptides/Biological activity
BACKGROUND: Wasp venoms are rich source of
interesting substances of different biological activities1.
Among these compounds, peptides play an important
role. A whole series of cationic wasp venom peptides were
isolated, chemically characterized and examined for their
biological activity2,3. Some of them were rather potent
antimicrobial factors. The emergence of bacterial pathogens resistant to conventional antibiotics stimulated the
search for new classes of antimicrobial agents from natural
sources. Antimicrobial peptides with unique mechanisms
of action provide a new path for developing medicines
in the fight against antimicrobial resistance. Two novel
peptides were recently isolated from the venom of the
social wasp Polistes major major4, one of the most common wasp in Dominican Republic. These two peptides
– mastoparan like one: H-Ile-Asn-Trp-Lys-Lys-Ile-Ala-SerIle-Gly-Lys-Glu-Val-Leu-Lys-Ala-Leu-NH2 (PMM, 1909.19
Da) and chemotactic like one H-Phe-Leu-Ser-Ala-Leu-LeuGly-Met-Leu-Lys-Asn-Leu-NH2 (PMM-CT, 1317.78 Da)
were synthesized using solid phase peptide methodology
and tested for antimicrobial activity against Bacillus subtilis and Escherichia coli (MIC determination), hemolytic
88
Table 1. Activities of Polistes Major Major Peptides.
Peptide
PMM
PMM-1
PMM-2
PMM-3
PMM-4
PMM-5
PMM-6
PMM-7
PMM-8
PMM-9
PMM-10
PMM-CT
Antimicrobial activity
MIC
MIC
anti E.c.
Anti B.s.
μM
μM
8.5
35
5.5
27.2
46
21
125
62
75
>>100
>>40
>>100
75
70
>>100
100
100
>>100
>>100
>>100
15
35
100
45
activity (rat blood cells) and mast cell degranulating activity (rat mast cells). As the first one, PMM, showed rather
potent antimicrobial activity, we have synthesized several
structural analogues for structure activity study and determined their qualities.
METHODS: Solid-phase peptide syntheses were carried outmanually in polypropylene syringes with a bottom
Teflon filter using standard protocol of Nα-Fmoc chemistry on 100 mg of Rink Amide MBHA resin. Crude peptides were purified by preparative HPLC using Thermo
Separation Product instrument on a Vydac C-18 column
(250 x 10 mm) at a flow rate 3.0 mL/min. The identities
of purified peptides were verified by MALDI-MS.
Determination of the hemolytic activity5: Peptides
were incubated with rat red blood cells for 1 h at 37 °C
in a final volume 0.2 ml (final erythrocyte concentration
5%, v/v, final peptide concentration 1–100 μM). The samples were then centrifuged for 5 min at 250 × g and the
absorbance of the supernatant was determined at 540 nm.
Controls for zero hemolysis (blank) and 100% hemolysis
consisted of supernatants of red blood cells suspended
in physiological solution and 0.2% TRITON X100, respectively. Each peptide was tested at least 2 times in
duplicates in two independent experiments.
Mast cell degranulation test6: Mast cell degranulation
was determined by measuring the activity of released betaD-glucosaminidase, which is co-localized with histamine.
Mast cells obtained by peritoneal washing of adult Wistar
rats were incubated in the presence or absence (zero degranulation) of the peptide or in the presence of 0.1 %
TRITON X100 (100 % lysis) for 15 min at 37 °C and
centrifuged. After centrifugation, samples of the supernatant (50 μl) were added to the solution of a substrate
(p-nitrophenyl-N-acetyl-beta-D-glucosaminidine, SIGMA)
and incubated for 6 h at 37 °C. The reaction was stopped
by the addition of 150 μl of 0.2M TRIS buffer (pH = 9)
and the absorbance determined at 405 nm.
Antimicrobial activity determination2,7: The minimal
Hemolytic
activity
IC50
μM
100
65
170
>>100
>>100
>360
>270
>>100
>>100
>>100
>100
45
Degranulation
activity
IC50
μM
7
10
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
inhibitory concentrations (MIC) were established by following the growth of bacteria Bacillus subtilis 168 and
Escherichia coli B in multiwell plates in LB broth. The
bacteria in mid exponential phase were added into individual wells (cca. 1 million colony forming units) to solutions of different concentrations of tested peptides (final
volume of 0.2 mL, final concentration of peptides in the
range of 1 to 100 μM) in the LB broth and incubated at
37 °C for 20 h under continuous shaking in Bioscreen C
instrument (Finland). The absorbance was measured at
540 nm every 15 min for 20 h. Each peptide was tested at
least 3 times in duplicates.
RESULTS: The following peptides were synthesized
and studied:
H-Ile-Asn-Trp-Lys-Lys-Ile-Ala-Ser-Ile-Gly-Lys-Glu-Val-LeuLys-Ala-Leu-NH2, PMM; [Ile17]PMM, PMM-1;
des-Ile1-PMM, PMM-2; des-Ile1-Asn 2-PMM, PMM-3;
des-Ile1-Asn2-Trp3-PMM, PMM-4;
des-Ile1- Asn2-Trp3-Lys4-PMM, PMM-5;
des-Leu17-PMM, PMM-6; des-Ala16-Ile17-PMM, PMM-7;
des- Lys15-Ala16-Ile17-PMM, PMM-8;
des-Leu14-Lys15-Ala16-Ile17-PMM, PMM9;
[Trp 2 ,Asn 3 ,Ile 5 ,Ala 6 -Lys 7,Val 12 ,Leu 13 ,Glu 14 ]-PMM,
PMM10.
Their biological activities are given in the Table 1. The
mother peptide PMM (isolation and synthesis described
in ref.3) was found to be rather potent antimicrobial
agent with MIC value 8.5 μM and 35 μM for E.c. and
B.s., respectively. This peptide showed the mast cell degranulation activity lower than mastoparan and negligible
hemolytic activity (50% hemolysis at 100 μM concentration). Even higher antimicrobial activity however showed
its analogue having Ile as the last amino acid (PMM-1).
Analogues of the PMM peptide truncated either on the
N- or C-end of the peptide chain showed gradual decrease
in potency depending on the degree of truncation (see
Table 1). Removal of only 1 amino acid from the C end
resulted in higher decrease of activity than the same
change on the N-end. Changing the sequence of amino
acids of PMM to better correspond to the alfa helical
wheel with hydrophobic and hydrophilic amino acids on
opposite sites (analogue PMM-10) resulted in a peptide
having comparable activities to the mother peptide PMM.
The chemotactic like peptide PMM-CT showed also some
degree of antimicrobial activity, however relatively high
hemolytic activity.
CONCLUSIONS: The potent antimicrobial peptides
described in this paper showed MIC in the range of
5–40 μM. This corresponds to the activity of most antimicrobial peptides described in literature. As structural
features that might underline antimicrobial activity should
be: (i) the ability to form alfa helical structure and (ii)
amphipaticity, we have synthesized and studied peptides
which would have better prerequisite to form alfa helix
and be more amphipatic. We also tried to formulate the
“active core” of our peptides for the antimicrobial activity.
As can be seen from the Table, truncation of the mother
peptide from both ends by one amino acid only slightly decreased the activity, however further shortening from both
ends resulted in strong decrease of the activity. Interesting
finding is, that if we have synthesized a peptide where the
important amino acids were placed into positions that
should result in theoretically better amphipathic properties, no improvement of the activity was found (analogue
PMM-10).
89
ACKNOWLEDGEMENT
Financial support by research project of the IOCB AVCR,
v.v.i., Z40550506 has been gratefully acknowledged.
REFERENCES
1. Habermann E. Bee and wasp venoms. Science 1972; 117:314-22.
2. Mendes MA, De Sousa BM, Marques MR, Palma MS. Structural
and biological characterization of two novel peptides from the venom of the neotropical social wasp Agelaia pallipes palipes. Toxicon
2004; 44:67–74.
3. Turillazi S, Mastrobuoni G, Dani FR, Moneti G, Pieraccini G,
la Marca G, Perito B, Lambardi D, Cavallini V, Dapporto L.
Dominulin A and B: two new antimicrobial peptides identified on
the cuticle and in the venom of the social paper wasp Polistes dominulus using MALDI-TOF, MALDI-TOF/TOF, and ESI-Ion trap.
J Am Soc Mass Spectrom 2006; 17:376–83.
4. Cerovsky V, Pohl J, Alam N, Yang Z, Attygalle AB. Identification
of three novel peptides isolated from the venom of the neotropical
social wasp Polistes major major. J Peptide Sci 2000; 13:445–50.
5. Oren Z, Shai Y. Selective lysis of bacteria but not mammalian
cells by dia-stereoisomers of mellitin: structure function study.
Biochemistry 1997; 36:1826–35.
6. Mendes MA, Palma MS. Two new bradykinin-related peptides
from the venom of the social wasp Protopolybia exigua (Saussure).
Peptides 2006; 27: 2632–9.
7. Lequin O, Ladram A, Chabbert L, Bruston F, Convert O, Vanhoye
D, Chassaing G, Nicolas P, Amiche M. Dermaseptin S9, an alfa/
helical antimicrobial peptide with a hydrophobic core and cationic
termini. Biochemistry 2006; 45:468–80.
MESENTERIC ISCHAEMIA/REPERFUSION – INDUCED INTESTINAL AND
VASCULAR INJURY: EFFECT OF H1 ANTIHISTAMINES
Ružena Sotnikova, Viera Nosalova, Katarina Drabikova, Viera Jancinova,
Margita Petrikova, Radomir Nosal
Key words: Ischaemia/reperfusion/Reactive oxygen species/Vessel reactivity/ Chemiluminescence/Antihistamines
BACKGROUND: Restoration of blood flow after preceding ischaemia results in reactive oxygen species (ROS)
formation and various biochemical and morphological
alterations, leading to endothelial dysfunction and neutrophil mediated intestinal tissue injury. Tissue ischaemia
is accompanied also by the activation of mucosal mast
Table 1. Effect of H1 antihistamines on extent of intestinal injury (in %) and on chemiluminescence (CL)
of intestinal and vascular samples (peaks in cm/100mg ww) induced by ischaemia/reperfusion.
Experimental
group
CONTROL
DMSO + I/R
LOR + I/R
H2O + I/R
DIT + I/R
extent of intestinal injury
Mean ± SEM
–
52.77 ± 6.35
22.91 ± 3.27*
51.74 ± 6.98
22.37 ± 5.40*
CL intestine
Mean ± SEM
1.12 ± 0.39
1.67 ± 0.30
3.17 ± 0.85+*
3.64 ± 0.64+
6.06 ± 1.53+*
*P<0.05 H1 antihistamines + I/R versus vehicle + I/R, +P<0.05 versus control, n = 4–10
CL SMA
Mean ± SEM
2.5 ± 0.60
3.24 ± 0.58
5.18 ± 0.58
5.31 ± 0.78+
5.34 ± 1.46
90
120
sham
I/R
100
I/R+loratadin
I/R+ditiaden
% of PE
80
60
40
*
20
0
8
7
6
Acetylcholine (-log mol/l)
5
Fig. 1. Effect of ischaemia/reperfusion on endothelium-dependent relaxation of SMA,
expressed as % of phenylephrine (PE)-induced contraction. Values are mean
± SEM, n =10-12. * P<0.05 vs sham controls.
cells with the release of histamine1. The aim of this work
was to assess the effect of H1 antihistamines dithiaden
(DIT) and loratadine (LOR) on intestinal as well as vascular injury induced by ischaemia/reperfusion (I/R) following occlusion of the superior mesenteric artery (SMA)
in rats.
METHODS: Male Wistar rats weighing 230-280 g
were used. All procedures received approval from the
State Veterinary and Food Administration of the Slovak
Republic. In thiopental anaesthesised rats, ischaemia was
induced by occluding the SMA for 60 min followed by 30
min reperfusion. H1 antihistamines (10 mg/kg i.p) were
administered 60 min before SMA occlusion and immediately before reperfusion. Sham operated animals were
used as controls. The extent of intestinal injury was assessed macroscopically and expressed as percentage of
the intestinal length. Endothelial function of SMA was
evaluated using isometric tension measurements in vitro. Arterial rings were precontracted with phenylephrine
(1 μmol) and at the plateau of contraction acetylcholine
(AC)-induced relaxation was tested. ROS production was
assessed by the luminol enhanced chemiluminescence
(CL) response of the SMA and ileal samples2. Number
of neutrophils in 1 μL of blood was determined. Statistical
analysis was assessed by ANOVA followed by Tukey’ s and
Student’s t- test.
RESULTS: Following I/R a pronounced intestinal
injury was observed. The blockade of H1 receptors by
both LOR and DIT reduced the extent of intestinal injury, suggesting the involvement of mast cells and their
major mediator histamine in this process (Table 1). I/Rinduced vascular injury was manifested as decreased endothelium-dependent relaxation of SMA. Whereas control
arterial rings responded to AC with maximal relaxation
reaching 4.9 ± 2.5 %, after I/R maximal relaxation was
30.5 ± 3.6 % of contraction. H1 antihistamines improved
endothelium-dependent relaxation with values similar to
sham controls (Fig. 1). Intestinal and vascular biopsy
specimens from I/R rats produced more CL than those
from control rats. A further increase in CL response of
the intestinal samples but not SMA by H1 antihistamines
was observed (Table 1). PMNL count in the whole blood
(in 1 μL x 1000) significantly increased after reperfusion
and showed the respective values before ischaemia and
after reperfusion: 9.4 ± 0.8 and 20.2 ± 2.5 in the DMSO
group, 10.3 ± 1.4 and 20.2 ± 3.4 in LOR, 9.5 ± 0.6 and
19.8 ± 1.6 in H2O, and 8.9 ± 0.3 and 9.2 ± 1.8 in DIT
group. DIT was able to prevent the increase of PMNL,
while LOR was ineffective.
CONCLUSIONS: A significant protective effect of H1
antihistamines against I/R induced intestinal and vascular damage was observed. The effect of DIT and LOR
was virtually the same, however, the mechanisms of their
action seem to be different. DIT appears to operate by
interference with activated PMNL, while the action of
LOR is less clear.
ACKNOWLEDGEMENT
Financial support by the VEGA grants No 2/5009/25
and No 2/4003/04 is gratefully acknowledged.
REFERENCES
1. Kimura T, Fujiyama Y, Sasaki M, Andoh A, Fukuda M, Nakajima
S, Bamba T. The role of mucosal mast cell degranulation and freeradical generation in intestinal ischemia-reperfusion injury in rats.
Eur J Gastroenterol Hepatol 1998; 10:659–66.
2. Nosalova V, Drabikova K, Sotnikova R, Nosal R. Enhanced chemiluminescence of rat small intestine after mesenteric ischaemia/
reperfusion. Biologia: section cellular and molecular biology. 2005;
60, suppl.17:141–43.
91
DEGRADATION OF OXIDIZED CALMODULIN (CAM) BY 20S PROTEASOME
AS POSSIBLE PROTECTION AGAINST OXIDATIVE STRES
Miriam Strosovaa,b, Lubica Horakovaa, Peter Vossb, Tilman Gruneb
a
Institute of Experimental Pharmacology, Slovak Academy of Sciences, Bratislava
Research Institute for Environmental Medicine, Heinrich Heine University, Düsseldorf
b
Key words: Calmodulin/Proteasomal degradation/Oxidative stress/Human fibroblast
BACKGROUND: Calmodulin1,2 (CaM) plays a key
role in intracellular signaling and is functionally sensitive
to conditions of oxidative stress. CaM oxidation results in
large alterations in cellular function as a result of changes in both calcium signaling and energy metabolism3.
Restoration of cell function may require degradation of
oxidized CaM (oxCaM) and other signaling proteins. The
20S proteasome4 has been suggested to play a critical role
in mediating the degradation of crucial proteins under conditions of oxidative stress. The aim of our present study is
to contribute to the explanation of oxCaM degradation by
20S proteasome. In addition, we tried to establish whether
CaM is oxidized and degraded by proteasome in vivo in
VHF93 cells after incubation with H2O2.
METHODS: Calmodulin (CaM) from bovine brain
was purchased from Sigma (Germany). CaM (1mg/mL)
was oxidized with various concentrations of H2O2 for
2 h at 37 °C in 20 mM PBS (pH 7.4) in the presence
of 1 mM CaCl2 or 1 mM EGTA. Native or oxidized
CaM (0.5 mg/ml) were degraded by 20S proteasome
(0.1 mg/mL) for 6 h at 37 °C in proteasome buffer. The
20S proteasome was isolated from erythrocytes of human
blood5. Cell Culture VHF93 (foreskin-fibroblasts) mycoplasmafree were grown at 37 °C on DMEM supplemented
with 10 % fetal bovine serum, 1% glutamax and 1% streptomycin under 5 % CO2. VHF93 cell lysates were exposed
to 100 μmol H2O2/mg prot. in PBS for 2h at 37 °C and
then degraded by 20S proteasome (0.1 mg/mL) for 6h at
37 °C in proteasome buffer. VHF93 cells (106 per flask)
were exposed to 4 μmol/mg prot. of H2O2 in PBS for
30 min at 37 °C. The rate of oxidation and of proteolysis
was demonstrated by SDS-PAGE with Coomassie Blue
D. Degradation of OxCaM by proteasome in the presence of Ca 2+
(Coomassie staining)
A. CaM oxidation in the presence of Ca2+
20 kDa
PS
20 kDa
oxCaM
15 kDa
CaM
oxCaM
15 kDa
CaM
E. Degradation of oxCaM by proteasome in the absence of Ca2+
B. CaM oxidation in the absence of Ca2+
20 kDa
20 kDa
PS
oxCaM
CaM
15 kDa
oxCaM
CaM
15 kDa
Proteasome
C. CaM oxidation in the presence of Ca2+
(Anti-CaM – immunoblot)
-
H2O2 (μmol/mg)
20 kDa
oxCaM - 18.25 kDa
CaM - 15.50 kDa
15 kDa
+
0
-
+
-
15
+
-
30
+
-
50
+
100
F. Time dependent degradation of oxCaM by the 20S proteasome
(Anti-CaM - immunoblot)
20 kDa
Signal intensity
(%)
oxIII - 14.25 kDa
oxIV - 12.75 kDa
20
15
10
5
0
H2O2 (μmol/mg)
oxCaM
I
II
III
15 kDa
III
oxIII
IV
IV
oxIV
Time (h)
Proteasome
H2O2
0
15
30
50
0
+
0
+
+
1
+
+
2
+
+
3
+
+
4
+
+
5
+
+
6
+
+
24
+
+
100
Fig. 1. Oxidative modification of CaM by H2O2 and proteolytic degradation of oxCaM by the 20S proteasome. The
polyacrylamide gels A, B, D, E and blot C, F show one representative of three independent experiments.
CaM is Calmodulin, oxCaM is oxidized Calmodulin and PS is proteasome. oxIII and oxIV are fragments of
oxCaM. I, II, III, IV are fragments of proteolytic degradation of oxCaM. The densitometric quantification of
the oxidation products by FluorChem 8900 Software (Version 3.2.3., Alpha Innotech Corporation) is shown
in graph in panel C. First columns in absence of H2O2 in all panels represent native non-oxidized CaM.
92
A.
Oxidation and degradation of cell lysates
B.
Cells VHF93 treated with H2O2
20 kDa
20 kDa
oxCaM - 18.25 kDa
oxCaM - 18.25 kDa
CaM - 15.50 kDa
15 kDa
CaM - 15.50 kDa
15 kDa
H2O2 (100 μmol/mg)
Proteasome
Time (h)
fragments III ox - 14.25 kDa
IV ox - 12.75 kDa
0
+
2
+
2+6
+
+
2+6
H2O2 (4 μmol/mg)
-
+
Fig. 2. VHF93 cell lysates and cells treated with H2O2. Immunoblots show one representative of two independent
experiments.
staining along with immunoblotting6 or by measurement
of protein carbonyl content by ELISA7.
RESULTS: The electrophoretic mobility of oxCaM
depended on the degree of oxidation and on the presence or absence of Ca2+ ions (Fig. 1A and B). This may
be induced by increasing molecular weight of oxCaM
with increasing H2O2 concentration. Two fragments of
oxCaM were distinguished by the immunoblot techniques
(Fig. 1C). The extent of protein carbonyl formation in
oxCaM increased concentration-dependently on H2O2 and
was not dependent on the presence of Ca2+. 20S proteasome degraded oxCaM concentration- and time-dependently, without affecting unoxidized CaM (Fig. 1D and E).
Oxidized CaM was degraded by 20S proteasome to four
proteolytic fragments (Fig. 1F).
Oxidation of VHF93 cell lysates by H2O2 induced generation of a band corresponding to oxCaM (Fig. 2A). In
the presence of proteasome under the same conditions,
degradation of oxCaM was observed similarly as in the
experiments in vitro on isolated CaM. 20S proteasome is
able to recognize oxidized CaM not only in vitro but also
in cell lysates. The bands induced in VHF93 cell culture
by H2O2 were comparable to the bands indicated by CaM
oxidized with H2O2 (Fig. 2B) and degraded with proteasome in vitro.
CONCLUSIONS: CaM oxidized by H2O2 was degraded by the 20S proteasome without ubiquitin conjugation
and without affecting the unoxidized CaM. Similarly as in
vitro, CaM may be oxidatively modified under conditions
of oxidative stress induced by H2O2 in vivo.
ACKNOWLEDGEMENT
Financial support by grants VEGA 2/5012/26,
APVV 51017905, DAAD and COST B35 is gratefully
acknowledged.
REFERENCES
1. Gao J, Yin, D, Yao Y, Williams, TD, Squier TC. Biochemistry 1998;
37:9536-48.
2. Yin D, Kuczera K, Squier TC. Chem Res Toxico. 2000; 13:103–
10.
3. Hühmer AFR, Gerber NC, Ortiz de Montellano PR, Schöneich
Ch. Chem Res Toxicol 1996; 9:484–91.
4. Ferrington DA, Sun H, Murray KK, Costa J, Williams TD, Bigelow
DJ, Squier TC. J Biol Chem 2001; 276:937–43.
5. Hough R, Pratt G, Rechsteiner M. J Biol Chem 1987; 262:8303–
13.
6. Laemmli UK. Nature. 1970; 227:680–5.
7. Buss H, Chan TP, Sluis KB, Domigan NM, Winterbourn CC. Free
Radic Biol Med 1997; 23:361–6; Erratum in: Free Radic Biol Med
1998; 24:1352.
DETERMINATION OF MICROMOLAR CONCENTRATIONS OF
NEUROTRANSMITTER AMINO ACID GLYCINE BY FLUORESCENCE DETECTION
IN RAT MICRODIALYSIS PERFUSATES
Marie Soukupovaa, Vladimir Kubicekb, Martin Janovskya
a
Department of Pharmacology, 3rd Faculty of Medicine, Charles University in Prague, Czech Republic
Department of Biophysics and Physical Chemistry, Faculty of Pharmacy in Hradec Kralove, Charles University in Prague,
Czech Republic
b
Key words: Glycine/HPLC/NBD-Cl/Microdialysis
BACKGROUND: Glycine is a presumed nocioception
inhibitory transmitter in specific brain regions, like for
example in the spinal cord, the thalamic nuclei and the
periaqueductal gray matter (PAG)(ref.1). Measuring quan-
titative changes in glycine release in these brain regions
during nocioception in awake animals might be useful.
The aim of the present study was to develop a method
combining pre-column derivatization and reverse-phase
HPLC for achiral assay of glycine in very small biological
samples. The method has been applied to a determination
of glycine in microdialysis perfusates from the rat brain.
METHODS: Chemicals: Glycine was obtained from
Herbacos, a.s. 7-Chloro-4-nitrobenzo-2-oxa-1,3-diazole
(NBD-Cl), a product of Fluka, was employed as the derivatizing reagent. Acetonitrile for HPLC, gradient grade,
was supplied from Fluka. Phosphate buffer was made
using potassium dihydrogen phosphate p.a. (Lachema
Brno) and phosphoric acid p.a. (Lachema Brno). Borate
buffer was made using boric acid p.a. (Sigma) and potassium chloride p.a. (Lachema Brno). Biological material:
brain microdialysates of male Wistar rats (250–300 g).
Chromatographic analysis was carried out using Shimadzu
LC Chromatograph equipped with fluorimetric detector
RF-10AXL and CSW 1,7 analytical software. The mobile
phase consisted of phosphate buffer (0.025M) and acetonitrile (75:25, v/v). The analytical column was Discovery
HS C18 (250 x 4,6 mm I.D.). The peaks were detected at
470 nm excitation and 535 nm emission wavelengths. At
a flow rate 1.0 mL/min the whole analysis lasted during
20 min. For construction of calibration curves a stock
solution of glycine in ultra pure water was prepared by
dissolving 0.75 g of the substance in 100 mL. The solution was diluted to prepare appropriate solutions of lower
concentrations.
RESULTS: Calibration curves were constructed for glycine samples in ultra pure water. A linear calibration curve
was obtained in a concentration range from 0.5 μmol.L–1
to 10.0 μmol.L–1. Calibration curve was linear with coefficient of determination 0.999. The value 0.574 μmol.L–1 is
a limit of quantitation (LOQ). All calibration samples and
biological samples (20 μL) were pre-column derivatized
93
with NBD-Cl and borate buffer. Glycine reacts readily
with the reagent in aqueous solution2 and the derivative
is stable almost 24 hours. Chromatographic resolution
of glycine peak and the nearest peaks on the chromatograms of perfusates was perfect. Glycine peak exhibits
retention time t = 8.640 min. The average value of determined glycine levels in four microdialysate perfusates
was 3.696 μM.
CONCLUSIONS: Determination of micromolar concentrations of neurotransmitter glycine in microdialysates
by HPLC with fluorescence detection has been feasible
with this method. Optimization of chromatographic separation and fluorescence detection of NBD-Cl derivatized
amino acid provides sufficient sensitivity for its quantification at levels up to 1.0 μM. Chromatographic conditions
can be easily modified to allow the simultaneous analysis
of another neurotransmitter amino acids like a gammaaminobutyric acid or glutamate. Further enhancements
to lower the detection limit for glycine is desirable to
enhance the response at basal levels if measured during
nocioception and permit the collection of smaller microdialysis fractions to increase the temporal resolution.
ACKNOWLEDGEMENT
The study was supported by the Research Grants VS
MSM0021620816 and IGA NR/9072-3.
REFERENCES
1. Maione S, Marabese I, Rossi F SCA, Berrino L, Palazzo E, Trabace
L, Rossi F. Effects of persistent nocioception on periaqueductal
gray glycine release. Neuroscience 2000; 97:311–6.
2. Ghosh PB, Whitehouse MW. 7-Chloro-4-nitrobenzo-2-oxa-1,3-diazole: A new fluorigenic reagent for amino acids and other amines.
J Biochem 1968; 108:155–6.
CURRENT TREATMENT
OF RHEUMATOID ARTHRITIS
David Suchy
Department of Clinical Pharmacology, Teaching Hospital Pilsen, Czech Republic
Key words: Rheumatic arthritis/Antirheumatic drugs/Combination therapy/Biological treatment
BACKGROUND: Rheumatoid arthritis (RA) is
a chronic, systemic inflammatory disorder of unknown
etiology, characterized by symmetric polyarticular pain
and swelling, morning stiffness and fatigue. RA has variable course with periods of exacerbations and less frequent remisions. Prevalence of RA is 0.5–1 % of the adult
population.
METHODS AND RESULTS: Ultimate goals of RA
management are reduction of pain, prevention of structural progression, maintaining normal daily life activities, quality of life and capacity to work. Maintaining
normal joint function and structure can be achieved by
controlling the disease before any irreversible damage has
occured. This is the reason for the use of disease modifying antirheumatic drugs (DMARDs) early; agressive
treatment can improve signs and symptoms of disease
and slow radiografic progression. All patiens with RA
are candidates of DMARDs therapy. Corticosteroids
and nonsteroidal anti-inflammatory drugs (NSAIDs)
are used, when symptomatic effect or rapid control of
disease activity is desired. The therapy is often initiated
in patients with active disease while awaiting the full
therapeutic effect of DMARDs. The most commonly
used DMARD is methotrexate (MTX) as a result of his
favorable toxicity, good efficacy and low price. More than
50 % of patients taking methotrexate continue with the
94
treatment after 5 years, which is longer than with any
other DMARD. Sulfasalazine (SAS) is other frequently
used DMARD probably with similar efficacy and good
tolerability. Antimalarials (chloroquine and hydroxychloroquine-HCQ) are commonly used drugs with a favorable
toxicity/benefit profile. There are not data for reduction of
disease progression (prevention of radiological damage),
therefore these drugs should be used in patiens with low
activity of disease. Leflunomide, an isoxazole derivative
and inhibitor of de novo synthesis, is a novel DMARD .
Several randomized controlled clinical trials have established leflunomide as an alternative to methotrexate especially for patients, who cannot tolerate MTX or have an
inadequate response to MTX. Cyclosporin A (CSA) is an
immunosupresive agent used in patiens with innadequate
response to first line DMARDs (mainly MTX), but high
frequency of adverse events is the major limiting factor
of its use. The use of combinations of DMARDs in case,
when a single agent fails to control symptoms and progression of RA is generally accepted, althougt the most effective way of providing combination therapy is still under
debate (step up, step down approach). Effective and ben-
efical combinations are: HCQ+MTX+SAS, MTX+SAS,
LEF+MTX or CSA+MTX.
Treatment with biological agents is recommended for
patiens with persistent disease activity despite DMARDs
treatment. In the past years, three biological response
modifiers capable of neutralising TNF alpha have been approved for patiens with active RA (infliximab, etanercept,
adalimumab). In combination with methotrexate or alone
they produced significant improvement in all measures in
disease activity and they also retard radiographfic progression. Careful evaluation which rules out the underlying
infections, including tuberculosis, should be performed.
Recently, anti CD20 therapy has been approved in patients with inadequate response (intolerance) to anti TNF
alpha treatment.
CONCLUSIONS: The activity of RA should be controlled as completelly as possible, as soon as possible and
as long as possible using DMARDs alone or in combinations. Patients with persistent activity despite DMARDs
therapy are candidates for biological treatment of RA with
or without methotrexate.
SMOKING OF TOBACCO AND CANNABIS IN STUDENTS OF SEVERAL
UNIVERSITIES IN CZECH REPUBLIC AND ITALY
Alena Trojackovaa, Lenka Spacilovab, Hana Klusonovab, Peter Visnovskyc
a
Office of the Governement of the Czech Republic, Prague
Department of Biological and Medical Science, and
c
Department of Pharmacology and Toxicology Charles University in Prague, Faculty of Pharmacy in Hradec Kralove,
Czech Republic
b
Key words: Tobacco/Cannabis/University students
BACKGROUND: The use of both licit and illicit
drugs is a great social, health, economic, legal and politic problem worldwide. In recent years, mainly Central/
Eastern European countries have reported stable increase
in the use of these substances. In the Czech Republic,
most surveys on smoking and illicit drug use have been
restricted to elementary and secondary school students
(e.g. ESPAD). However, research has revealed that some
university students smoke tobacco and experiment with
illicit drugs of abuse both worldwide.
METHODS: The aim of this study was to evaluate
experience and opinions of university students on smoking of tobacco and/or cannabis in several Czech and
Italian university students, to estimate the prevalence rates
and to examine changing patterns among these students,
and to compare results with previous local, multi-regional
and international studies among university students. The
evaluation of the use was performed through the questionnaire-based survey. This survey was supplemented by
toxicological urinalysis.
RESULTS: In academic years 2002/3 – 2005/6, the
questionnaire-based survey was performed at thirteen faculties of eight universities in seven regions of the Czech
Republic. Anonymous standardized questionnaires were
personally administered to students during their registration at the beginning of the academic year or during seminars with obligatory participation of students.
A total number of 3.484 university students completed
and returned the distributed questionnaires. The number
of females was higher (more than 70 %) than that of
males. Average age of students was 20.8 years (range:
18–48). 8.6 % and 20.2 % of Czech university students
were regular or occasional tobacco smokers, respectively.
The life-time prevalence of illicit drug use was 51.4 %. The
most frequently used and available illicit drug of abuse
was marihuana with the life-time prevalence 49.6 % of
students, followed by hashish (14.2 %). We found sex differences in both licit and illicit drug use. Illicit drug use
as well as regular tobacco smoking was reported by higher
proportion of males than females. A significant positive
association between financial situation and drug use of
students was found. Students with greater financial support smoked tobacco and used illicit drugs more often and
more frequently than their indigent colleagues.
In 2004, the questionnaire-based survey was supplemented by toxicological urinalysis with the main aim to
distinguish regular users of illicit addictive substances
among university students from the students who had only
experimented with these substances before entering the
university. Urine specimens were anonymously collected
from the students of Charles University in Prague, Faculty
of Pharmacy in Hradec Kralove. Cannabinoids (THC),
methylenedioxymethamphetamine (MDMA) and methamphetamine (MET) were searched for by simple, onestep, immunochromatografic assay for the rapid qualitative
detection. Preliminary positive analytical results were then
confirmed by gas chromatography/ mass spectrometry.
150 urine specimens were collected, 67 students (i.e. almost one third of addressed students) refused to participate in the study. The average age of students was 21.7
years (range: 20–29). The number of females was higher
(74.7 %) than that of the males (25.3 %). The presence of
THC was detected in 6 samples (i.e. 4.0 %), no evidence
of MDMA and MET was found. A significant sex difference was estimated in the use of THC, male samples being
more positive than the female ones. The samples’ positive
findings were lower than the life-time prevalence of cannabis, MDMA and MET obtained from the questionnairebased study in the same group of university students. This
discrepancy can be explained by several factors; mainly by
the high number of students refusing participation in the
study and the supposed irregular pattern of cannabis use
in the group of pharmacy students (the use of other illicit
drugs was very low in this group of students).
95
Tab. 1. Prevalence of use of marihuana in 1995–1999
and in 2002–2006 in %.
University
FPTUL
FTT UL
PdFU HK
FaFUK HK
FaFVFU Brno
LFUP Olomouc
Total
1995–1999
42.1
52.6
27.2
27.8
31.4
36.5
31.7
2002–2006
54.2
53.6
51.7
45.4
41.7
39.6
49.6
FaFUKHK – Farmaceutická fakulta v Hradci Králové,
Univerzita Karlova v Praze; FaF VF UBrno – Farmaceutická fakulta, Veterinární a farmaceutická univerzita
v Brně; FPTUL – Pedagogická fakulta, Technická univerzita Liberec; LFUP Olomouc – Lékařská fakulta, Univerzita Palackého v Olomouci; Pd FUHK – Pedagogická
fakulta, Univerzita Hradec Králové
In Italy, in our study in University of Camerino, in
2006 28.2 % students smoked tobacco regularly.
CONCLUSIONS: With regards to the results of both
questionnaire-based survey and toxicological urinalysis, it
can be concluded that in the Czech Reublic more than one
half of university students have tried illicit addictive substances during their life. However, these illicit substances
(mainly cannabis) belong to the group of substances with
acceptable health and social risk and students had usually
tried them mainly before entering the university.
EFFECT OF DEXAMETHASONE AND BETAMETHASONE ON
11β-HYDROXYSTEROID DEHYDROGENASE TYPE 2 EXPRESSION IN HUMAN
CHORIOCARCINOMA JEG3 CELLS
Zuzana Vackova, Antonin Libra, Martina Ceckova, Petr Pavek, Frantisek Staud
Department of Pharmacology and Toxicology, Faculty of Pharmacy in Hradec Kralove, Charles University in Prague, Czech
Republic
Key words: 11β-Hydroxysteroid dehydrogenase/JEG3/glucocorticoids/RT-PCR
BACKGROUND: Placental 11β-hydroxysteroid dehydrogenases type 2 (11β-HSD2) is a NAD+-dependent
oxidase which catalyses the conversion of endogenous
glucocorticoids (cortisol and corticosterone) into their
inactive keto-derivates (cortisone and 11-dehydrocorticosterone, respectively)1,2. The activity of 11β-HSD2 in the
placenta is believed to be the key regulatory mechanism
that prevents the excessive transplacental passage of glucocorticoids from mother to fetus.
The expression and activity of placental 11β-HSD2
is not constant during gestation and displays marked
variability among species3–5. The difference is presumably caused by distinct regulation of 11β-HSD2 by endogenous compounds, particularly sex steroid hormones6.
Moreover, several recent studies suggested that 11β-HSD2
is subjected to regulation by antenatal therapy with synthetic glucocorticoids. However, there is some controversy
among the results obtained from different experimental
96
Fig. 1. The effect of DXM and BTM on the expression
of 11β-HSD2 in JEG3 cells. Incubation periods
were 12 (A), 24 (B) and 48 (C) hours.
models7–9. In humans the effect of glucocorticoids on
11β-HSD2 expression was previously studied in cultured
human placental trophoblast cells 9 and preterm human
placenta obtained from mothers who received betamethasone treatment before delivery10. In our study we used human choriocarcinoma JEG3 cells to investigate the effect
of dexamethasone (DXM) and betamethasone (BTM) on
the expression of 11β-HSD2. The changes in 11β-HSD2
mRNA expression were analyzed by quantitative real-time
RT-PCR.
METHODS: Human choriocarcinoma JEG3 cells
purchased from the European Cell Culture Collection
(ECACC; Salisbury, Wiltshire, UK) were maintained
in EMEM culture medium (Sigma, St.Luis, Missouri,
USA) supplemented with 10% fetal bovine serum, 2mM
L-glutamine, 1% NEAA and 1mM sodium pyruvate.
Cells seeded on 12-well plates at a density of 1.2x105
were exposed to DXM (ICN Biomedicals Inc., Aurora,
Ohio, USA) or BTM (Sigma, St.Luis, Missouri, USA)
in concentrations ranging from 10–9 to 10–6 nM for 12,
24 or 48 hours. Controls were treated similarly but without the addition of glucocorticoids. All the experiments
were carried out in fenol red free EMEM medium (Gibco
Invitrogen, Paisley, UK). Total RNA isolation and reverse
transcription were performed as described previously11.
Real-time RT-PCR analysis was performed on iCycler
iQ (Bio-Rad, Hercules, CA) with HotStart Taq DNA
polymerase (AB gene, Epsom, UK). Primers for amplification of segments of target genes were designed using
the Vector NTI Suite software (Infomax, Bethesda, MD,
USA) and are given in Table 1. Expression of 11β-HSD2
was calculated using previously described mode12 and expressed as a percentage of control.
RESULTS: Our results revealed that the effect of synthetic glucocorticoids on the mRNA expression of 11βHSD2 is time and concentration dependent. In the case of
BTM we observed marked but not significant increase in
11β-HSD2 mRNA expression at low BTM concentrations
followed by slight drop of 11β-HSD2 at higher concentrations of BTM. The induction of 11β-HSD2 was particularly apparent in cells treated with BTM for 12 (Fig. 1A).
24- and 48-hour incubations seemed to cause inhibition
rather than induction of 11β-HSD2, especially at high
BTM concentrations (Fig. 1B, C). In DXM treated JEG3
cells the expression of 11β-HSD2 tended to decrease with
rising concentration of DXM and length of incubation
(Fig. 1A, B, C).
CONCLUSIONS: Antenatal glucocorticoid therapy has been suggested to affect the barrier function of
11β-HSD2 (ref.8,9). This phenomenon could be of high
clinical importance since glucocorticoids are often administered to pregnant women to treat either the mother
or the fetus13, 14. In baboons, prenatal administration of
BTM lead to increased expression of 11β-HSD2 in the
placenta8. These results indicate the presence of a potent
regulatory mechanism which protects the fetus in the
environment of high glucocorticoid levels. Nevertheless,
inverse results were obtained in ewes premedicated with
single or repeated dose of DXM (ref.7). In the study with
cultured human placental trophoblast cells van Beek et
al. demonstrated that DXM enhances the transcription
and activity of 11β-HSD2 in the concentration and time
dependent manner9. On the other hand, no changes in
11β-HSD2 expression were observed in preterm placentas
obtained from women premedicated with BTM (ref.10).
By employing an experimental model of human placental
JEG3 cells we tested the influence of DXM and BTM on
11β-HSD2 mRNA expression. We noted a tendency to depression of 11β-HSD2 transcription with increasing steroid concentration and incubation period, however, these
changes were not statistically significant. Considering
the necessity to understand regulatory mechanisms of
97
Table 1. Sequences of primers used in real-time RT-PCR.
Gene
NCBI sequence
Sequence 5’→3’
Product length
(bp)
h β2M
NM_004048
CGT GTG AAC CAT GTG ACT TTG TC (f)
CAT CTT CAA ACC TCC ATG ATG C (r)
85
h 11β-HSD2
NM_000196
TGG CTG CTT CAA GAC AGA GTC (f)
CGT AGC GAG TGC AGG AAC TG (r)
150
11β-HSD2 in the placenta and their possible impact on
healthy development of a new individual, further investigation in this field is required.
ACKNOWLEDGEMENT
The work was supported by grant 102/2006/C/FaF from
Grant Agency of Charles University, Czech Republic.
REFERENCES
1. Albiston AL, Obeyesekere VR, Smith RE, Krozowski ZS. Cloning
and tissue distribution of the human 11 beta-hydroxysteroid dehydrogenase type 2 enzyme. Mol Cell Endocrinol 1994; 105:R11–7.
2. Zhou MY, Gomez-Sanchez EP, Cox DL, Cosby D, Gomez-Sanchez
CE. Cloning, expression, and tissue distribution of the rat nicotinamide adenine dinucleotide-dependent 11 beta-hydroxysteroid
dehydrogenase. Endocrinology 1995; 136:3729–34.
3. Staud F, Mazancova K, Miksik I, Pavek P, Fendrich Z, Pacha J.
Corticosterone transfer and metabolism in the dually perfused rat
placenta: effect of 11beta-hydroxysteroid dehydrogenase type 2.
Placenta 2006; 27:171–80.
4. Pepe GJ, Babischkin JS, Burch MG, Leavitt MG, Albrecht ED.
Developmental increase in expression of the messenger ribonucleic acid and protein levels of 11beta-hydroxysteroid dehydrogenase types 1 and 2 in the baboon placenta. Endocrinology 1996;
137:5678–84.
5. Shams M, Kilby MD, Somerset DA, et al. 11Beta-hydroxysteroid dehydrogenase type 2 in human pregnancy and reduced expression in
intrauterine growth restriction. Hum Reprod 1998; 13:799–804.
6. Sun K, Yang K, Challis JR. Regulation of 11beta-hydroxysteroid
dehydrogenase type 2 by progesterone, estrogen, and the cyclic
adenosine 5’-monophosphate pathway in cultured human placental
and chorionic trophoblasts. Biol Reprod 1998; 58:1379–84.
7. Kerzner LS, Stonestreet BS, Wu KY, Sadowska G, Malee MP.
Antenatal dexamethasone: effect on ovine placental 11beta-hydroxysteroid dehydrogenase type 2 expression and fetal growth.
Pediatr Res 2002; 52:706–12.
8. Ma XH, Wu WX, Nathanielsz PW. Gestation-related and betamethasone-induced changes in 11beta-hydroxysteroid dehydrogenase
types 1 and 2 in the baboon placenta. Am J Obstet Gynecol 2003;
188:13–21.
9. van Beek JP, Guan H, Julan L, Yang K. Glucocorticoids stimulate the expression of 11beta-hydroxysteroid dehydrogenase type 2
in cultured human placental trophoblast cells. J Clin Endocrinol
Metab 2004; 89:5614–21.
10. Johnstone JF, Bocking AD, Unlugedik E, Challis JR. The effects
of chorioamnionitis and betamethasone on 11beta hydroxysteroid
dehydrogenase types 1 and 2 and the glucocorticoid receptor in preterm human placenta. J Soc Gynecol Investig 2005;12: 238–45.
11. Novotna M, Libra A, Kopecky M, et al. P-glycoprotein expression
and distribution in the rat placenta during pregnancy. Reprod
Toxicol 2004;18: 785–92.
12. Pfaffl MW. A new mathematical model for relative quantification
in real-time RT-PCR. Nucleic Acids Res 2001; 29:e45.
13. Crowley P. Prophylactic corticosteroids for preterm birth. Cochrane
Database Syst Rev 2000:CD000065.
14. Namazy JA, Schatz M. Pregnancy and asthma: recent developments. Curr Opin Pulm Med 2005; 11:56–60.
DEGRADATION OF HYALURONAN SAMPLES WITH THE ADDITION
OF ASCORBIC ACID, CU (II), Fe (II) AND Mn (II) IONS
Katarina Valachovaa, Ladislav Soltesa, Peter Gemeinerb, Katarina Bauerovaa
a
Institute of Experimental Pharmacology, SAS, 84104 Bratislava, Slovak Republic
Institute of Chemistry, SAS, 84538 Bratislava, Slovak Republic
b
Key words: Hyaluronan/Viscosimetry/Metal ions
BACKGROUND: Hyaluronan (HA) is a polysaccharide composed of D-glucuronic acid and N-acetylglucosamine linked with β-1,4 a β-1,3 bonds. It is present
in synovial fluid, where it acts as a lubricant by preventing friction between bones. In healing osteoarthritis it is
applied in the form of viscosupplementation. Ascorbate
is well-known for its antioxidant properties in vivo, but in
vitro in the presence of metal ions, e.g. copper or iron ions
it behaves as a prooxidant.
METHODS: Dynamic rotational viscometry of 10 HA
samples (20 mg/8 mL of 0.15 M NaCl) was applied. All
the measurements were carried out at 25 ± 0.1 °C. The
samples viscosity was monitored in 3 min intervals for
up to 5 h at a rotational spindle speed of 180 rpm. In the
next series of experiments the efficacy of ascorbate at the
concentration 100 μM to HA samples was tested.
RESULTS: Addition of ascorbate resulted in an instant change of the HA solution η value. Viscosity of
98
the HA solutions could be reduced much more by trace
amounts of metal cations, i.e. Cu(II) and Fe(II) ions in
the presence of ascorbate. The tested metal ions concentrations were 0.1, 1.0, 5.0 μM CuCl2, 0.5, 5.0, 25.0 μM
FeCl2 and 0.5, 5.0 μM MnCl2 in the system HA+ascorbate.
In contrast, Mn(II) ions known for their ability to inhibit
lipid peroxidation were demonstrated to be also effective
in inhibiting HA samples degradation.
CONCLUSIONS: When tested only hyaluronans,
increasing of their viscosity, i.e. rheopexy was observed.
Ascorbate acts as a powerful reducing agent, so it reduces
traces of transition metals present in the samples. Under
aerobic conditions a ternary system comprising HA mac-
romolecules, ascorbate and the traces of Fe(II) or Cu(II)
ions reveal gradual decrease of the viscosity of the HA
solution as a result of the fragmentation/degradation of
HA macromolecules. Among the tested metal ions, Cu(II)
ions plus ascorbate are found to be the most powerful HA
degrading system. HA samples of higher molar mass were
degraded more intensively compared to HA samples of
lower molar mass.
ACKNOWLEDGEMENT
Supported by the VEGA grants 2/5002/5 and 2/7028/27
and the grants APVV-51-017905.
RESVERATROL AND ITS POTENTIAL ANTICANCER EFFECT
Zuzana Vidova, Lenka Varinska, Jan Mojzis
Department of Pharmacology, Faculty of Medicine, P.J. Safarik University, Kosice, Slovak Republic
Keywords: Resveratrol/Angiogenesis/Cancer
BACKGROUND: Resveratrol (3,4’,5-trihydroxystilbene) is a natural polyphenol, synthesized by a wide variety of plant species, including aliments such as grapes,
peanuts, mulberries, in response to injury, UV irradiation
and fungal attack. It was identified in 1963 as the active
constituent of the dried roots of Polygonum cuspidatum,
used since ancient times against suppurative dermatitis, gonorrhea, favus, athlete’s foot and hyperlipemia1.
Chemoprevention is a promising approach to control human cancer. Resveratrol has been shown to have a potent
chemopreventive effect in multiple carcinogenesis models2. It affects a broad range of intracellular mediators
involved in the initiation, promotion and progression of
cancer3. Resveratrol, a red wine polyphenolic compound,
is able to inhibit several key events of the angiogenic process such as proliferation and migration of endothelial cells
and vascular smooth muscle cells. Moreover, the expression of two major proangiogenic factors, vascular endotelial growth factor (VEGF) and matrix metalloproteinase-2
(MMP-2) was observed4.
METHODS: The colorimetric MTT test was used to
analyze the cytotoxic effect of resveratrol using human
cancer cell lines (HeLa, CEM, Jurkat, MDA, MCF-7,
A-549) at various concentrations (10–4–10–8 mol.L–1) of
resveratrol. Ability of resveratrol to inhibit colony formation of cancer cells was observed by colony formation assay. The potential antiangiogenic effect of resveratrol was
determined using the migration of human umbilical vein
endothelial cells (HUVECs). Matrix metalloproteinases
(MMP-2 and MMP-9) activity was assessed by gelatine
zymography.
RESULTS: Significant cytotoxic effect of resveratrol
on cancer cells was detected at concentration 10–4 mol.L–1
(Table 1). Promising antiangiogenic activity was observed
at concentrations of resveratrol from 10–4 to 10–7 mol.L–1.
Resveratrol significantly inhibited activity of MMP-9 at
concentrations 10–4–10–7 mol.L–1 and MMP-2 at concentrations 10–4–10–5 mol.L–1. Antiangiogenic activity of resveratrol was examined by MTT test using HUVECs. Low
cytotoxicity against HUVECs was observed (Table 1).
Table 1. Effect of reveratrol on the viability [%] of different cancer cell lines and HUVECs.
Concentration
of resveratrol (mol.L–1)
A549
MDA
MCF
Jurkat
HeLa
CEM
HUVEC
10–4
73.1*
47.4**
22.0***
19.0***
14.2***
10.9***
59.6**
10–5
86.2
97.1
97.3
83.0*
91.4
71.0*
97.6
–6
10
89.1
98.2
98.9
91.1
94.6
84.6
100.0
10–7
97.6
99.7
99.5
91.6
99.0
95.9
100.0
10–8
100.0
100.0
100.0
96.0
100.0
90.2
100.0
99
inhibited migration of endothelial cells at concentrations 10–4–10–6 mol.L–1. Finally, resveratrol also inhibited
the activity of matrix metalloproteinase-9 (MMP-9) and
matrix metalloproteinase-2 (MMP-2) at concentrations
10–4–10–7 mol.L–1 and 10–4–10–5 mol.L–1, respectively.
ACKNOWLEDGEMENT
Financial support by the grants VEGA 1/3365/05, VVGS
6/2006 and VEGA 1/4236/07 is greatfully acknowledged.
REFERENCES
Fig. 1. Resveratrol.
CONCLUSIONS: We observed antiproliferative effect of resveratrol on cancer and endothelial cells. At
the highest (10–4 mol.L–1) concentration, significant effect was observed in all cell types examined. At 10–5 mol.
L–1, significant cytotoxicity was observed only in Jurkat
and CEM leukemic cells. Furthermore, resveratrol also
1. Gusman J, Malonne H, Atassi G. A reappraisal of the chemopreventive and chemotherapeutic properties of resveratrol. Carcinogenesis
2001; 22:1111–7.
2. Dong Z. Molecular mechanism of the chemopreventive effect of
resveratrol. Mutat Res 2003; 523–524:145–50.
3. Signorelli P, Ghidoni R. Resveratrol as an anticancer nutrient :
molecular basis, open questions and promises. J Nutr Biochem
2005; 16: 449–66.
4. Oak M, Bedoui J, Schini-Kerth V. Antiangiogenic properties of
natural polyphenols from red wine and green tee. J Nutr Biochem
2005; 16:1–8.
CHALCONES AND THEIR POTENTIAL ANTIANGIOGENIC EFFECTS
Lenka Varinskaa, Robert E. Verloopb, Pal Perjesic, Jan Mojzisa, Pieter Koolwijkb
a
Department of Pharmacology, Faculty of Medicine, P. J. Safarik University, , Kosice, Slovak Republic
Department for Physiology,VU University Medical Center, Amsterdam, The Netherlands
c
Institute of Pharmaceutical Chemistry, Faculty of Medicine, University of Pecs, Pecs, Hungary
b
Key words: Angiogenesis/Chalcones/Endothelial cells/In vitro
BACKGROUND: Antiangiogenic treatment is presently one of the strategies for fighting cancer. Endothelial
cells play a pivotal role in the process of angiogenesis.
Numerous bioactive plant compounds have recently been
tested for their potential clinical application. One of the
most frequently studied substances are flavonoids. We
focused our research work on chalcones that are the first
intermediates in biosynthesis of flavonoids. Chalcones are
unique templates that are associated with several biological processes including antiinflammatory, antimicrobial,
antifungal, chemopreventive and cytotoxic effects1. In recent years, the interest of research groups has raised to
use these compounds as the antiangiogenic agents2–4. To
test the antiangiogenic properties in vitro, we have studied
the effect of chemical analogues of naturally occurring
chalcones.
METHODS: Human foreskin microvascular endothelial cells (HMVEC) were used in in vitro tube formation
model (described by Koolwijk et al.5, 6) to study the effects
of chalcones on angiogenesis. The simultaneous addition
of vascular endothelial growth factor (VEGF) and tumour
necrosis factor α (TNFα) resulted in the outgrowth of
tubular structures invading the fibrin matrix and forming a capillary network (Fig. 1A). Briefly, in this model
we tested the ability of human microvascular endothelial
cells to invade a three-dimensional human fibrin matrix
and form capillary-like tubular networks. An important
feature of this model is that it does not allow endothelial
cells to proliferate, thereby excluding an antiproliferative
effect of the studied compounds as the main cause of their
antiangiogenic action. Instead, cells migrate and invade
the underlying matrix.
RESULTS: According to results obtained from this
model we divided the tested compounds into three groups.
In the first group the compounds possessed cytotoxic effect at the tested concentration 100 μM (Fig. 1B). In contrast, in the second group a significant inhibition in the
formation of tubular structures was observed (Fig. 1C).
The morphology of the endothelial monolayer covering
the fibrin matrix was not affected. The compounds in the
third group inhibited tube formation significantly but the
tubes were still present. It was observed that only the cells
inside the tubes were dead (Fig. 1D).
CONCLUSIONS: In conclusion, we observed different effects of newly synthesized chalcones on the ability of
human microvascular endothelial cells to invade a threedimensional fibrin matrix and form capillary-like tubular
structures. The next study will be focused on elucidating
100
A
C
B
Control
Control
Q510 10-4M
Q797 10-4M
D
Q766 10-4M
Fig. 1. Capillary-like tube formation is influenced by newly synthesized chalcones. HMVEC cultured on the top of
a three-dimensional fibrin matrix were stimulated with 25 ng.mL–1 VEGF and 10 ng.mL–1 TNFα (A), or with
VEGF and TNFα in the presence of 100 μM Q510 (B), Q797 (C), and Q766 (D). After 6 days of culture
representative phase contrast photographs were taken. Data shown are representative of 3 experiments performed.
the mechanism of action of these compounds. Present
research is focusing on chalcone-induced repression of
proteolytic enzymes and inhibition of proliferation.
ACKNOWLEDGEMENT
The authors thank Ester M. Weijers for excellent technical assistance.
REFERENCES
1. Go ML, Wu X, Liu XL. Chalcones: an update on cytotoxic and
chemoprotective properties. Curr Med Chem 2005; 12:481–99.
2. Lee YS, Lim SS, Shin KH, Kim YS, Ohuchi K, Jung SH. Anti-angiogenic and anti-tumor activities of 2‘-hydroxy-4‘-methoxychalcone.
Biol Pharm Bull 2006; 29:1028–31.
3. Nam NH, Kim Y, You YJ, Hong DH, Kim HM, Ahn BZ. Cytotoxic
2‘,5‘-dihydroxychalcones with unexpected antiangiogenic activity.
Eur J Med Chem 2003; 38:179–87.
4. Zhu XF, Xie BF, Zhou JM, Feng GK, Liu ZC, Wei XY, Zhang FX,
Liu MF, Zeng YX. Blockade of vascular endothelial growth factor
receptor signal pathway and antitumor activity of ON-III (2‘,4‘dihydroxy-6‘-methoxy-3‘,5‘-dimethylchalcone), a component from
Chinese herbal medicine. Mol Pharmacol 2005; 67:1444–50.
5. Koolwijk P., van Erck MG, de Vree WJ, Vermeer MA, Weich
HA, Hanemaaijer R et al. Cooperative effect of TNFalpha, bFGF,
and VEGF on the tube formation of tubular structures of human
microvascular endothelial cells in a fibrin matrix. Role of urokinase
activity. J Cell Biol 1996; 132:1177–88.
6. Kroon ME, Koolwijk P, van Goor H, Weidle UH, Collen A, van
der Pluijm G et al. Role and localization of urokinase receptor in
the formation of new microvascular structures in fibrin matrices.
Am J Pathol 1999, 154:1731–42.
CHANGES IN VASCULAR REACTIVITY OF DIABETIC RATS WITH AND WITHOUT
SULODEXIDE TREATMENT
Robert Vojtko, Silvia Liskova, Viera Kristova
Department of Pharmacology, School of Medicine, Comenius University, Bratislava, Slovak Republic
Key words: Vessel/Diabetes/Sulodexide/Noradrenaline/Acetylcholine
BACKGROUND: Endothelial dysfunction and other
vascular disorders of diabetes mellitus play a crucial role
in its pathogenesis. Sulodexide is known as an agent with
antithrombotic, fibrinolytic and endothelium-protective
properties. The objective here was to evaluate a possible
influence of sulodexide on vascular reactivity of diabetic
rats vessel segments by responses to noradrenaline and
acetylcholine in vitro.
METHODS: Adult male Wistar rats were divided into
4 groups: control, a group with streptozotocine induced
diabetes mellitus (dose 25 mg/kg i.p. administered in
3 consecutive days), a group with i.p. administered sulodexide (dose 100 LS units/kg daily) and last group with
diabetes and administration of sulodexide. Sulodexide
treatment lasted 8 weeks. Thereafter, renal arteries were
isolated and their preparations were tested in vitro by perfusion method. Vessel segments were perfused by Tyrode’s
solution and stimulated to contractions by consecutive
increasing bolus doses of noradrenaline (50, 100, 500 ng,
1, 3, 6, 10 μg). Thereafter, vessel segments were precontracted by incubation with stable concentration of noradrenaline (10–5 M) and relaxations were tested by bolus
dose of acetylcholine (20 μg).
RESULTS: Evaluated vessels did not exhibit a significant difference in contraction amplitudes between
the groups for any dose of noradrenaline. Relaxation responses of precontracted renal arteries were expressed by
101
average value 16.7 % in the control group, while those in
the diabetic group were reduced to 6.8 % (signicant difference). The arteries of diabetic animals treated by sulodexide showed the ability of relaxation sligtly shifted towards
to control by 11.9 % (non-significant difference).
CONCLUSIONS: The results suggest that diabetes
mellitus in our model did not influence the contractions
of rat vessel segments compared to control and sulodexide
group. On the other side, streptozotocine induced diabetes significantly impaired the endothelium-dependent
relaxation and sulodexide treatment of diabetic rats was
linked with partial recovery of arterial relaxation values
towards to control group, but this remained in a range
under significance.
ACKNOWLEDGEMENT
Financial support by the grant Vega 1/2292/05 is gratefully acknowledged.
PYCNOGENOL® DOSE-DEPENDENTLY DECREASED FASTING AND
POSTPRANDIAL BLOOD GLUCOSE CONCENTRATIONS AND IMPROVES NERVE
CONDUCTION VELOCITY IN STZ- INDUCED DIABETIC RATS
Diana Yaghia, Stanislava Jankyovaa, Jan Klimasa, Jana Navarovab, Svorad Stolcb,
Zoltan Goldenbergc, Pavol Kucerac, Stefan Matyasa
a
Department of Pharmacology and Toxicology, Faculty of Pharmacy UK, 83232 Bratislava, Slovak Republic
c
1st Neurology Clinic, Faculty of Medicine, Comenius University, 813 69 Bratislava, Slovak Republic
b
Key words: Neuropathy/Pycnogenol®/Oxidative stress/Blood glucose/Diabetes
BACKGROUND: Diabetes mellitus is a metabolic disorder characterized with hyperglycemia and insufficiency of secretion or effect of endogenous insulin. While
exogenous insulin and other medications can control
many aspects of diabetes very well, some complications,
including injury of vascular system, retina, lens, kidney,
skin and peripheral nerves, are common and extremely
costly, thus limiting the longevity and quality of diabetic‘s
life1. The oxidative stress plays a major role in pathogenesis of diabetes and its progression. Diabetes is usually
accompanied with increased production of free radicals2
or impairment of antioxidant defenses1. Not only oxygen
radicals are involved in the etiology of diabetes, but there
is an extensive evidence that implicate these radicals in
the pathogenesis of diabetes complications3, including peripheral neuropathy4. Peripheral neuropathy is one of the
most frequent and troublesome complications of diabetes
mellitus. It is a major reason for morbidity and mortality
among diabetic patients. The most responsible pathogenic
factor for development of peripheral neuropathy is probably long-term hyperglycemia5 and increased oxidative
stress. Increased production and/or attenuated clearence
of reactive oxygen species (ROS) can be postulated as
a possible mechanism, which might contribute both to defective nerve blood supply and to endoneurinal oxidative
damage6–8. The animal experiments showed, that oxidative
stress (as the developer of diabetic neuropathy) could be
deacreased with antioxidants and that they can prevent
or reverse hyperglycaemia-induced nerve dysfunction5.
Pycnogenol® is a direct strong antioxidant extracted from
French maritime pine bark (Pinus maritima)9. Treatment
with Pycnogenol® significantly reduces blood glucose in
diabetic rats and restores the elevated expression of some
prooxygenic enzymes10. The aim of our study was to determine whether Pycnogenol® exhorts on fasting and
postprandial blood glucose concentrations and on motor
nerve conductivity in STZ-induced diabetic rats.
METHODS: In our experiment we used male Wistar
rats. Rats were divided randomly into five groups. Four
groups (n = 8) were made diabetic by streptozotocin in
dose 25 mg/kg i.p. for three days. The fifth group (n = 8)
was used as a control. After 14 days of induction of diabetes three groups were treated with Pycnogenol® once daily
(in doses 10, 20 and 50 mg/kg p.o.) for 8 weeks. In the 8th
102
+++
40
+++
xx
hladina glc (mmol/l)
35
*
*
30
K
D
DP10
DP20
DP50
*
25
20
***
***
15
10
5
0
preprandiálna
postprandiálna
Fig. 1. Fasting and postprandial serum glucose levels (KControl, D-Diabetes, DP10 – Diabetes treated with
Pycnogenol® in dose 10 mg/kg, DP20 – dose 20
mg/kg, DP50 – dose 50 mg/kg )+++ (p < 0.001) K vs
D fasting and postprandial, *** (p < 0.001) D vs
DP20 , *** D vs DP50 fasting ; xx (p < 0.01) DP10 vs
DP50 fasting ; * (p < 0.05) D vs DP10 fasting, D vs
DP10 postprandial, D vs DP20 postprandial. The
values are expressed as mean ± standard error of
the mean.
++
xxx
60
velocity (m/s)
50
*
***
Pycnogenol® in the dose of 10 mg/kg reduced fasting and
postprandial blood glucose significantly (p < 0.05). Also
Pycnogenol® in the dose of 20 mg/kg significantly decreased fasting (p < 0.001) and postprandial (p < 0.05)
blood glucose compared to diabetic rats. Pycnogenol® in
the dose of 50 mg/kg significantly reduced fasting blood
glucose (p < 0.001) compared to diabetic group and to
treated rats with dose 10 mg/kg (p < 0.01) (Fig. 1). In
diabetic rats the nerve conduction velocity (NCV) was significantly (p < 0.01) decreased compared to control group.
Compared to diabetic group, treatment with Pycnogenol®
dose-dependently elevated the depressed NCV: in dose
10 mg/kg does not increase NCV significantly, while other
both, 20 mg/kg and 50 mg/kg increased NCV significantly
(in dose 20 mg/kg p < 0.05, in dose 50 mg/kg p < 0.001)
(Fig. 2).
CONCLUSIONS: The fasting and postprandial blood
glucose was elevated in diabetic and treated rats compared
to control group. Pycnogenol® significantly reduced elevated fasting and postprandial blood glucose compared
to diabetic group in all doses. There were no significant
changes in fasting and postprandial blood glucose between
different doses of Pycnogenol®, except in 50 mg/kg dose
compared to 10 mg/kg in fasting blood glucose (p < 0.01).
In diabetic group the NCV was decreased compared to
control group. Pycnogenol® in the doses of 20 mg/kg and
50 mg/kg dose-dependetly increased the NCV.
40
ACKNOWLEDGEMENT
Financial support by the grant VEGA SR č.2/5129/25,
VEGA SR 2/5009/27 is gratefully acknowledged.
30
20
10
REFERENCES
0
K
D
DP 10
DP 20
DP 50
Fig. 2. The nerve conduction velocity (K- Control,
D- Diabetes, DP 10 – Diabetes treated with
Pycnogenol ® in dose 10 mg/kg, DP 20 – dose
20 mg/kg, DP 50 – dose 50 mg/kg ) xxx (p <
0.001) DP10 vs DP50, *** (p < 0.001) D vs DP50;
* (< 0.05) D vs DP20; ++ (p < 0.01) K vs D. The
values are expressed as mean ± standard error of
the mean.
week the fasting and postprandial glucose concentrations
were measured using the commercial glucose kit (Sigma,
St. Louis, MO, USA). Then the motor nerve conductivity
was measured under halothane anaesthesia. The results
were evaluated with nonequal Student’s t-test with mean
± standard error of the mean.
RESULTS: Compared to control group, the fasting
and postprandial serum blood glucose was elevated in
diabetic rats significantly (p < 0.001). The fasting and
postprandial blood glucose in treated rats was significantly increased compared to control group (p < 0.001)
in all doses. Compared to diabetic group, treatment with
1. Maritim AC, Sanders RA, Watkins III JB. Diabetes, oxidative
stress, and antioxidants: A Review. J Biochem Mol Toxicol 2003;
17:24–38.
2. Baynes JW, Thorpe SR. Role of oxidative stress in diabetic complications: A new perspektive on an old paradigm. Diabetes 1991;
48:1–9.
3. Oberley LW. Free radicals and diabetes. Free Radic Biol Med 1988;
5:113–24.
4. Van Dam PS. Oxidative stress and diabetic neuropathy: pathophysiological mechanisms and treatment perspectives. Diab Metab Res
Rev 2002; 18:176–84.
5. Dobretsov M, Romanovsky D, Stimers JR. Early diabetic neuropathy: Triggers and mechanisms. World J Gastroenterol 2007;
13:175–91.
6. Van Dam PS., Bravenboer B. Oxidative stress and antioxidant treatment in diabetic neuropathy. Neurosci Res Commun 1997; 21:
41–8.
7. Van Dam PS., Van Asbeck BS, Erkelens DW, Mrxs JJM, Gispen
WH, Bravenboer B. The role of oxidative stress in neuropathy and
other diabetic complications. Diabetes Metab Rev 1995; 11:181–
92.
8. Greene DA, Stevens MJ, Obrosova I, Feldman EL. Glucose- induced oxidative stress and programmed cell death in diabetic neuropathy. Eur J Pharmacol 1999; 375:217–23.
9. Liu X., Zhou HJ, Rohdewald P. French pine bark extract
Pycnogenol® dose-dependetly lowers glucose in type 2 diabetic
patiens. Diab care 2004; 27:839.
10. Maritim AC, Dene BA, Sanders RA, Watkins III JB. Effects of
Pycnogenol® treatment on oxidative stress in streptozotocin-induced diabetic rats. J Biochem Mol Toxicol 2003; 17:193–8.
103
THE INFLUENCE OF CANNABINOID METHANANDAMIDE ON THE ACTIVITY
OF HEPATIC CYP2D2 IN RATS
Lucia Zahradnikovaa, Ondrej Zendulkaa,b, Jan Juricaa,c, Eva Hadasováa
a
Department of Pharmacology, Faculty of Medicine, Masaryk University, 662 43 Brno, Czech Republic
Department of Preventive Medicine, Faculty of Medicine, Masaryk University, 662 43 Brno, Czech Republic
c
Department of Biochemistry, Faculty of Medicine, Masaryk University, 662 43 Brno, Czech Republic
b
Key words: (R)-(+)-Methanandamide/CYP 2D2/Isolated perfused rat liver
BACKGROUND: Anandamide, an endogenous ligand
for brain cannabinoid CB1 receptor, produces many behavioral effects similar to those of Δ9-tetrahydrocannabinol
(THC), the main psychoactive ingredient in marijuana.
Reinforcing effects of THC have been demonstrated in
experimental animals1. (R)-(+)-Methanandamide is a synthetic long-lasting arachidonylethanolamide (anandamide)
analog that is metabolically stable and displays higher affinity for the cannabinoid receptor. Four chiral congeners
of anandamide have been synthesized and evaluated for
their ability to bind to the cannabinoid receptor and their
pharmacological potency. The aim of this work was to
investigate the effect of repeated administration of (R)(+)-methanandamide on cytochrome P 450 (CYP450)mediated biotransformation in rat. The enzymatic system
of CYP450 is a part of phase I of enzymatic biotransformation. It consists of many isoenzymes characterized
by specific substrates and organ localization. CYP450 is
involved in metabolism of many endogenous as well as
exogenous substrates. Both, the induction and inhibition
of specific CYP450 isoenzymes are important in terms
of the efficacy or toxicity of drugs that are substrates for
this system. CYP450 metabolizes many drugs including
the major psychoactive cannabinoid present in marijuana
THC (ref.2). We have studied the influence of (R)-(+)methanandamide on rat liver CYP2D2 isoenzyme using
dextromethorphan as a specific marker3.
METHODS: The experiment was carried out on male
Wistar rats (weighing 250 ± 40 g, BioTest, Konarovice,
Czech Republic) with free access to food and water,
housed under the controlled conditions. Animals were
randomly divided into 2 groups with 10 animals. Control
group (CG) animals was treated with TocrisolveTM 100
(Tocris Cookson Ltd.) (1mg/kg/day intraperitoneally)
and (R)-(+)-methanandamide group (R-MG) animals
was treated with the drug at the dose of 1 mg/kg/day
intraperitoneally in TocrisolveTM 100 (Tocris Cookson
Ltd.) for 7 days. The rat liver was isolated from donors
using a standard surgical technique. Cannulas were introduced into the portal vein and inferior cava vein, the
Fig. 1. The influence of 7-day pretreatment with R-methanandamide on the
activity of CYP2D2 isoenzyme in rats. Columns represent concentrations of DOR in the perfusate (means and SD), in the 30th, 60th
and 120th minute of perfusion (*significant difference p<0,05) in
CG and R-MG.
104
liver was shortly washed out by a tempered (38 °C) saline
which was changed for the perfusion medium (120 ml
of Williams medium E) equilibrated with a mixture of
95 % O2 and 5 % CO2 in a short time. The recirculating
perfusion apparatus was constructed according to the
principles originated by Hugo Sachs Gnith (SRN). After
20 min pre-perfusion, a specific marker – dextromethorphan (DEM) (10.0 mg.L–1) was added as a bolus into
the perfusion medium. Samples of perfusate (1.0 mL)
were collected at the 30th, 60th and 120th min of perfusion and were stored at –75 °C until analysis. Quantitative
analysis detecting DEM and it’s metabolite dextrorphan
(DOR) was performed by HPLC method (Shimadzu,
Japan). For statistical calculations F-test and Student’s
t-test (Microsoft Excel 2000) were used, p < 0.05 considered to be statistically significant difference.
RESULTS: In the CG, the levels of CYP2D2 – dependent metabolite DOR were significantly higher compared to the R-MG (Fig. 1). The levels of DOR were
increased in 30th (42 %), 60th (25 %) and 120th (30 %)
minutes in CG than in R-MG. Levels of the parent drug
DEM was significantly higher during the whole perfusion
in R-MG animals.
CONCLUSIONS: The model used is suitable for investigation on the activity of hepatic CYPs 450 and bio-
transformation processes represented by the isolated and
perfused liver. The main advantage of this model, in comparison with other methods used for measuring the CYP
activity, is that conditions resemble physiological situation
in the organism. As we expected, the activity of CYP2D2
differed due to pretreatment with methanandamide. In
our experiments the influence of this compound on rat
CYP2D2 was inhibitive and metabolic activity of studied
isoenzyme CYP2D2 in male rats was significantly higher
in the control group than in (R)-(+)-methanandamide
group.
ACKNOWLEDGEMENT
Financial support by the grant MSM 002162240 is gratefully acknowledged.
REFERENCES
1. Justinova Z, Solinas M, Tanda G, Redhi GH, Goldberg SR. The
Endogenous Cannabinoid Anandamide and Its Synthetic Analog
R(+)-Methanandamide Are Intravenously Self-Administered by
Squirrel Monkeys. J Neurosci 2005; 5645–50.
2. Bornheim LM, Kelly KY, Chen B, Correia A. The effect of cannabidiol on mouse hepatic microsomal Cytochrome P450-dependent Anandamide metabolism. Biochem Biophys Res Commun
1993; 197:740–6.
3. Dostalek M, Sliva J, Hadasová E. Effect of methamphetamine and
fluoxetine combination on metabolic activity of CYP2D1 in isolated perfused liver. Homeostasis 2003; 42:86–90
THE INFLUENCE OF POLYPHENOLIC EXTRACT FROM ANDRE AND BLAUER
PORTUGAISER GRAPE BYPRODUCTS ON THE ACTIVITY OF LIVER CYP1A2
ISOENZYME
Ondrej Zendulkaa, b, Lucia Zahradníkovaa, Jan Juricaa, c, Jiri Totusekb
a
Department of Pharmacology, Masaryk University, 662 43 Brno, Czech Republic
Department of Preventive Medicine, Masaryk University, 662 43 Brno, Czech Republic
c
Department of Biochemistry, Masaryk University, 662 43 Brno, Czech Republic
b
Key words: Polyphenolic extracts/Wine byproduct/Isolated perfused rat liver
BACKGROUND: The enzymatic system of cytochrome P 450 (CYP450) is one of the most important
enzymatic systems participating in drug metabolism.
Both, the induction and inhibition of specific CYP450
isoenzymes are important in terms of the efficacy or toxicity of drugs that are substrates for this system. Within
the genetic polymorphism as an essential definition of
activity of CYP450 many other factors influence liver
oxidative metabolism (i.e. age, sex, administered drugs,
diseases). Nutrition state, components of diet and exposure to chemical elements belongs to the most important
exogenous ones. Polyphenolic compounds are biologically active substances with many protective effects1, 2.
Polyphenols are widely distributed in plants and are
a common part of human diet. As well as other compo-
nents of diet polyphenols can also influence the activity of
liver CYP450 and can interfere with administered drugs
metabolized by hepatic isoenzymes of CYP450 (ref.3).
The aim of our present study was to investigate the influence of polyphenolic extract from grape byproducts and
hypercholesterolemic diet on the activity of hepatic CYP
1A2 isoenzyme in rats.
METHODS: Grape byproducts (mixture of Andre
and Blauer Portugaiser) were obtained from Vinné sklepy Lechovice s.r.o. (Czech Republic). Byproducts were
dried (20 °C), then homogenised and extracted with
methanol and water (w/w=1/1.5). Solvents were evaporated and extract was diluted in water. Total polyphenolic
content was determinated with Folin- Ciocalteu’s reagent
and gallic acid as standard. The total polyphenolic con-
tent is presented as gallic acid equivalents (GAE).The
experiment was carried out on male Wistar rats (weighing
450 ± 100 g, BioTest, Konarovice, Czech Republic) with
free access to food and water. Animals were randomly
divided into 3 groups with 8 animals. Control group animals (CG) were treated with water and fed with standard
diet. Control hypercholemic group (CHG) animals were
treated with water and fed with hypercholesterolemic diet
(1.7% of cholesterol) as well as animals from polyphenolic extract group (PEG), which were treated with extract
by intragastric sond in the dose of 5 mg GAE/kg/day
for 40 days. The rat liver was isolated from donors by
a standard surgical technique. Cannula was introduced
into the portal vein and the liver was shortly washed
out by a tempered (38 °C) saline which was changed for
the perfusion medium (120 ml of Williams medium E)
equilibrated with a mixture of 95 %O2 and 5 % CO2 in
a short time. After 20 min pre-perfusion, specific marker – phenacetine (PHEN) (10.0 mg.L–1) was added as
a bolus into the perfusion solution. Samples of perfusate
(1.0 mL) were collected in the 30th, 60th, 90th and 120th
min of perfusion and were stored at –75 °C until analysis.
Quantitative analysis was performed after extraction of
markers and their metabolites from perfusion solution by
HPLC methods (Shimadzu, Japan). Data were analysed
using F-test and unpaired Student’s t-Tests.
RESULTS: Hypercholesterolemic diet had no effect
on the activity of hepatic CYP1A2 isoenzyme and concentrations of marker PHEN and its metabolite paracetamole (PAR) were comparable in cholesterol and control
animals. The administration of extract caused induction
105
of CYP1A2 isoenzyme and was manifested as increased
levels of PAR in perfusion medium in PEG animals in the
30th minute of perfusion.
CONCLUSIONS: As we predicted, hypercholesterolemic diet had no effect on the activity of hepatic
CYP1A2. Our opinion is that the only effect of long-lasting hypercholesterolemic diet on the activity of CYP can
be caused by steatosis of hepatocytes and thus a decrease
of their metabolic activity. Administration of polyphenolic
extract caused induction of CYP1A2 isoenzyme and was
manifested as increased levels of PAR in perfusion medium. One should expect that inducers of CYP isoenzyme
would cause in the treated animals an increase in metabolite levels and decrease of marker levels compared to the
controls. In our case however administration of polyphenolic extract lead to statistically significant increase of
both, PAR and PHEN in the 30th minute of perfusion.
In our opinion, this can be caused by the changes in the
binding capacity of liver for PHEN, as liver of control rats
showed rather a big portion of PHEN bound, too.
ACKNOWLEDGEMENT
Supported by the grant GACR 525/06/1757.
REFERENCES
1. Hao HD et al. Mechanisms of Cardiovascular Protection by
Resveratrol. J Med Food 2004; 7:290–8.
2. Curin Y et al. Cellular Mechanisms of the Protective Effect of
Polyphenols on the Neurovascular Unit in Strokes. Cardiovasc
Hematol Agents Med Chem 2006; 4:277–88.
3. Moon YJ et al. Dietary flavonoids: Effects on xenobiotics and carcinogen metabolism. Toxicol in Vitro 2005; 20:187–210.
THE RELATIONSHIP BETWEEN POTASSIUM ION CHANNELS AND DEFENCE
REFLEXES OF THE AIRWAYS
Martina Sutovska, Gabriela Nosalova
Department of Pharmacology, Jessenius Faculty of Medicine, Sklabinska street 26, 03753, Martin, Slovak Republic
Key words: Cough/Potassium channels/Airways/Sensory nerves/Pinacidil
BACKGROUND: It is generally accepted that potassium ion channels, widely expressed on sensory nerves,
playing the main pathophysiological as well as regulatory
role in defence reflexes of the airways. K+ATP ion channels
link membrane excitability to metabolism. The several
endogenous agonists (e.g. intracellular nucleotides and
calcitonin – gene related peptide; CGRP) activate K+ATP
resulting to hyperpolarization and relaxation, a response
that is similar to effect of K+ATP openers pinacidil or mogusteine1. Pinacidil relaxed guinea pig isolated trachea
tone was either spontaneous or induced by a range of
airway contractile mediators (histamine, PGF2α, leucotrienes or carbachol) enforcing in asthma pathogenesis2.
The ASM relaxation produced by pinacidil was selectively
blocked by the antidiabetic glibenclamide. Morita and
Kamei3 demonstrated suppression of capsaicin – induced
cough in guinea pigs on mogusteine administration prevented by glibenclamide. BK+Ca have been shown to be
present on ASM from a number of species and may be
important regulators of the membrane potential and intrinsic tone of smooth muscle4. Furthermore, selective
opener of BK+Ca, NS 1619, inhibits the activity of myelinated and nonmyelinated sensory nerves of guinea pigs
airways in vitro as well as citric acid – induced cough5.
BK+Ca probably represent a common mechanism, which
mediate inhibition of airway reflexes evoked by number
of sensory nerves receptors (e.g. μ – opioid or 5-HT1).
The aim of presented study was complex evaluation of
potassium ion channels role in defence reflexes of the
airways.
106
Fig. 1. The antitussive effects of pinacidil (A) and NS 1619 (B) on the number of citric acid- induced cough assessed
0.5, 1, 2 and 5 h after s.c. administration of pinacidil (1 mg.kg–1 b.w.) or inhaled NS 1619 (200 μM for 10 min).
The effect of selective blockers glibenclamide (dose 3 mg.kg–1 b.w. i.p., A) and TEA (dose 8 mg.kg–1 b.w., i.p.,
B) on antitussive effects of potassium channels openers. The values labelled as N represents baseline measurements results before any agents application. ∗ p< 0,05; ∗∗ p< 0,01 vs. N values.
METHODS: The adult male TRIK strain guinea pigs,
weighing 150-350 g used in the studies, were obtained
from Department of Experimental Pharmacology, Slovak
Academy of Sciences, Dobra Voda, Slovakia and were
housed in approved animal holding facility. The function
of both, ATP or calcium – sensitive potassium ion channels was tested on two groups of guinea pigs: A, “Agonist”
group of animals (n= 8) – received K+ATP opener pinacidil
in the dose 1 mg.kg -1 b.w. by subcutaneous route (s.c.)
or were exposed to an aerosol of BK+Ca agonist NS 1619
(200 μM) for 10 min interval. B, “Antagonist/agonist”
group of animals received combined medication consisting of pretreatment by potassium ion channels antagonist
glibenclamide (K+ATP) in dose 3 mg.kg-1 b.w. intraperitoneally, i.p. or tetraethylammonium – TEA (BK+Ca) in dose
8 mg.kg-1 b.w. i.p., followed by agonist medication after 20
min interval described previously. The “negative control
group” of guinea pigs received solvents used in experiments – DMSO (dimethylsulfoxide) in the dose 1 ml.kg
-1
b.w. i.p. and was tested under same conditions. For comparative purposes in measurement of cough response the
codeine in the dose 10 mg.kg-1 b.w. i.p. was applied to
“positive control group” of guinea pigs.
Method of chemically induced cough reflex: Awaken
guinea pigs were individually placed in a bodypletysmograph box (HSE type 855, Hugo Sachs Elektronik,
Germany) and were exposed to citric acid aerosol in
concentration 0.3 M for 3 min. The number of coughs
evaluated on the basis of sudden enhancement of expiratory flow accompanied by a typical cough movement was
counted. The cough response was measured in all groups
of animals before administration of vehicle, codeine, ion
channels agonists or medication by antagonists/agonists
(N) and than after their application in confirmed time
intervals (0.5, 1, 2 and 5 h).
The evaluation of ASM reactivity – in vitro conditions:
Four strips (two of tracheal and two of pulmonary smooth
muscle) obtained from each animal were placed into
30 ml organ bath chambers filled with Krebs-Henseleit’s
buffer, saturated by pneumoxide (95 % O2 + 5 % CO2),
the temperature was held at 36 ± 0.5 °C, maintained at
pH 7.5 ± 0.1. The amplitude of isometric contraction
(mN) of tracheal and pulmonary smooth muscle to cumulative doses of contractile mediator acetylcholine and
histamine (Sigma – Aldrich, Slovakia) at concentrations
10–8–10–3 mol.L–1 were used for reactivity evaluation.
The evaluation of ASM reactivity – in vivo conditions:
The ASM reactivity was expressed as specific airway resistance (R.V) values calculated by Pennock6. Conscious
adult male TRIK strain guinea pigs were individually
placed in a double chambers bodypletysmograph box for
laboratory animals consisting of head and body chambers.
The nasal airflow is registered in head chamber and the
thoracic airflow in body chamber. The value of specific
airway resistance either extent of bronchoconstriction is
proportional to phase difference between nasal and thoracic respiratory airflow.
All data are expressed as mean ± SEM. Significance of
difference was statistically evaluated using the Student’s ttest for paired data. P< 0,05 was considered significant.
RESULTS: Evaluation of registered data showed that
both potassium ion channels agonists reduced the number
of citric acid-induced coughs (Fig. 1). Pinacidil, K+ATP
opener, administered s.c. lead to statistically significant
decline of citric acid-induced cough values in all measured
time intervals. Pretreatment by glibenclamide partially
reduced pinacidil influence on number of coughs. Agonist
of BK+Ca, agent NS 1619, significantly decreased number
of coughs in 0.5 and 1 h time interval in comparison
with baseline measurement. This effect was completely
abolished by pretreatment of selective BK+Ca inhibitor,
tetraethylammonium, before NS 1619 application. Our
experiments demonstrated that both potassium ion
channels agonists influenced specific airway resistance
in conscious guinea pigs. Pinacidil administration caused
decline of R.V in whole time intervals of experiment. NS
1619 significantly reduced values of this parameter in 0.5
and 1 h time interval in comparison with baseline measurement. The effects of both K+ ion channels openers
were completely antagonized by pretreatment of selective inhibitors of these structures activity. We found that
administration of K+ATP agonists resulted in statistically
significant decrease of tracheal smooth muscle response
on cumulative doses of contractile mediators’ histamine
and acetylcholine. The reactivity of pulmonary smooth
muscle on pinacidil application was reduced mildly and
furthermore, BK+Ca agonist NS 1619 did not changed the
pulmonary smooth muscle contractile response.
CONCLUSIONS: Thus, it is presumable that BK+Ca
and K+ATP ion channels are involved in the mechanisms of
107
cough, antitussive activity of more agents as well as airway
defence reflexes based on smooth muscle reactivity and
represent an excellent target for the new drugs treated
airways diseases.
ACKNOWLEDGEMENT
The study was supported by Grant of Agency for Science
(VEGA) No. 1/3375/06 and Grant of Ministry of Health No.
2005/13-MFN-05.
REFERENCES
1. Rodrigo GC, Standen NB. ATP-sensitive potassium channels. Curr
Pharm Des 2005; 11:1915–40.
2. Nielsen–Kudsk JE. Potassium channel modulation: a new drug
principle for regulation of smooth muscle contractility. Studies on
isolated airways and arteries. Dan Med Bull.1996; 43:429–47.
3. Morita K, Kamei J. Involvement of ATP-sensitive K(+) channels
in the anti-tussive effect of moguisteine. Eur J Pharmacol. 2000;
395:161–4.
4. Zhu FX, Zhang XY, Olszewski MA, Robinson NE. Mechanism of
capsaicin-induced relaxation in equine tracheal smooth muscle.
Am J Physiol Lung Cell Mol Physiol, 1997; 273:997–1001.
5. Fox AJ, Barnes PJ, Venkatesan P, Belvisi MG. Activation of large
conductance potassium channels inhibits the afferent and efferent
function of airway sensory nerves in the guinea pig. J Clin Invest.
1997; 99:513–9.
6. Pennock BE, Cox CP, Rogers RM, Cain WA, Wells JH. A noninvasive technique for measurement of changes in specific airway
resistance. J Appl Physiol 1979; 46:399–406.
THE ANTITUSSIVE AND IMMUNOMODULATORY ACTIVITIES OF PECTIN
AND HEMICELLULOSE POLYSACCHARIDES ORIGINATED
FROM SALVIA OFFICINALIS L.
Martina Sutovskaa, Gabriela Nosalovaa, Sona Franovaa, Peter Capekb, Vera Hribalovac
a
Department of Pharmacology, Jessenius Faculty of Medicine, Sklabinska 26, 037 53, Martin, Slovak Republic
Institute of Chemistry, Slovak Academy of Sciences, Dubravska 9, 845 38, Bratislava, Slovak Republic
c
National Institute of Public Health, Srobarova 48, 100 42, Praha 10, Czech Republic
b
Key words: Cough/Sage/Antitussive activity/Plant polysaccharides/Cats
BACKGROUND: Salvia officinalis L., so-called sage,
is a popular medicinal plant worldwide cultivated for a
large scale of bioactive compounds, e.g. diterpenoids, flavonoids, phenolic acids or phenolic glycosides, many of
which possess a variety of biological activities including
antioxidant, antiplatelet, antitumor and antiviral. Sage is
used in traditional medicine for treatment of numerous
range of diseases including cough, for culinary purposes
and antioxidant properties1. The ethanolic tinctures and
decoctions have been long known for their curing effects
in various inflammations of oral cavity, digestive and intestinal tracts. Despite of the well known antitussive effect of sage, their experimental evaluation is still missed
in actual scientific literature. Though the curing effects
of sage have often been ascribed to a low – molecular
mass components contained in the herb in relative large
amounts, but high – molecular mass compound polysaccharides have not been investigated for biological activities as well as for compositional analyses of individual
polymers2,3. According above mentioned, our study was
aimed on immunomodulatory and antitussive abilities of
sage polysaccharides.
METHODS: Salvia officinalis L. was purchased from
Slovakofarma, Medicinal Plants, Malacky, Slovakia. The
crude polysaccharide fractions, rich in arabinogalactans
associated with rhamnogalacturonans (A), pectin material containing smooth and hairy regions (B) and glucuronoxylan polymers (D) have been obtained by sequential
extraction with distilled water, ammonium oxalate and
1M potassium hydroxide from aerial parts of Salvia offici-
108
Fig. 1. The antitussive activity of Salvia officinalis L.
polysaccharide complex (A) evaluated on the
base of mechanically – induced cough parameters changes compared with cough suppression
on codeine and dropropizine administration.
nalis L. (ref. 4). The crude polysaccharide fractions were
purified by anion – exchange and gel chromatography.
Individual polysaccharide fractions were examined for
their immunomodulatory activity in the in vitro comitogenic thymocyte test. The antitussive activity of water–soluble
polysaccharide complex A (dose 50 mg kg–1 body wt.)
was tested on non-anaesthetized cats with body weight
between 2000–4000 g. Previous to mechanical induction
of cough reflex, surgical implantation of chronic endotracheal cannula was executed under general anesthesia using
thiopental administered intraperitonealy. Experimental
cough was elicited by five consequents stimulation of nylon fiber 0.35 mm in diameter inserted to laryngopharyngeal and tracheobronchial airway mucous area through
implanted cannula. The cough-related parameters were
recorded on a Biograph 12-03 electromanometer as intraluminal pressure changes. We evaluated the number
of cough efforts (NE), the cough frequency (NE min–1),
intensity of maximal cough effort and intensity of cough
attacks during expiration (IME+, IA+) and inspiration
(IME-, IA-)5. Prior to application of the tested substance,
the induced-cough parameters were recorded to have the
control values for each animal. The same parameters in
the 0.5, 1, 2 and 5 hours intervals were registered after
administration of agent. For comparative purposes ability of opiate agonist codeine in the dose 10 mg kg–1 body
wt. and cough suppressive effect of peripherally acting
dropropizine (50 mg kg-1 body wt.) were followed under
same conditions. All cough – related data are expressed as
mean ± SEM. Significance of difference was statistically
evaluated using the Wilcoxon – Wilcox test. P < 0,05 was
considered significant.
RESULTS: Water–extractable polysaccharides (A)
were rich in arabinose, galactose, 3-O-methylgalactose and
glucose, while the acidic polysaccharides (B) were consisted mainly of galacturonic acid along with rhamnose,
arabinose and galactose residues indicating the pectin
type of polysaccharides with rhamnogalacturonan type I
backbone. Hemicellulose polysaccharides (D) solubilized
with 1M potassium hydroxide solution were rich mainly in
xylose, whereas glucuronic acid, arabinose and galactose
were present as minor components indicating the presence of arabinoglucuronoxylan or glucuronoxylan type of
polymers. All polysaccharide fractions tested possessed
the capacity to induce rat thymocyte proliferation in the
order D>B>A. Besides, fraction D possessed a significant
comitogenic effect, and Sicomit/Simit ratio 3-4 indicates
potential adjuvant properties of this carbohydrate material. The Salvia officinalis L. polysaccharide complex A
significantly decreased number of the cough efforts (NE)
and the intensity of inspiratory and expiratory cough attacks (IA– and IA+) of mechanically – induced cough reflex from both, laryngopharyngeal and tracheobronchial
areas of airways. Antitussive activity tests with some classic drugs, narcotic codeine and non-narcotic dropropizine
performed under same experimental conditions demonstrated antitussive potency of sage polysaccharide complex
two fold exceeded cough suppressive effect of peripheral
antitussive agent and effectiveness only by 13 % lower
than opioid receptors agonist (Fig. 1). Furthermore, side
effects resulting from application of sage extract have not
been observed.
CONCLUSIONS: We can report that all polysaccharides showed a broad molecular – mass distribution pattern. All fractions of isolated polysaccharides possessed
ability to increase rat thymocyte proliferation, which
confirmed their immunological property. Moreover, water–soluble polysaccharide fraction A showed impressive
cough suppression without any side effects in conscious
cats. This result supported also our previous studies in
which antitussive activities of more plant polysaccharides
rhamnogalacturonan type were verified6. The high antitussive effect and rare adverse reactions are the reason
why the Salvia officinalis L. polysaccharides might be considered as prospective drugs able to influence the cough
reflex.
ACKNOWLEDGEMENT
The study was supported by VEGA No. 2/6131/26 and
APVT 2/017304 grants.
REFERENCES
1. Dittmann K, Gerhauser C, Klimo K, Hamburger M. HPLC-Based
Activity Profiling of Salvia miltiorrhiza for MAO A and iNOS
Inhibitory Activities. Planta Med 2004; 70:909–13.
2. Janicsak G, Hohmann J, Zupko I, Forgo P, Redei D, Falkay G,
Mathe I. Diterpenes from the aerial parts of Salvia candelabrum
and their protective effects against lipid peroxidation. Planta Med
2003; 69:1156–9.
3. Chavkin C, Sud S, Jin W, Stewart J, Zjawiony JK, Siebert DJ, Toth
BA, Hufeisen SJ, Roth BL. Salvinorin A, an active component of
hallucinogenic sage salvia divinorum is a highly efficacious kappaopioid receptor agonist: structural and functional considerations.
J Pharmacol Exp Ther 2004; 308:1197–203.
4. Capek P, Hribalova V. Water-soluble polysaccharides from Salvia officinalis L., possesses immunomodulatory activity. Phytochemistry
2004; 65:1983–92.
5. Nosalova G, Strapkova A, Korpas J, Criscuolo D. Objective assessment of cough suppressants under normal and pathological
experimental conditions. Drugs Exptl Clin Res 1989; 15:77–81.
6. Nosalova G, Capek P, Sutovska M, Franova S, Matulova M.
Antitussive Active Polysaccharides from 7. Ornamental-Medicinal
Plants. Floriculture, Ornamental and Plant Biotechnology, 2006;
4:471–80.
109
AUTHOR INDEX
Adamcova, Michaela 57, 69, 86
Adameova, Adriana 3, 27
Andrasina, Igor 19
Anzenbacher, Pavel 51, 53
Anzenbacherova, Eva 51, 53
Bajuszova, Zuzana 4, 6, 27, 42
Balaz, Matej 74
Bartosik, Tomas 7, 64
Bartosikova, Lenka 7, 64
Bartosova, Ladislava 9, 24, 31, 45
Bauerova, Katarina 97
Bela, Radek 58, 60
Benedekova, Marta 66
Berc, Alexander 19
Blahos, Jaroslav 11
Blechova, Renata 12
Bobrovova, Zuzana 13
Borovickova, Lenka 87
Brabec, Viktor 51
Brcakova, Eva 14, 57
Brtko, Julius 14
Cacanyiova, Sona 65
Capek, Peter 107
Ceckova, Martina 16, 95
Ceral, Jiri 25
Cermanova, Jolana 14, 57
Cerna, Olga 81
Cerna, Pavla 17
Cerovsky, Vaclav 87
Cervinkova, Zuzana 14
Chladek, Jaroslav 14, 29, 35
Chovancova, Iveta 66
Cizmarikova, Martina 19
Curillova, Zuzana 68
Cygalova, Lenka 16
Daniel, Catherine 80
Dolezal, Tomas 73
Doppler, Edith 77
Drabikova, Katarina 89
Eybl, Vladislav 17
Farsa, Oldrich 64
Foltanova, Tatiana 21, 71
Franova, Sona 23, 107
Frydrych, Marek 9, 24, 31
Fucik, Vladimir 87
Fuksa, Leos 14, 57
Gajdosik, Andrej 39
Gajdosikova, Alena 39
Gazova, Andrea 46
Gemeiner, Peter 97
Gersl, Vladimír 69, 86
Goldenberg, Zoltan 101
Gonec, Tomas 45
Grim, Jiri 35
Grune, Tilman 91
Guncova, Ivana 69
Habalova, Viera 19
Habrdova, Vilma 25
Hadasová, Eva 103
Hajkova, Jitka 14, 57
Hamakova, Barbora 26
Harcarova, Anna 27, 66
Havlinova, Zuzana 29
Herout, Vladimir 47
Hlavackova, Veronika 11
Holanova, Katerina 9
Holeckova, Magdalena 13
Horakova, Lubica 91
Horka, Klara 9, 24, 31
Hrdina, Radomir 13
Hribalova, Vera 107
Hrncir, Tomas 80
Hroch, Milos 14, 35
Hruba, Sabina 33
Hruby, Radovan 33, 41
Hubl, Mojmir 13
Hudcovic, Tomas 80
Hudec, Roman 46
Hutter-Paier, Birgit 77
Jahodar, Ludek 44
Jancinova, Viera 89
Janicek, Frantisek 23
Jankyova, Stanislava 26, 37, 101
Janovsky, Martin 92
Jedlickova, Anna 81
Jurica, Jan 38, 103, 104
Juskova, Maria 39
Kaiserova, Helena 86
Kajo, Karol 75
Karpisek, Michal 45
Kerna, Valeria, jr. 33, 41
Klecakova, Jana 44
Klimas, Jan 4, 6, 26, 37, 42, 65, 101
Klusonova, Hana 94
Kmecova, Jana 26, 42, 43
Kohut, Anton 19
Kollar, Peter 45
Kolouchova, Gabriela 57
Koolwijk, Pieter 99
Kotolova, Hana 45
Kottova, Martina 44
Kotyzova, Dana 17
Kovacs, Laszlo 66
Kovarikova, Petra 86
Kozakova, Hana 80
Krcmar, Josef 24, 45
Krenek, Peter 4, 6, 27, 37, 42, 43,
65
Kriska, Milan 46
Kristek, Frantisek 65
Kristova, Viera 100
Krizanova, Ludmila 39
Kroslakova, Miroslava 4, 27, 42
Krsiak, Miloslav 73
Kubant, Pavel 47
Kubatka, Peter 75
Kubicek, Vladimir 92
Kucera, Pavol 101
Kucerova, Dana 4
Kucharska, Jarmila 3
Kunes, Martin 47
Kutschy, Peter 61, 68
Kuzelova, Magdalena 3, 27, 66, 71
Kuzmina, Galina 7, 64
Kvetina, Jaroslav 47
Kyselovic, Jan 4, 6, 26, 27, 65
Lamka, Jiri 58, 60
Landa, Leos 50
Lassanova, Monika 46
Laznicek, Milan 55
Laznickova, Alice 55
Libra, Antonin 16, 95
Lietava, Jan 21
Liskova, Silvia 100
Lotkova, Halka 14
Luza, Jiri 7, 64
Macek, Karel 25
Machova, Marketa 51
Mackovicova, Katarina 43, 65
Malik, Ivan 74
Martinkova, Jirina 14, 29, 35, 57,
84
Masek, Vlastimil 51
Matal, Jaroslav 53
Matyas, Stefan 26, 37, 101
Mazurova, Yvona 69
Melicharova, Ludmila 55
Mezencev, Roman 68
Micuda, Stanislav 14, 29, 57
Mirossay, Andrej 68
Mirossay, Ladislav 19, 61, 68
Mlada, Jana 58, 60
Mladenka, Premysl 13
Moessler, Herbert 77
Mojzis, Jan 61, 68, 98, 99
Mojzisova, Gabriela 61
Mokry, Juraj 63
Mokry, Petr 9, 24
Navarova, Jana 37, 101
Necas, Jiri 7, 64
Nobilis, Milan 47, 53
Nosal, Radomir 89
Nosalova, Gabriela 23, 33, 41, 63,
75, 105, 107
Nosalova, Viera 89
Ochodnicky, Peter 6, 42, 43, 65
Olvedy, Michael 65
Ondrejka, Igor 33, 41
Ondrejkova, Jana 43
Ondriasova, Elena 66
Opatrilova, Radka 9
Palicka, Vladimir 13, 25
Patockova, Jitka 77
Pavek, Petr 16, 95
Pavlik, Martin 7
Perjesi, Pal 99
Petrik, Milos 55
Petrikova, Margita 89
Pilatova, Martina 61. 68
Pin, Jean-Philippe 11
Plackova, Silvia 66
Plandorova, Jana 4
Pokorna, Pavla 84
Ponka, Premysl 86
Popelova, Olga 69, 86
Pot, Bruno 80
Pourova, Jana 44
Prezeau, Laurent 11
Priesolova, Elena 26
Prochaska, Juraj 71
Prochazkova, Michaela 73
Prokesova, Ludmila 73
Prokopova, Jana 29
Racanska, Eva 74
Raganova, Andrea 46
Rajec, Jan 46
Repa, Andreas 80
Repovska, Maria 68
Riedel, Rudolf 66
Sadlonova, Vladimira 75
Sarissky, Marek 61, 68
Sedivy, Josef 81
Selke-Krulichova, Iva 84
Schauer, Evelyn 77
Schwarzer, Martin 80
Simkova, Marie 35
Simunek, Tomas 69, 86
Slaninova, Jirina 87
Smidova, Ilona 47
Snirc, Vladimir 39
Soltes, Ladislav 97
Sotnikova, Ružena 89
Souckova, Martina 80
Soukupova, Marie 92
Spacilova, Lenka 94
Staud, Frantisek 14, 16, 57, 95
Stefek, Milan 39
Stepankova, Renata 80
Sterba, Martin 69, 86
Stolc, Svorad 101
Strnadova, Vera 9
Strosova, Miriam 91
Suchy, David 93
Suchy, Pavel 9, 31, 45
Sumbalova, Zuzana 3
Sutovska, Martina 23, 105, 107
Svec, Pavel 3, 21, 71
Svetlikova, Zuzana 25
Svoboda, Zbynek 47
Thurzo, Martin 21
Tlaskalova-Hogenova, Helena 80
Totusek, Jiri 104
Trojackova, Alena 94
Tumova, Ingrid 21, 71
Vackova, Zuzana 95
Vaja, Vaclav 6
Valachova, Katarina 97
Vaneckova, Jaroslava 35
Varinska, Lenka 98, 99
Vavrinec, Peter 43
Vavrova, Jaroslava 13
Veinlichova, Alena 53
Verloop, Robert E. 99
Vidova, Zuzana 98
Visnovsky, Jozef 23
Visnovsky, Peter 94
Vojtko, Robert 100
Voprsalova, Marie 44
Vorisek, Viktor 25
Voss, Peter 91
Wagnerova, Maria 19
Wiedermann, Ursula 80
Windisch, Manfred 77
Wronski, Robert 77
Yaghi, Diana 26, 37, 101
Zahora, Jiri 84
Zahradnikova, Lucia 103, 104
Zanvit, Peter 73
Zendulka, Ondrej 103, 104
Zivny, Pavel 25
BIOMEDICAL PAPERS
Volume 151, Supplement 1
Published semiannually
MK ČR E 12793
Published by Palacký University, Olomouc
Křížkovského 8, 771 47 Olomouc, IČO 61989592
Printed by Papírtisk, s. r. o.,
Lindnerova 5, 779 00 Olomouc
Olomouc 2007
ISSN 1213-8118

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