1

65. ČESKO-SLOVENSKÉ FARMAKOLOGICKÉ DNY

Sborník abstraktů

16. – 18. Září 2015 Praha, Česká republika

2

OBSAH

Obsah ....................................................................................................................................................... 1

Přednášky ................................................................................................................................................ 3

Postery – prezentovány 16.9.2015 ....................................................................................................... 15

Postery – prezentovány 17.9.2015 ....................................................................................................... 28

Vydavatel:

Farmakologický ústav 1. LF UK a VFN v Praze

Albertov 4, 128 00 Praha 2

ISBN 978-80-260-8027-5

3

PŘEDNÁŠKY

MEDICAL POTENTIAL OF SNAKE VENOMS Antoliková N.1, Chripková M.1, Petrilla V.2, Mojžiš J.3, Pirnik Z.1

1 University of Veterinary Medicine and Pharmacy, Institute of Human and Clinical Pharmacology, Košice 2 University of Veterinary Medicine and Pharmacy, Institute of Physiology, Košice 3 Pavol Jozef Šafárik University in Košice, Institute of Pharmacology

Snake venoms exhibit a multi-component character, representing a mixture of biologically active components; ions;

polysaccharides; protein, polypeptide, peptide toxins and enzymes specific to a wide range of tissue receptors. They are used

for diagnosis and the treatment of various human diseases in the cardiovascular, haematological, neurological and oncological

fields. In addition, with respect to its composition and chemical properties, its ability for immunogenicity is used in the

production of antisera. Medically the most important venom in contemporary literature has been particularly recorded in snakes

of the Elapidae and Viperidae family. In Europe the Viperidae family is the best known and most widely used. The main

component of the venom is a substance that affects blood coagulation and the release of histamine. In addition it contains, to a

lesser extent, cytotoxic agents. Great potential has been recorded in the integrins or disintegrins representing a group of

cysteine-rich peptides. It influences the process of wound healing, cancer metastasis and the formation of thrombosis. α2β1

integrin plays an important role, while the inhibition of this integrin-binding protein is used. An alternative anti-carcinogenic

mechanism consists of the influence of the NF-kB inhibition on the apoptotic process and the influence of the phosphorylation

reduction on the STAT3 signal pathway. The Elapidae family represents a large group of poisonous snakes, which are among

the most dangerous and venomous animals. Despite the threat they exhibit an interesting toxicological profile. It is assumed

that the cytotoxic potential is due to the presence of heat-stable forms of oxidase, which has very significant anti-proliferative

activity in human tumor cells. The apoptotic process is triggered through the effect of the caspase cascade. Thanks to molecular

or biological diversity and the ability to selectively affect different body functions, studying snake venoms therefore has great

potential and can be the basis for the development of new drugs.

Microbiota and drug metabolism Anzenbacher P.1, Matušková Z.1, Hudcovic T.2, Tlaskalová-Hogenová H.2, Anzenbacherová E.3

1 Department of Pharmacology, Faculty of Medicine and Dentistry, Palacky University Olomouc, Czech Republic, 2 Institute of Microbiology, Academy of Sciences, Prague, Czech Republic 3 Department of Medical Chemistry and Biochemistry, Faculty of Medicine and Dentistry, Palacky University Olomouc, Czech Republic

Microbiome is an omnipotent and versatile system of our microbial companions. As the majority of drugs are

administered per os, it may be expected that it is the intestinal microbiome which may possibly interfere with drug metabolism.

There are in the literature examples of reactions of drugs, mostly reductions, in which the role of intestinal microbiota has been

implicated. However, the majority of drug metabolism is based on oxidative biotransformations through cytochromes P450

which are present thorough the body incl. the intestine. Hence, there are two systems in the intestine which are participating in

drug metabolism: Intestinal drug metabolizing enzymes and intestinal microbiota. We will focus on the models (rats, mice) to

which the probiotic bacterium E. coli Nissle 1917 (EcN) has been administered and 1. its effect on liver and intestinal drug

metabolizing enzymes cytochromes P450 has been studied and 2. the effect of the EcN on the pharmacokinetics of model

drug, amiodarone, has been followed. Also, the difference between germ free and conventionally colonized mice in their ability

to metabolize drugs will be discussed. The authors gratefully acknowledge the financial support through the Grant Agency of

the Czech Republic grant P303/12/0535 and from the students´ project of the Palacky University IGA UPOL LF_2015_004.

Contribution of ABCB1 and CYP2D6 polymorphisms to the outcome of tamoxifen adjuvant treatment in

premenopausal women with breast cancer Argalácsová S.1,2, Slanař O.2, Vítek P.4, Tesařová P.1, Bakhouche H.2, Dražďáková M.3, Zima T.3, Pertuželka L.1

1 Department of Oncology, First Faculty of Medicine, Charles University and General Teaching Hospital, Prague, Czech Republic 2 Institute of Pharmacology, First Faculty of Medicine, Charles University and General Teaching Hospital, Prague, Czech Republic 3 Institute of Clinical Biochemistry and Laboratory Diagnostics, First Faculty of Medicine, Charles University and General Teaching

Hospital, Prague, Czech Republic 4 Institute of Radiotherapy, First Faculty of Medicine, Charles University and Hospital Na Bulovce, Prague, Czech Republic

Key words: P-glycoprotein; polymorphism; pharmacogenetics; tamoxifen; CYP2D6

Recent pre-clinical evidence suggests that the active metabolite of tamoxifen, endoxifen, is a substrate for efflux pump

P-glycoprotein (P-gp). The aim of our study was to evaluate, if the polymoprhisms within ABCB1 gene alter tamoxifen

adjuvant treatment efficacy in premenopausal women. Totally 71 premenopausal women with estrogen receptor positive breast

cancer indicated for tamoxifen adjuvant treatment were followed retrospectively for median period of 56 months. The gentic

polymorphisms of CYP2D6 and ABCB1 were analysed and potential covariates as tumor grading, staging, age at the diagnosis,

comedication, quantitative positivity of ER or PR were also evaluated. The patients carrying at least one variant allele in MDR1

rs1045642 had significantly longer time to event survival with hazard ratio of 0.69 (95% CI 0.21-2.31) compared to patients

carrying wild type allele. Non-significant trend was noted for better treatment outcome of patients carrying at least one variant

allele in the SNP rs2032582, while for the CYP2D6 polymorphism poor metabolizer phenotype resulted in worse outcome in

comparison to extensive metabolizers subjects with HR of 4.04 (95% CI 0.31-52.19). Similarly, patients using CYP2D6

inhibitors had non-significantly shorter time-to-event as compared to never users resulting in hazard ratio of 2.06 (95% CI

4

0.40-10.63). ABCB1polymorphisms may affect outcome of tamoxifen adjuvant treatment in premenopausal breast cancer

patiens. This factor should be taken into account in addition to the CYP2D6 polymorphism or phenotypic inhibition of CYP2D6

activity.

Charakterizácia kmeňových buniek z kostnej drene a tukového tkaniva metódou prietokovej cytometrie Bačkayová T., Gažová A., Lešková Z., Danišovič Ľ., Maďarič J., Musil P., Sprušanský O., Kyselovič J.

Farmaceutická fakulta Univerzity Komenského Bratislava, Katedra farmakológie a toxikológie; Lekárska fakulta Univerzity Komenského Bratislava, Ústav farmakológie a klinickej farmakológie; Oddelenie kardiológie a angiológie, Kardiologická klinika NÚSCH, a.s., a LF SZU,

Bratislava; Lekárska fakulta Univerzity Komenského Bratislava, Ústav lekárskej biológie, genetiky a klinickej genetiky

Najčastejšie skúmaným zdrojom kmeňových buniek je kostná dreň. Ako sľubný alternatívny zdroj sa ukazuje aj možnosť

využiť tukové tkanivo, v ktorom sa šetrným zákrokom získava veľké množstvo adipóznych kmeňových buniek. Cieľom práce

bolo porovnávanie povrchovej charakterizácie buniek izolovaných z kostnej drene a tukového tkaniva metódou prietokovej

cytometrie. Vzorky tukového tkaniva sme získali enzymatickou digesciou z lipoaspirátu a po ich kultivácii v 5% atmosfére

CO2 pri 37°C sme metódou prietokovej cytometrie zanalyzovali prvých päť pasáži. Vzorky kostnej drene sme získali od

pacientov s kritickou končatinovou ischémiou, ktorým bola indikovaná autológna transplantácia kmeňových buniek. Po

odobratí aspirátu z bedrovej kosti boli bunky premyté, erytrocyty zlyzované a hneď analyzové. Prietokovou cytometriou sme

u oboch zdrojov stanovili pozitivitu buniek na markery mezenchymálnych buniek (MSC) CD90, CD105 a CD73 a zároveň

negativitu na markery CD14, CD20, CD34 a CD45. U buniek z kostnej drene sme okrem čerstvých vzoriek obdobne

analyzovali aj tretiu pasáž kultivovaných buniek. Morfológiu buniek v rôznych pasážach sme pozorovali pomocou inverznej

mikroskopie. Priemerná viabilita buniek izolovaných z tukového tkaniva bola 90,67%. Množstvo buniek z tohto zdroja

pozitívnych na markery MSC stúpalo od nultej (50,20%) po štvrtú (95,20%) pasáž a zároveň pozitivita na markery CD14,

CD20, CD34 a CD45 postupne klesala od 24,69% v nultej pasáži až po 0% v piatej pasáži. Najväčšia pozitivita v priemere bola

na marker CD73 (93,13%). Pasážovaním získavali bunky natiahnutý vretenovitý tvar s jadrom a cytoplazmou. Priemerná

viabilita buniek izolovaných z aspirátov kostnej drene bola 92,88%. Pozitivita buniek na markery MSC sa pohybovala od

0,031% po 0,135%. Vzorka kultivovaných buniek v tretej pasáži obsahovala 56,124% buniek pozitívnych na markery MSC.

Morfológia kultivovaných buniek bola blízka bunkám izolovaných z tukového tkaniva. Mnohé štúdie charakterizovali a

porovnávali imunofenotyp kultivovaných kmeňových buniek z tukového tkaniva (ATSC) v skorých a neskorších pasážach a

spozorovali, že profil expresie ATSC sa počas kultivačného času mení. Expresia špecifických povrchových markerov CD29,

CD90 a CD166 sa počas kultivácie zvykne zvyšovať, zatiaľ čo expresia ďalších markerov znižovať (Baer et al., 2012). V našej

práci sme pozorovali tendenciu nárastu pozitivity ATSC na marker CD90, zatiaľ čo pozitivita na markery CD105 a CD73

narastajúcimi pasážami kolísala. Percentuálne zastúpenie buniek pozitívnych na MSC markery vo vzorkách kostnej drene bolo

v priemere 0,08±0,04%. Vo všetkých vzorkách prevládala pozitivita na markery buniek kostnej drene CD14, CD20, CD34 a

CD45 a pasážovaním stúpala pozitivita na markery MSC. Kultúry buniek kostnej drene v porovnaní s ATSC až po dlhšom čase

dosahovali 80% konfluenciu. Toto pozorovanie je v súlade so zisteniami Ahmed Lofty, aj keď ich štúdia prebiehala so vzorkami

experimentálnych zvierat (Lotfy et al., 2014). Metódou prietokovej cytometrie sme stanovovali prítomnosť povrchových

markerov mezenchymálnych buniek na bunkách vyizolovaných z tukového tkaniva a kostnej drene. Práca bola podporená

grantom VEGA 1/0905/14, APVV-14-0416, UK/541/2015

ABCB1 and OPRM1 polymorphisms alter maternal efficacy and neonatal safety of remifentanil in women

undergoing ceasaraen section Bakhouche H.1, Noskova P.2, Svetlik S.1, Bartosova O.1, Ulrichova J.2, Kubatova J.1, Marusicova P.1 , Parizek A.3, Blaha J.2,

Slanar O.1

1 Institute of Pharmacology, First Faculty of Medicine and General Teaching Hospital, Charles University in Prague, Albertov 4, 128 00

Prague 2, Czech Republic 2 Department of Anesthesia, Resuscitation and Intensive Medicine, First Faculty of Medicine, Charles University and General University

Hospital, Apolinářská 18, 128 51 Prague 2, Czech Republic 3 Department of Gynaecology and Obstetrics, 1st Faculty of Medicine, Charles University and General University Hospital in Prague,

Apolinarska 18, 128 51 Praha 2, Czech Republic.

Remifentanil is a rapid onset, ultra-short acting opioid that displays stabilizing effect on maternal circulation during

caesarean section in general anaesthesia while its effect on postnatal adaptation of the neonate are usually only modest. Women

undergoing general anesthesia for caesarian section were administred remifentanil bolus (1µg/kg i.v.) 30s prior to the induction

to standardized general anesthesia . The ABCB1 (rs2032582, rs1045642) and OPRM1 (rs1799971) polymorphisms were

analyzed from maternal peripheral blood. The basal hemodynamic and demographic parameters in the study population (n=54)

were similar in the subgroups. The median ± SD increase of systolic blood pressure at 5 minutes from the baseline was

practically completely abolished in homozygous carriers of ABCB1 variants in comparison with wild-type subjects -2.67 ±

25.0 vs. 16.57 ± 15.7 mmHg, p<0.05 for rs2032582, and 2.00 ± 23.9 vs 22.13 ± 16.8 mmHg, p<0.05, for rs1045642. There

was a trend towards better stabilization of the hemodynamic parameters in OPRM1 wild-type homozygous subjects in

comparison with carriers of the variant allele carriers. The neonatal safety was not statistically diferent among genotype

supgroups, however, clinical diference was clearly pronounced. While no neonate belonging to ABCB1 wild-type homozygous

or OPRM1 variant allele carrying mothers needed any resuscitative measure, 10.5% of the neonates belonging to OPRM1 wild-

type homozygous mothers received early resuscitative support similarly as 11.1%, and 12.5% of neonates belonging to mothers

carrying variants of rs2032582, and rs1045642, respectively. Significantly decreased stabilizing effects of remifentanil has

been observed in ABCB1 wild type mothers, while the adaptation of their neonates was clinically worse in ABCB1 variant

allele carriers. Similar trend was noted for OPRM1 wild-type homozygote mothers in both hemodynamic affects and neonatal

safety.

5

OPRM1 and ABCB1 polymorphisms and their effect on postoperative pain relief with piritramide BARTOŠOVÁ O.1, POLANECKÝ O.2, PERLÍK F.1, ADÁMEK S.2, SLANAŘ O.1

1 Institute of Pharmacology, First Faculty of Medicine, Charles University in Prague and General University Hospital in Prague, Czech

Republic 2 3rd Department of Surgery, First Faculty of Medicine, Charles University in Prague and University Hospital Motol, Prague, Czech Republic

Genetic factors contribute to the differential response to opioids. The aim of this study was to evaluate the association

between the rs1799971 (OPRM1) and rs1045642, rs2032582 (ABCB1) SNPs on postoperative pain relief with piritramide in

51 patients with patient-controlled analgesia. OPRM1 variants were associated with significant differences of sum of pain

intensity difference in the 0-6 hours postoperative period (SPID0-6), (F = 3.27, P = 0.029). Mean (SD) SPID0-6 was observed

in the 118GG 10.0 (4.4) mm, which was significantly different (P < 0.05) both from the 118GA 20.5 (7.4) mm and 118AA

22.9 (6.1) mm. The highest cumulative dose was recorded in 118GG 36.6 (6.1) mg, which was significantly (P < 0.05) different

both from the 118GA 31.3 (20.8) mg and 118AA 19.1(9.8) mg. The rate of opioid – induced adverse effects was 11 % in

118AA, 30 % in 118AG and 100 % in 118GG (P < 0.05). No significant effect of ABCB1 polymorfism on postoperative pain

relief with piritramide was proved. The variant OPRM1 118G allele is associated with decrease in acute postoperative pain

relief and increase in consumption of piritramide and adverse effects. This study has been supported by Charles University

project PRVOUK P25/LF1/2 and UNCE 204022.

MicroRNA as biomarkers of cardiovascular disease Gažová A.2, Polakovičová M.2, Eugen L.2, Kyselovič J.3, Kyselovičová O.2

1 Faculty of Medicine, Comenius University, Bratislava, Slovakia 2 Faculty of Physical Education and Sports, Comenius University, Bratislava, Slovakia 3 Faculty of Pharmacy, Comenius University, Bratislava, Slovakia

MiRNAs are single-stranded RNAs with approximately 22 nucleotides in length that repress the expression of specific

proteins by annealing to complementary sequences in the 3' untranslated regions of target mRNAs. There are estimated to be

up to 1000 miRNAs encoded by the human genome. Most miRNAs are transcribed as separate genes or as miRNA clusters

that are generated from large precursor transcripts. Numerous miRNAs display characteristic changes in expression during

pathogenesis of the heart and blood vessels and may serve as markers for cardiovascular disease progression. MiRNA have

been implicated in virtually every cardiovascular disorder in which they have been examined, including heart failure, cardiac

hypertrophy, remodeling after myocardial infarction, arrhythmias, atherosclerosis, atrial fibrillation, and peripheral disease.

Virtually all of the basic cellular processes involved in cardiovascular development and disease, such as cell proliferation,

differentiation, apoptosis, fibrosis, angiogenesis, and inflammation are subject to miRNA control (1,2). We analyzed plasma

levels concentration miRNA-29, miRNA-499, miRNA-1 and miRNA-133a with method real time polymerase chain reaction

in physiological – modern gymnastic, sport gymnastic and sport aerobic and pathological models – oncology treated patients

with or without work out. We analyzed plasma levels concentration miRNA-29, miRNA-499, miRNA-1 and miRNA-133a

with real time polymerase chain reaction. One of the most validated miRNAs with respect to its role in cardiovascular disease

in miRNA-29, which targets a broad collection of mRNAs encoding collagens and other extracellular matrix proteins involved

in fibrosis. Members of the miRNA-29 family are down-regulated in a variety of fibrotic disorders, which has been proposed

to result in excessive extracellular matrix production. Strategies to enhance miRNA-29 expression would be expected to

suppress fibrosis. Inhibition of miRNA-29 has been proposed as a way to enhance extracellular matrix production as a

therapeutic intervention for treatment aneurysms. In our research we observed up-regulation in sports models such as modern

and sport gymnastic and down-regulation in sport aerobic. In our other model we observed up-regulation in oncological patients

without sport activity before and after the treatment. In group oncological patients with sport activity we observed up-regulation

in the same time schedule. The other a muscle-specific miRNA, miRNA-499, has been reported to alter mRNA targeting,

leading to subtle changes in the cardiac proteome, at least when overexpressed in mice. In our models we determinate up-

regulation in modern gymnastics and down regulation in sport gymnastic and aerobic. In model oncology patients with or

without work out we cannot determinate levels miRNA-499. In particular, it has been clearly shown that miRNA-133 and

miRNA-1 promote myoblast proliferation and differentiation, respectively, and that miRNA-499 enhances the differentiation

of cardiac progenitor cells into cardiomyocytes. However, it is currently unknown whether these miRNA may act

synergistically. In level miRNA 133a we analyzed up-regulation in modern gymnastic and down-regulation in sport gymnastic

and aerobic. However, we observed counter levels of miRNA-1, down-regulation in modern gymnastic and up-regulation in

sport gymnastic and aerobic. In oncology patients we determinate down-regulation miRNA-1 and miRNA-133 in the group

with work out and up-regulation in the group without work out. Recently, it has been suggested that certain miRNA are

powerful regulators of cardiac differentiation processes, and it has been shown that miRNA-1, miRNA-499, miRNA-133a and

miRNA-29 are highly expressed in plasma levels. Our results shown up or down regulation of these miRNAs plasma levels

concentration as the answer of muscle work out in physiological or pathological process. Supported by grants: APVV-0518-

12, VEGA-1/0882/14

References:

1, Federica Pisano, Claudia Altomore, Elisabetta Cervio et al. 2015. Combination of miRNA499 and miRNA133 Exerts a

Synergic Effect on Cardiac Differentiation. STEM CELLS, 2015, 33:1187-1199

2, Eric N.OLson. 2014. MicroRNAs as Therapeutic Targets and Biomarkers of Cardiovascular Disease. Sci. Transl. Med.,

2014, Vol 6, Issue 239ps3

6

Identifikácia nových alosterických inhibítorov cyklín dependentnej kinázy 2 Chripková M.1, Antoliková N.1, Zigo F.2, Mojžiš J.3, Pirník Z.1

1 Ústav humánnej a klinickej farmakológie, Univerzita veterinárskeho lekárstva a farmácie v Košiciach 2 Ústav chovu zvierat, Univerzita veterinárskeho lekárstva a farmácie v Košiciach 3 Ústav farmakológie, Lekárska fakulta, Univerzita P. J. Šafárika v Košiciach

Cyklín dependentné kinázy (CDK) sú členmi serín/treonín kinázovej rodiny a sú kľúčovými enzýmami v progresii

bunkového cyklu a transkripcie. Deregulácia CDK je často spojená so vznikom mnohých zápalových, neurodegeneratívnych a

nádorových ochorení. Inhibítory CDK sú intenzívne skúmané ako protinádorové liečivá. Najnovšou generáciou sú alosterické

inibítory kináz, viažuce sa na alosterické miesto v blízkosti αC helixu, ktorý je centrálnym mediátorom aktivácie resp.

inaktivácie proteínkinázy. Indukujú zmenu orientácie alfa C helixu, ktorá je dôležitá pre interferenciu s cyklínom, tým pádom

nedochádza k fosforylácii substrátu. Cieľom práce bolo identifikovať nové nízkomolekulové ligandy CDK2 ako potenciálne

alosterické inhibítory. Na ich identifikáciu sme využili ANS skríningovú metódu. Molekulovým dokovaním bolo vybratých 7

potenciálnych alosterických ligandov. Niektoré z nich inhibovali rast nádorových buniek prsníka v mikromolárnych

koncentráciách. ANS metóda vykonaná v prítomnosti ATP-kompetitívneho inhibítora staurosporínu potvrdila 7 ligandov, ktoré

sú alosterickými inhibítormi.

Effect of simvastatin treatment of various forms of neurodegeneration in rat models by in vivo magnetic

resonance Kašparová S., Tušková R., Lipták B., and Dubovický M.

The aim of the study was multimodal investigation and quantitative evaluation of neurodegenerative changes in the brain

in the animal models before and after application of the therapy using drug Simvastatin by non-invasive in vivo MRS and MRI

techniques. 1H MRS enables investigation of the brain metabolism in the well-defined region of the brain. The proton magnetic

resonance spectroscopy (1H MRS) was used for the non-invasive study of the brain metabolism and phosphorous magnetic

resonance spectroscopy (31P MRS) plays an important role in the understanding of high energy and membrane metabolism. We

studied also reaction kinetics of a reversible exchange of the phosphate group in the reaction catalyzed by creatine kinase (CK),

using in vivo a saturation transfer (31P MRS) technique. A model of age-related dementia was induced in adult rats by D-

galactose treatment (120 mg/kg) every day during 8 weeks and D-gal+NaNO2 during 30 and 90 days (second model). The D-

gal rats were treated with simvastatin (40 mg/kg), a drug with pleiotropic properties including anti-oxidant effects. First model:

A quantification of absolute concentrations of metabolites from 1H MRS in the D-gal group versus the control group showed a

significant decrease in the concentration of NAA (p<0.002, marker of neurons), as well as a significant decrease in the

concentration of mIns (p<0.03, marker of glial cells). However, the rate constant of CK reaction in D-gal increased. Second

model: significant lower NAA+NAAG (p<0.002), mIns (p<0.008) and Glu+Gln (p<0.02). The rate constant of CK reaction in

D-gal increased (p<0.003). These findings were in accordance with behavioral tests and volumetric studies. Most interesting

findings of our studies is recovery of neuronal marker NAA after simvastatin treatment in D-gal rat model. In the D-gal/Simv

group, we found a significant increase of NAA (p<0.04) compared to that in the D-gal group. We studied also reaction kinetics

of a reversible exchange of the phosphate group in the reaction catalyzed by creatine kinase (CK), using in vivo a saturation

transfer (31P MRS) technique. CK play a key role in energy homeostasis of the brain and a central role in energy transfer in

cells and is highly susceptible to oxidative inactivation and inflammation. Brain isoform CK regulates the ATP level in neural

cells. It can be expected that activity of the CK reaction in the brain in vivo is significantly changed under the conditions of the

above mentioned animal models of dementia or during oxidative stress. In conclusion, in vivo MRS is a very suitable method

for studies of various forms of neurodegeneration because of its noninvasiveand repeatable nature, and in pathophysiological,

metabolic models of dementia. The MRS could substantially accelerates discovery of new drugs and treatment strategies for

neurodegenerative disorders. The study was supported by VEGA 2/0177/14.

BRONCHODILATAČNÝ A ANTITUSICKÝ ÚČINOK MORINU V PODMIENKACH

EXPERIMENTÁLNE VYVOLANÉHO ALERGICKÉHO ZÁPALU Kazimierová I., Jošková M., Pappová L, Šutovská M., Fraňová S.

Ústav farmakológie, Biomed JLF UK, Malá Hora 10701/4A, 03601 Martin

The aim of our study was to evaluate possible effect of ABCB1, and OPRM1 polymorphisms on the therapeutic efficacy

and neonatal safety of remifentanil in women undergoing elective caesarian section under general anesthesia. Parametre

bronchiálnej reaktivity a kašľového reflexu boli hodnotené po skončení 21- dňovej senzibilizácie morčiat ovalbumínom. V

prípade sledovania akútneho účinku bol morin podávaný jednorázovo, pri dlhodobom denne v priebehu celej doby

senzibilizácie v dávke 30 mg/kg p.o. Špecifický odpor dýchacích ciest a kašľový reflex boli vyšetrené in vivo metódou.

Reaktivita hladkého svalu trachey bola hodnotená metódou in vitro. Bronchodilatačný a antitusický účinok morinu bol

porovnávaný s referenčnými liečivami (kodeín, salbutamol, salmeterol). Akútne podanie morinu viedlo k signifikantnému

zníženiu bronchiálnej reaktivity a k supresii kašľového reflexu. Podobný obraz zmien bol zaznamenaný aj pri chronickej terapii.

Dlhodobá terapia morinom vykazovala výraznejší bronchodilatačný účinok v porovnaní so salmeterolom. Na základe našich

výsledkov môžeme zhrnúť, že morin pozitívne moduluje bronchiálnu hyperreaktivitu a má antitusický účinok. APVV 0305-

12, VEGA 1/0165/14 a MZ 2012/35- UK, CERK II

7

Pretreatment with sirtuin 1 allosteric activators ameliorates D-Galactosamine/Lipopolysaccharide-induced

hepatotoxicity Kemelo M., Kutinová Canová N., Farghali H.

Institute of Pharmacology, 1st Faculty of Medicine, Charles University, Prague, Czech Republic; Tel (224968106); Email (hfarg@lf1.cuni.cz)

We showed that polyphenols such as resveratrol and quercetin are protective against D-GalN/LPS-induced

hepatotoxicity. The notion that their health benefits are SIRT1-mediated is still controversial. In this study, we examined

whether the synthetic allosteric SIRT1 activator, SRT1720, may similarly ameliorate D-GalN/LPS-induced hepatotoxicity.

Male Wistar rats were randomly divided into four groups; the normal control group and three treatment groups (SRT1720, D-

GalN/LPS, SRT1720 + D-GalN/LPS). After twenty-four hours, the effects of these treatment were evaluated by biochemical

studies, real-time PCR and Western blotting. On its own, SRT1720 did not have any significant effects on the measured

biochemical parameters relative to the normal control group. D-GalN/LPS was able to induce hepatotoxicity and dramatically

increase transaminases, catalase and bilirubin levels. SRT1720 pretreatment consistently attenuated the cyctotoxic effects of

D-GalN/LPS. Both SRT1720 and D-GalN treatments modulated SIRT1 protein/gene expression. The allosteric SIRT1

activator, SRT1720, exhibits comparable liver-protective effects to natural polyphenols. SUPPORTED BY PRVOUK-

P25/LF1/2 & GAUK-916314.

Úloha napäťovo-riadených sodíkových kanálov v neuronálnej regulácii aktivity hladkej svaloviny

bronchov u morčiat Kocmálová M., Meeker S. , Ru F., Kollárik M., Šutovská M., Undem B.

Univerzita Komenského Bratislava, Ústav farmakológie JLF UK Martin, BioMed Martin, Slovensko

Division of Allergy and Clinical Immunology, Johns Hopkins School of Medicine, Baltimore, MD, USA

Cholínergické nervové vlákna hrajú dominantnú úlohu pri regulácii kontrakčnej aktivity dýchacích ciest (DC). Hoci je

známe, že akčný potenciál v týchto nervoch je senzitívny na tetrodotoxín (TTX), existuje veľmi málo informácii o tom, ktoré

TTX-senzitívne napäťovo-riadené sodíkové kanály (Nav) sú do tohto procesu zapojené. Hodnotili sme účinok viacerých

blokátorov Nav na kontrakciu hladkého svalu (HS) DC vyvolanú stimuláciou elektrickým poľom u morčiat. Následne sme

dosiahnuté výsledky konfrontovali s génovou analýzou Nav v parasympatikových gangliách izolovaných z bronchov morčiat.

TTX antagonizoval atropín-senzitívne kontrakcie HSDC s – log (M) IC50 7.95 ± 0.01. Látka A, blokátor s vysokou potenciou

k Nav 1.1, 1.3 a nízkou afinitou k Nav1.6, nemal takmer žiadny účinok na kontrakčnú aktivitu HS bronchov. Látky B a C,

blokátory izoforiem Nav 1.7 a Nav 1.6, výrazne potlačili kontrakčnú odpoveď HSDC. Pomer účinnosti látky B ku TTX bol

0.97 a látky C 0.73. Génovou analýzou sme potvrdili prevažujúcu expresiu izoforiem Nav 1.7 a Nav 1.9. Tieto údaje sú v súlade

s hypotézou, že Nav 1.7 hrajú kľúčovú úlohu v regulácii aktivity parasympatikových nervových vlákien a tým ovplyvňujú

tonus HSDC u morčiat. Práca vznikla za podpory grantov VEGA 1/0165/14, MZ 2012/35-UKMA-12 a APVV 0305-12.

Method of genotyping of GSTM1 and GSTT1 null alleles. Possible predictor of azathioprine-induced

pancreatitis? Kolorz M.1,2, Bartosova L.1, Navas-Lopez V.M.3, Sierra-Salinas C.3

1 Department of Human Pharmacology and Toxicology, Faculty of Pharmacy, UVPS Brno, Czech Republic 2 Department of Clinical Pharmacy, Hospital Pharmacy, The University Hospital Brno, Czech Republic 3 Hospital Materno Malaga, Pediatric Gastroenterology and Nutrition Unit, Malaga, Spain

Glutathion-S-transferase (GST) facilitates elimination of many toxic substrates. In human GSTM1-null and GSTT1-null

alleles leads to lack of enzyme activity when homozygous. The aim of our study was to evaluate whether GST null genotype

is associated with azathioprine-induced pancreatitis. We optimized multiplex PCR for the detection of homozygous GSTM1-

null and GSTT1-null genotype. Protocol was optimized for amplification of both loci in one reaction. Null allele was confirmed

by lack of amplification and presence of amplification of internal standard. We genotyped 166 patients treated with azathioprine

in standard doses (2,5 mg/kg/day). Four patients experienced acute pancreatitis. 31 of non-pancreatitis patients (19.1%) were

homozygous for GSTT1-null. One of four patients experienced pancreatitis was GSTT1-null homozygote (25%; P=0.579). 74

non-pancreatitis patients (45.7%) were homozygous for GSTM1-null. All four patients with pancreatitis were GSTM1-null

homozygotes (100%; P=0.047, RR 0.46, CI 95% 0.386-0.540). The initial step of azathioprine bioactivation is conversion to

6-mercaptopurine. However this occurs spontaneously, GST facilitates and increase this reaction. Mechanism of azathioprine-

induced pancreatitis remains unknown and occurs within 3-4 initial weeks of therapy. Although rare, its occurrence leads to

necessity of azathioprine withdrawal. However, due to small number of patients we suggest that GSTM1-null homozygous

genotype correlates with azathioprine-induced severe pancreatitis.

8

Manifestace nežádoucích metabolických účinků atypických antipsychotik u pacientů s první epizodou

schizofrenie Kotolová H.1, Horská K.1,2, Ustohal L.3, Mayerová M.3, Kašpárek T.3

1 Ústav humánní farmakologie a toxikologie, FaF VFU Brno, Brno, Česká republika 2 Nemocniční lékárna – úsek klinické farmacie, FN Brno, Brno, Česká republika 3 Psychiatrická klinika LF MU a FN Brno, Brno, Česká republika

Základem pro udržení stabilizovaného stavu u nemocných schizofrenií je dlouhodobé podávání antipsychotik. Léčba

atypickými antipsychotiky (AAP) je i přes výborný bezpečnostní profil oproti starším generacím nadále spojena s řadou

nežádoucích, především metabolických účinků. Sledovali jsem drug-naïve pacienty s první epizodou schizofrenie a dynamiku

změn základních tělesných a biochemických parametrů v prvních týdnech léčby. Pacienti byli léčeni olanazapinem, nebo

risperidonem, pacienti neužívali žádná jiná farmaka, které by mohli mít vliv na vzestup hmotnosti. Základní tělesné parametry

byly měřeny pomocí lékařské váhy OMRON BF511-T s monitorem skladby lidského těla. Nejčastějším nežádoucím účinkem

atypických antipsychotik velmi úzce souvisejícím s rozvojem metabolického syndromu je vzestup hmotnosti. Během 4 týdenní

léčby jsme zaznamenali u všech pacientů vzestup hmotnosti, zvětšení objemu pasu, nárůst BMI, snížení svalové tkáně a

zvětšení objemu tukové tkáně. Zvýšené riziko vzniku obezity, diabetu mellitu II.typu, hyperglykémie, hypertenze a

dyslipidémie vedoucí v konečném důsledku až k rozvoji metabolického syndromu patří k nejčastějším hrozbám dlouhodobé

terapie AAP. Vysoká prevalence NÚ AAP vede často ke změně účinné medikace a z hlediska pacienta k non conpliance. Každý

pacient by proto měl mít vytvořen plán frekvence a rozsahu monitorování tělesného zdraví včetně laboratorních vyšetření.

Jedna z možností, jak oddálit manifestaci kardiometabolických poruch bez nutnosti změny zavedené medikace, je kromě

fyzické aktivity a dietní intervence i důsledné léčení všech složek metabolického syndromu. Monitorování tělesných parametrů

a skríning rizikových jedinců z hlediska kardiálních, metabolických a endokrinologických je přes dostupnost řady vodítek a

doporučení stále nedostatečné a kardiovaskulární mortalita u schizofreniků nadále stoupá.

PRECLINICAL STUDY IN PIGS: IMPORTANCE OF WIRELESS CAPSULE ENDOSCOPY FOR THE

RESEARCH OF ABSORPTION WINDOW OF XENOBIOTICS IN INTESTINAL TRACT Květina J.1, Tachecí I.1, Kuneš M.2, Nobilis M.3, Kopáčová M.1, Bureš J.1

1 2nd Department of Internal Medicine - Gastroenterology, Charles University Faculty of Medicine and University Teaching Hospital.

Hradec Králové, Czech Republic, 2 Department of Pharmaceutical Chemistry and Drug Control, Faculty of Pharmacy, Charles University in Hradec Králové, Czech Republic 3 Biomedical Research Centre, University Hospital Hradec Králové, Czech Republic

The identification of intestinal segment, in which a xenobiotic (drug) is predominantly absorbed, is important both for

orally administered solid dosage forms treatment (tablets) as well as for patients with some intestinal discomfort (with irritable

bowel syndrome, ulcerative colitis etc.). The aim of this study is to create the experimental study scheme that would be useful

in pre-clinical research for predictive applications of more general character. For fulfilling this purpose were used: a)

experimental pigs as an omnivorous representative, physiologically similar to a man, b) a combination of two coupled

diagnostic techniques (bioanalytics for monitoring systemic levels of the model drug and capsule endoscopy for determining

the disintegration kinetics of drug formulation), c) a model drug (intestinal anti-inflammatory drug 5-aminosalicylic acid=5-

ASA) in two different technologically prepared tablet forms (from granulate = "G", from pellets = "P"). Tablets were

administered endoscopically and simultaneously with the capsule microcamera into duodenum (behind the pyloric sphincter).

Animals were in general anesthesia during the whole experiment. Blood samples were withdrawn from incannulated vena cava

(via vena jugularis externa) into heparinised tubes. Blood samples (blood plasma) were frozen (-70°C) until HPLC analysis.

The disintegration of tablets “G“ is gradual and steady (without any peak), starting from the 1st hour after administration

(proximal part of jejunum) with the disintegration of the largest tablet between the 3rd and the 4th hour (middle jejunum). 5-

ASA is biotransformed predominantly in N-acetyl-5-ASA. In the case of tablet “G“, the plasma time profiles correspond with

dissolution kinetics, i.e. without expected plasmatic cmax. On the other hand, the disintegration of tablets “P” starts at the 2nd

hour after administration (ca 100 - 200 cm after pylorus), the peak is reached at the 3rd hour (in the middle jejunum). Suspension

form (completely disintegrated) was found at the 4th hour (in distal part of jejunum). Plasma time profiles of N-acetyl-5-ASA

are usual, i.e. the peak of maximal drug concentrations is achieved between the 2nd and the 3th hour after administration and

then continual elimination phase follows until the 4th hour. Comparative studies have pointed to the possibility of using wireless

capsule endoscopy - one of the clinical non-invasive diagnostic methods - as a part of the preclinical pharmacological tests in

innovative pharmaceutical research and possibly even in the bioequivalence testing of generic products. Supported by grant

IGA NT/14270.

Vplyv vybraných liečiv na kultiváciu a diferenciáciu kmeňových buniek izolovaných z tukového tkaniva pre

možné aplikácie v rámci regeneratívnej medicíny kardiovaskulárnych ochorení Lešková Z. 1, Gažová A.2, Bačkayová T. 1, Musil P. 1, Sprušanský O. 1, Maďarič J.3, Kyselovič J. 1

1 Farmaceutická fakulta Univerzity Komenského Bratislava, Katedra farmakológie a toxikológie, 2 Lekárska fakulta Univerzity Komenského Bratislava, Ústav farmakológie a klinickej farmakológie, 3 Oddelenie kardiológie a angiológie, Kardiologická klinika NÚSCH, a.s., a LF SZU, Bratislava

Klinické použitie mezenchymálnych kmeňových buniek má veľký potenciál v rámci regeneratívnej medicíny

kardiovaskulárnych ochorení. Na diferenciačné schopnosti týchto buniek vo veľkej miere vplýva ich kultivačné prostredie.

Cieľom práce bolo vyhodnotiť vplyv kyseliny askorbovej, magnézia a vitamínu D3 na kultiváciu a diferenciáciu kmeňových

buniek izolovaných z tukového tkaniva sledovaním zmeny expresie miRNA metódou real-time polymerázovej reťazovej

9

reakcie (RT-PCR). Bunky boli kultivované za štandardných podmienok. V priebehu kultivácie sme charakterizovali ich

povrchové markery špecifické pre mezenchymálne kmeňové bunky metódou prietokovej cytometrie. Následne boli inkubované

24 hodín v médiách s prídavkami rôznych koncentrácií kyseliny askorbovej, magnézia a vitamínu D3. Prípadné štrukturálne a

morfologické zmeny sme zaznamenávali pod inverzným mikroskopom. Metódou RT-PCR sme stanovili zmenu miery expresie

cieľových kardio-špecifických miRNA (miRNA-1, miRNA-133a, miRNA-499, miRNA-29b). Po 24 hodinách kultivácie

buniek s vitamínom D3 nedošlo k výrazným štrukturálno-morfologickým zmenám, ale štatisticky významne sa zvýšila expresia

všetkých sledovaných miRNA. Ani použité koncentrácie magnézia výrazne neovplyvnili kvalitatívnu charakteristiku buniek

ale signifikantne sa zvýšila expresia miRNA-1 a miRNA-29b. Pri zvýšených koncentráciách kyseliny askorbovej bunky stratili

svoj typický tvar a vo výraznej miere bola oslabená ich schopnosť adherencie k plastu. Vplyvom kyseliny askorbovej došlo k

výraznému zníženiu expresie miRNA-499, bez výrazných zmien expresie miRNA-1 a miRNA-133a. Určité kardio-špecifické

mikroRNA (miRNA-1, miRNA-133a a miRNA-499) môžu v poškodenom myokarde reprogramovať asi 1% prítomných

fibroblastov na bunky podobné kardiomyocytom (Jayawardena, et al., 2012) a znížené hladiny miRNA-29 sú asociované so

zvýšenou expresiou a ukladaním kolagénových komponentov fibrotickej jazvy (van Rooji, et al., 2008). Rôzne kardiogénne

faktory a molekuly by mohli tento proces ovplyvniť. Niekoľko štúdií poukázalo na schopnosť kyseliny askorbovej (Takahashi,

et al., 2003), či vitamínu D (Hlaing, et al., 2014) podporiť proces srdcovej diferenciácie. V našich experimentoch kyselina

askorbová výrazne neovplyvnila expresiu miRNA-1 a miRNA-133a, ale štatisticky významne znížila expresiu miRNA-499.

Naopak, signifikantné zvýšenie expresie všetkých vybraných miRNA sme pozorovali v bunkách ovplyvnených vitamínom D3.

Vplyvom magnézia došlo k štatisticky významnému nárastu expresie miRNA-1 a miRNA-29b. Kultivačné médium obsahujúce

vitamín D3 malo najpozitívnejší vplyv na podporu difernciačného potenciálu kmeňových buniek smerom k srdcovým líniám,

ale potenciál pre ďalšie experimenty majú aj kyselina askorbová a magnézium. Grantová podpora: FaF UK/34/2015, VEGA

1/0905/14, APVV-14-0416.

DLHODOBÝ ÚČINOK INHALAČNE PODANÉHO BUDESONIDU, SALMETEROLU A ICH

KOMBINÁCIE NA CILIÁRNU FREKVENCIU V PODMIENKACH EXPERIMENTÁLNE

VYVOLANÉHO ALERGICKÉHO ZÁPALU Pappová L., Jošková M., Kazimierová I., Šutovská M., Fraňová S.

Ústav farmakológie, Biomed JLF UK, Malá Hora 10701/4A, 03601 Martin

Cieľom práce bolo sledovať vplyv dlhodobého podania salmeterolu, budesonidu a ich kombinácie na ciliárnu frekvenciu

(CBF) v podmienkach experimentálne vyvolaného alergického zápalu. V experimente bol použitý 21 a 28 dňový model

senzibilizácie morčiat ovalbumínom. Simultánne s prebiehajúcou senzibilizáciou boli zvieratá liečené aerosólom budesonidu

(1 mM), salmeterolu (0,17 mM) alebo ich kombináciou v polovičných dávkach. Pohyb cílií bol sledovaný svetelným

mikroskopom napojeným na vysokorýchlostnú kameru. Hladiny zápalových cytokínov (IL-4,IL-5, IL-13) boli stanovené v sére

a BAL metódou BioPlex. Alergický zápal, vyvolaný trojtýždňovou senzibilizáciou, viedol k zvýšeniu CBF. Za týchto

podmienok všetky terapeutické postupy vykazovali ciliostimulačný účinok v poradí salmeterol< budesonid< kombinovaná

terapia. Z hladín zápalových cytokínov sme pozorovali zvýšenie najmä IL-4 a IL-13, ktoré boli signifikantne potlačené

dlhodobým podaním budesonidu a čiastočne aj kombinovanou terapiou. Štvortýždňová senzibilizácia mala depresívny účinok

na CBF a ciliostimulačný účinok sa výrazne prejavil len pri kombinovanej terapii. Alergický zápal bol charakterizovaný

nárastom zápalových cytokínov, najmä IL-5. Štatisticky významné zníženie sledovaných cytokínov bolo pozorované v skupine

zvierat liečených budesonidom a v kombinácií so salmeterolom. Jedine kombinovaná terapia budesonidom a salmeterolom

preukázala výrazný ciliostimulačný účinok u oboch modelov alergickej astmy. Pozorované zvýšenie CBF je pravdepodobne

výsledkom zníženia stupňa zápalu a priaznivého pôsobenia budesonidu na expresiu β2 receptorov na ciliárnych bunkách.

APVV 0305-12, VEGA 1/0165/14 a MZ 2012/35- UK, CERK II

DISTRIBÚCIA EXPRESIE KOMPONENTOV CHOLINERGICKÉHO SYSTÉMU V POTKANEJ

AORTE Piváčková L., Šrankova J., Vetešková J., Dingová D., Szmicseková K., Klimas J., Hrabovská A., Křenek P.

Katedra farmakológie a toxikológie, Farmaceutická fakulta, Univerzita Komenského v Bratislave

Acetylcholínom navodená vazodilatácia je bežne používaná na charakterizáciu endotelovej funkcie. Acetylcholín

aktiváciou M3 receptorov stimuluje syntézu NO, prostanoidov a EDHF v endoteli, ktoré účinkom na hladký sval vyvolávajú

od endotelu závislú vazodilatáciu. O vaskulárnom cholinergickom systéme existuje veľmi málo údajov. Naším cieľom bolo

určiť lokalizáciu expresie acetylcholínesterázy (AChE), butyrylcholínesterázy (BChE), M3 muskarínového receptora (M3MR),

kotviacich proteínov PRiMA1 a ColQ1 v endoteli a v hladkom svalstve aorty. V našej práci sme použili torakálnu aortu

dospelých samcov potkanov kmeňa Wistar. Expresia cholinergických markerov AChE, AChE-T, BChE, M3MR, PRiMA1 a

ColQ1 bola analyzovaná pomocou RT-qPCR v prúžkoch aorty s intaktným endotelom (AoE+), v endotelových RNA frakciách

získaných perfúziou aorty s Tri-Reagentom (E1-E3) a v aorte zbavenej endotelu (AoE-). Endotelový a hladkosvalový pôvod

frakcií bol overený pomocou stanovenia endotelových markerov (endotelová NO syntáza, NOS3; preproendotelín-1; ET-1),

hladkosvalových markerov (L-typ vápnikového kanála, Cav1.2; endotelínový receptor typu A, ETA; hladkosvalový aktín,

ACTA2) a génov exprimovaných v endoteli aj v hladkom svalstve (endotelínový receptor typu B, ETB). Frakcie E1-E3

vykazovali výraznú expresiu endotelových markerov NOS3 a ET-1. Expresia hladkosvalových markerov Cav1.2 a ETA v

týchto frakciách bola nízka, smerom od E1 k E3 sa mierne zvyšovala a vo frakciách AoE- and AoE+ bola vysoká. Expresia

mRNA pre ETB bola podobná vo všetkých frakciách. V AoE+ frakcii sme detegovali všetky stanovované cholinergické

markery. BChE mala 30-násobnú expresiu v porovnaní s AChE. Expresia AChE, BChE, PRiMA1 a ColQ1 vykazovala

podobnú distribúciu vo frakciách ako hladkosvalové markery. M3MR bol podobne exprimovaný v endotelových ako aj v

hladkosvalových frakciách. Cholinergické markery sú v potkanej aorte hojne exprimované napriek neprítomnosti

10

parasympatickej inervácie, čo poukazuje na prítomnosť neneuronálneho cholinergického systému. 30-násobná expresia BChE

v porovnaní v AChE naznačuje jej dôležitosť pri regulácii cievnej odpovede na acetylcholín. Lokalizácia BChE prevažne na

hladkom svalstve by za fyziologických podmienok mohla umožniť vazodilatačné pôsobenie acetylcholínu na endotelové bunky

a zároveň zabraňovať jeho difúzii do tunica media a tým limitovať jeho vazokonstrikčné pôsobenie. V potkanej aorte je

prítomná expresia mRNA cholinergických markerov AChE, AChE-T, BChE, M3MR, PRiMA1 a ColQ1. Okrem M3MR sú

tieto markery lokalizované prevažne na hladkom svalstve, M3MR je exprimovaný v hladkom svalstve ako aj v endoteli. Z

cholínesteráz prevažuje expresia BChE, ktorá je až 30-násobne vyššia v porovnaní s AChE. Grantová podpora: VEGA

1/0294/15, 1/0855/15, VEGA 1/0564/13, APVV-0887-11.

Pharmacokinetics and pharmacodynamics of phenobarbital in asphyxiated newborns treated with or

without therapeutic whole body hypothermia Pokorná P.1, 2, 3, Černá O.1, Posch L.1, Srnský P.1, Šíma M.2, Tibboel D.1, 3, Slanař O.2, Vobruba V.1

1 Department of Pediatrics - PICU/NICU, General University Hospital, 1st Faculty of Medicine Charles University in Prague, Ke Karlovu 2, 128 02 Prague 2, Czech Republic

2 Department of Pharmacology, First Faculty of Medicine, Charles University in Prague and General University Hospital in Prague,

Albertov 4, 128 00 Prague 2, Czech Republic; 3 Intensive Care and Department of Pediatric Surgery, Erasmus MC - Sophia Childrens Hospital, Rotterdam, the Netherlands, Wytemaweg

80, 3015 CN - Rotterdam, the Netherlands

Primary aim was to determine the pharmacokinetics (PK) and pharmacodynamics (PD) of phenobarbital (PHE) in

asphyxiated newborns treated with (HT) or without hypothermia (nonHT). A secondary aim was to determine the role of

covariates. Prospective open-label PK study included 40 asphyxiated newborns (≥37 weeks) treated for hypoxic-ischemic

encephalopathy (HIE) undergoing HT (GroupHT 26/40); (33°C-34°C) or nonHT (GroupnonHT 14/40). Serum concentrations

of PHE were determined using a quantitative enzyme immunoassay. Clearance (Cl), volume of distribution (Vd) of PHE were

estimated using a non-compartment analysis. In newborns treated for HIE the time to reach normal EEG pattern (TnormaEEG)

was determined. CL [(0.0021±0.0023 vs 0.0027±0.0017)] L.h-1.kg-1; Vd [(0.5186±0.1363 vs 0.4090±0.2280)] L.kg-1 were not

significant different between HT and nonHT. A two-way ANOVA model showed significant impact of HT and asphyxia

interaction on CL (P=0.0377). TnormaEEG was significantly prolonged in HT compared to the nonHT [52.5 (5-280) vs 26.4

(0-74) h, (P=0.006)]. The treatment with PHE was discontinued in 58% of newborns before day 5 based on HIE and

therapeutical drug monitoring. Interaction of treatment modality (HT) and severe asphyxia determined CL of PHE as

explanatory variable and dosage regimen of PHE in this clearence covariance PK-model.

Inhibition of oxidative stress in brain during rat adjuvant arthritis by carnosine, trolox and novel trolox-

carnosine Poništ S.1, Slovák L.1, Kuncírová V.1, Fedorova T.2, Logvinenko A.2, Muzychuk O.2, Mihalová D.1 and Bauerová K.1

1 Institute of Experimental Pharmacology and Toxicology of Slovak Academy of Sciences, Dúbravská cesta 9, Bratislava, Slovak Republic,

katarina.bauerova@savba.sk 2 Research Center of Neurology, Moscow, Volokolamskoye shosse 80, 125367 Moscow, Russian Federation

Carnosine (CAR) is an anti-glycating agent able to quench superoxide, and to neutralize 4-hydroxynonenal. We evaluated

the impact of trolox (TRO), CAR and carnosine-trolox (CAR-T) on oxidative stress (OS) in brain during rat adjuvant arthritis

(AA). The experiments were done on healthy, control arthritic and arthritic animals with administration of CAR 150 mg/kg

b.w., TRO 41 mg/kg b.w. and CAR-T 75 mg/kg b.w. in a daily oral dose during 28 days. Antioxidants did not affect the body

weight on day 14, however on day 28 TRO enhanced the weight reduction. On days 14 and 28 CAR-T and TRO reduced hind

paw swelling (HPS). IL-1beta, MCP-1 and MMP-9 were measured in plasma on day 14. IL-1beta and MCP-1 were decreased

by CAR-T and TRO. All antioxidants reduced MMP-9 levels. Malondialdehyde, 4-hydroxynonenal and protein carbonyls were

increased in brain of rats suffering with AA. CAR, CAR-T and TRO prevented lipid and protein oxidation in brain. CAR and

CAR-T caused no weight reduction like TRO. Moreover these evaluated antioxidants had a similar therapeutic effect on HPS,

inflammation markers in plasma and OS in brain. Project VEGA 2/0044/15, bilateral SAS-RAMS project 2013-2015

coordinated by Dr. Bauerova (Slovakia) and Dr. Fedorova (Russia).

High doses of testosterone do not have apparent deleterious effects on heart ventricles in rats, even in

combination with spontaneous mild physical aktivity Radik M., Dóka G., Malíková E., Křenek P., Klimas J.

Comenius University in Bratislava, Faculty of Pharmacy, Department of Pharmacology and toxikology

Adequate physical activity has positive impact on cardiovascular system. Protective, or damaging effect of high

testosterone levels on heart is a controversial topic in scientific literature. The aim is to identify a possible damage to heart

ventricles caused by high doses of testosterone and voluntary, mild physical activity, or it’s combination. 10-12 weeks old male

Wistar rats were once a week subcutaneously administered testosterone depot in dose of 100 mg/kg (TES, n = 15) or vehiculum

(CON, n = 12). Next groups injected with testosterone (SPOTES, n = 12) or vehiculum (SPO, n = 12) were running in exercise

wheels ad libitum. Body characteristics and amount of physical activity of the animals were measured after 8 weeks. In left

and right ventricles of the myocardium were estimated expressions of genes essential in contractile apparatus of the heart

(myosin heavy chains, Myh6, Myh7), gene for regulation of cytoplasmic calcium (sarcoendoplasmic ATP-ase, Atp2a2), marker

of fibrosis and hypertrophy (collagen, Col3a1), marker of oxidative stress (subunit of NADPH oxidase 2, Cybb), marker of

non-specific heart damage (natriuretic peptide A Nppa) and androgen receptor (Ar) by RT-qPCR. Weight and naso-anal length

11

of rats administered with testosterone was lower compared to control groups (-84 g, decrease of 23 %; -1 cm, decrease of 5 %

respectively; p<0.05 for both parameters), absolute weights of the heart and individual ventricles remained unchanged in all

groups, independent from physical activity. Animals administered with testosterone spent on average 3x more time (35 hrs.)

and ran 5x longer distance (100 km) in exercise wheels than control rats. In left ventricles of testosterone groups was found a

mild growth in relative amount of Myh6 (+1,5 %; p<0.05) and higher expression of Cybb (+ 26 %; p<0.05) and decrease in

expression of Ar (-16 %; p<0.05). Voluntary, mild physical activity didn’t have a substantial effect on measured results.

Combination of supraphysiological doses of testosterone and increased physical activity was expected to have a cumulative

effect on expression of following genes and function of the heart itself, but it did not affect expression of selected genes.

Although most of gene expressions were higher in the right ventricle, than the left, recorded changes manifested mostly in the

left ventricle. According to recorded results, rats in our experiment administered with supraphysiological doses of testosterone

has shown increased physical activity, but function of the heart did not impair, on the contrary, it had tendency to improve. We

did not find a clear evidence, that high doses of testosterone are causing damage to myocardium of healthy rats. Our results

indicate no proof of hypertrophy and no significant changes in expression of genes involved in development of heart damage.

Voluntary physical activity did not have a measurable effect on heart.

Mykofenolát mofetil a cyklofosfamid snižují intenzitu zánětu na modelu experimentální autoimunitní

uveitidy u myší Seidler Štangová P.1, Klímová A.1, Svozílková P.1, Kučera T.2, Heissigerová J.1

Výzkum na experimentálních modelech autoimunitní uveitidy pomáhá najít nové léčebné strategie. Cílem této studie

bylo porovnat vliv terapie cyklofosfamidem (CPA), mykofenolát mofetilem (MMF) a golimumabem na intenzitu zánětu na

modelu experimentální autoimunitní uveitidy (EAU) u myši. EAU byla indukována u kmene myší C57BL/6J subkutánní

aplikací IRBP (interphotoreceptor retinoid binding protein) v kompletním Freundově adjuvans a intraperitoneální aplikací

pertusového toxinu. Léčba byla zahájena v den indukce uveitidy. CPA byl aplikován intraperitoneálně v jediné dávce 100

mg/kg, MMF intraperitoneálně denně v dávce 30 a 50 mg/kg, golimumab subkutánně jednou měsíčně v dávce 70 mg/kg.

Kontrolní skupiny zahrnovaly myši bez indukce EAU, neléčené myši s EAU a myši s EAU, kterým bylo aplikováno placebo

(aqua pro injectione). Bulby byly enukleovány post mortem 35. den po indukci EAU a intenzita zánětu byla hodnocena

histologickým barvením kryořezů metodou hematoxylin-eozin. Statistická analýza byla provedena metodou Mann-Whitney U

test. Srovnání intenzity zánětu na histologických řezech ve skupinách neléčených a léčených myší dokazuje, že MMF výrazně

potlačuje zánět (p˂0,05). Silné protizánětlivé účinky CPA jsou již dlouho známé, což jsme potvrdili absolutním potlačením

uveitidy (p˂0,0001). Golimumab zánět naopak potencoval. V této studii jsme potvrdili účinnost CPA a MMF v léčbě zadní

uveitidy, které podporují jejich použití v humánní medicíně. Tento projekt byl financován z podpory IGA MZ ČR NT/14017-

3/2013 a SVV UK 260148/2015.

Molekulární faktory prognózy a predikce výsledku terapie karcinomu prsu Souček P., Vaclavíková R., Brynychová V., Kunická T., Vrána D., Koževnikovová R., Trnková M., Kubáčková K.,

Měšťáková S., Mrhalová M., Gatěk J., Vernerová Z.

Státní zdravotní ústav Praha, Medicon a.s. Praha, Biolab Praha, FN Olomouc, FN a 2LF UK Motol Praha, FN a 3LF UK Královské Vinohrady

Praha, Nemocnice Atlas Zlín

Naše pracovní hypotéza vychází z předpokladu, že existují genetické, epigenetické a fenotypické markery, které korelují

s výsledkem chemoterapie nádorových onemocnění. Existence těchto markerů byla naznačena řadou studií za pomoci

modelových buněčných linií, transgenních zvířecích modelů a studií na tkáňových vzorcích pacientů. Cílem této studie bylo

najít souvislost mezi genotypem a fenotypem transportérů léčiv a prognózou a výsledkem terapie karcinomu prsu. Pomocí

kvantitatviní PCR v reálném čase jsme prostudovali genovou expresi všech 48 známých lidských genů ABC a vybraných SLC

transportérů v tkáních pacientek s karcinomem prsu. Srovnání rozdílů v expresních profilech mezi nádorovými a patologicky

nádorem nepostiženými tkáněmi pacientů a mezi pacienty rozdělenými dle klinických dat, včetně odpovědi na terapii a

přežívání ukázalo řadu výsledků, které ověřujeme funkčními studiemi. Jedním z příkladů je gen ABCC1 (OMIM: 158343),

který kóduje efluxní transportér MRP1 se širokým spektrem substrátů, mezi nimiž nechybí řada protinádorových léčiv.

Prostudovali jsme genetickou variabilitu v oblasti funkční domény NBD1 (nucleotide binding domain 1) tohoto kandidátního

genu u 540 pacientek s karcinomem prsu. Variabilita, vyjádřená sadou jednonukleotidových polymorfismů pokrývajících

kompletní haplotyp NBD1 s přiléhajícími oblastmi, korelovala s molekulárním i klinickým fenotypem karcinomu prsu. Nalezli

jsme vztah mezi některými polymorfismy a expresní hladinou ABCC1 v nádorové tkáni i vztahy polymorfismů k přežití

pacientek. Tato studie poprvé ukázala, že genetická variabilita ve funkční doméně NBD1 transportéru ABCC1, který in vitro

přenáší cytostatika (anthracykliny, taxany, apod.), významně ovlivňuje hladinu transkriptu v cílové tkáni i výsledek terapie

karcinomu prsu. Výše popsané pozorování činí z tohoto transportéru atraktivní kandidátní molekulu (biomarker) pro

farmakogenomiku a eventuálně i cíl pro vývoj léčby určené ke zlepšení odpovědi pacientů na terapii. Hlavní využití

prediktivních biomarkerů vidíme v oblasti stratifikace pacientů do jednotlivých léčebných režimů. Nalezení a validace

prognostických biomarkerů rovněž může přispět k vývoji terapeutik pro cílenou léčbu. Výzkum je podporován granty GAČR

13-25222J, IGA 13679-4, 14055-3, AZV 15-25618A, GAUK 1444313 a projektem CZ.1.05/2.1.00/03.0076 Evropského fondu

pro regionální rozvoj.

12

Effect of co-medication on the pharmacokinetic parameters of phenobarbital in asphyxiated newborns Šíma M.1, Pokorná P.1,2,3, Hronová K.1, Slanař O.1

1 Department of Pharmacology, First Faculty of Medicine, Charles University in Prague and General University Hospital in Prague,

Albertov 4, 128 00 Prague 2, Czech Republic; 2 Department of Pediatrics - PICU/NICU, General University Hospital in Prague and First Faculty of Medicine, Charles University in

Prague, Ke Karlovu 2, 128 02 Prague 2, Czech Republic; 3 Intensive Care and Department of Pediatric Surgery, Erasmus MC - Sophia Childrens Hospital, Rotterdam, the Netherlands, Wytemaweg

80, 3015 CN - Rotterdam, the Netherlands

Phenobarbital is an anticonvulsive drug widely used in newborns with hypoxic-ischemic encephalopathy. The objective

of our study was to describe possible effect of frequently co-administered medications (dopamine, dobutamine, noradrenaline,

furosemide, phenytoin, analgesics) on the phenobarbital pharmacokinetics in full term newborns with hypoxic-ischemic

encephalopathy. Phenobarbital pharmacokinetic parameters (i.v. loading dose was 10-20 mg /kg, maintenance dose 2 - 6

mg/kg/day) were computed using non-compartmental analysis. Co-medication was evaluated throughout the whole treatment

period up to 5 days. Volume of distribution, clearance, and half-life median values (95% CI) for phenobarbital in the whole

study population (n=37) were 0.48 (0.41 – 0.56) L/kg, 0.0034 (0.0028 – 0.0040) L/h/kg, and 93.7 (88.1 – 99.2) h, respectively.

Phenobarbital pharmacokinetic parameters were not significantly affected by vasoactive drugs (dopamine, dobutamine,

norepinephrine), furosemide, phenytoin, or analgesics. Furthermore, no dose-dependent alteration of phenobarbital

pharmacokinetic parameters was noted for vasoactive medication at doses equivalent to cumulative vasoactive-inotropic score

143.2 – 8473.6, furosemide at cumulative doses of 0.2 – 42.9 mg/kg, or phenytoin at cumulative doses of 10.3 – 46.2 mg/kg.

Phenobarbital pharmacokinetics was not affected by investigated co-administered drugs used in newborns with hypoxic-

ischemic encephalopathy in real clinical settings. The work was supported by the Charles University Project PRVOUK

P25/LF1/2.

Vplyv G-CSF a inhibítora Dpp4 na expresiu osi Sdf-1/Cxcr4 a markerov kmeňových buniek v srdci potkana

s daunorubicínovou kardiomyopatiou Šrankova J., Piváčková L., Dóka G., Vetešková J., Marušákova M., Klimas J., Křenek P.

Katedra farmakológie a toxikológie, Farmaceutická fakulta Univerzity Komenského v Bratislave

Úloha chemokínu Sdf-1 a jeho receptora Cxcr4 v migrácii a uchytení kmeňových buniek v srdci s antracyklínovou

kardiomyopatiou nie je dostatočne známa. Proteáza Dpp4 štiepi Sdf-1, preto inhibítor Dpp4 a mobilizačný factor G-CSF by

mohli prispieť k regenerácii srdca poškodeného antracyklínmi. Samcom kmeňa Wistar (12 týždňové) sme i.v. aplikovali

daunorubicín (7.5 mg/kg) (DAU, n=10) alebo fyziologický roztok (KON, n=8). Analóg G-CSF-pegfilgrastim (100 ug/kg, s.c.)

bol podaný 24h po DAU (DAU+GCSF, n=14). Inhibítor DPP4-linagliptín (5 mg/kg/d) bol podavaný v krmive tesne po aplikácii

DAU (DAU+LINA, n=10) po dobu 8 týždňov. Daunorubicínovej skupine DAU+G-CSF+LINA (n=14) bola podávaná

kombinácia oboch látok (G-CSF a LINA). Po 8. zvieratá boli utratené a odobraté vzorky srdca boli uchované pri -80°C.

Expresia génov atriálneho natriuretického peptidu (Nppa), α-myozínového reťazca (Myh6), Sdf-1, Cxcr4, Dpp4, faktora

kmeňových buniek (Scf), rastových faktorov (Vegf, Hgf, Igf-1), markerov endotelových progenitorových (Cd34 a Cd33),

srdcových progenitorových (c-kit a Sca-1) a mezenchymálnych kmeňových buniek (Cd44 a Cd105) na úrovni mRNA bola

sledovaná prostredníctvom qRT-PCR. Aplikácia DAU viedla k mortalite 55% zvierat (P<0.05 vs KON), podanie G-CSF znížilo

úmrtnosť o 35% (P<0.05 vs DAU). V skupine DAU+G-CSF+LINA bolo zaznamenané 100% prežívanie (P<0.05 vs DAU,

P<0.05 vs DAU+LINA). DAU zvýšil Nppa a znížil Myh6 (P<0.05 vs KON), zatiaľ čo terapia neviedla k zmenám oproti DAU

skupine. Expresia Sdf-1, Scf a Dpp4 bola downregulovaná v skupinách ovplyvnených DAU (P<0.05 vs. KON), zatiaľ čo

expresia Cxcr4 bola znížená iba v skupinách DAU a DAU+G-CSF (P<0.05 vs KON). Z rastových faktorov bolo zaznamenané

signifikantné zníženie Vegf vo všetkých DAU skupinách (P<0.05 vs KON), Igf-1 bol znížený v DAU-G-CSF (P<0.05 vs KON)

a LINA viedol k jeho zvýšeniu (P<0.05 vs DAU-G-CSF). Expresia Hgf bola downregulovaná v DAU-G-CSF (P<0.05 vs KON)

a upregulovaná v DAU-G-CSF-LINA oproti KON a ostatným DAU skupinám. V expresii markerov kmeňových buniek po

podaní DAU bola zaznamenaná downregulácia (Cd105, c-kit), upregulácia (Cd44) alebo neboli zaznamenané zmeny (Cd34,

Sca-1) oproti KON, zatiaľ čo terapia G-CSF+LINA viedla k zvýšeniu expresie markera mezenchymálnych kmeňových buniek

Cd44 (P<0.05 vs KON, P<0.05 vs všetky DAU skupiny). Podanie DAU bolo asociované s vysokou úmrtnosťou zvierat. Po 8.

týždňoch sa aplikácia DAU prejavila v zvýšení hladiny Nppa a znížení hladiny Myh6, čo poukazuje na poškodenie ľavej

komory srdca. Účinok DAU sa prejavil aj v expresii cytokínov (Sdf-1, Cxcr4, Scf) a čiastočne aj rastových faktorov (Vegf,

Igf-1), čo mohlo viesť v zníženiu chemoatrakcie kmeňových buniek. Downregulácia niektorých markerov kmeňových buniek

(Cd105, c-kit) môže indikovať zníženie uchytenie kmeňových buniek po podaní DAU. Benefičný účinok terapie G-CSF sa

prejavil v znížení mortality tesne po podaní DAU, zatiaľ čo kombinovaná terapia G-CSF+LINA ovplyvnením hladín Hgf a

Cxcr4 mohla viesť k zvýšenému uchyteniu kmeňových buniek exprimujúcich Cd44. Podanie DAU viedlo k zníženiu expresie

Sdf-1, Cxcr4 a Scf, čo môže prispievať k rozvoju DAU kardiomyopatie. Kombinovaná terapia G-CSF+LINA viedla k

upregulácii Hgf a čiastočnej obnove hladiny Cxcr4, čo by mohlo prispieť k zvýšeniu migrácie a uchytenia kmeňových buniek

v srdci, avšak zmeny v expresii markerov kmeňových buniek boli zaznamenané iba v prípade mezenchymálneho markera

Cd44. Táto praca bola podporená grantmi: FaF UK/54/2015, VEGA 1/0294/15, APVV-0887-11.

13

Cystatin-C appears to be better renal function marker for therapeutic drug monitoring based amikacin

dose adjustment in spinal cord injury (SCI) patients: Preliminary case series report Tesfaye H.1, Jedlickova B.1, Prusa R.1, Hakova R.2, Kriz J.2

1 Department of Medical Chemistry and Clinical Biochemistry, Division of Clinical Pharmacology, University Hospital Motol, 2nd Faculty of Medicine Charles University, Prague, Czech Republic

2 Department of Rehabilitation, Spinal Cord Unit, University Hospital Motol, 2nd Faculty of Medicine Charles University, Prague, Czech

Republic.

Serum creatinine (s-Cr) based estimation of the glomerular filtration rate (eGFR) is the most widely used test of renal

function. However, s-Cr levels are affected by a number of factors (age gender, muscle mass). Spinal cord injury itself is

associated with changes in volume of distribution, clearance, and drug half-life that can affect disposition of drugs including

aminoglycosides. Therefore, drug dosage based on s-Cr level in these patients can be very challenging. There is well founded

evidence that suggests s-Cr and various equations are not accurate for assessing renal function in SCI patients. Cystatin-C (cys-

C) may be a better option, although its use in routine practice is limited to recent date. Aim: In the present series of cases we

demonstrate the visibility of cys-C as better marker for renal function estimation and amikacin dose adjustment in SCI patients.

Case series of three Caucasian male (age 38ys 62ys, 63ys) plegic patients, who were on amikacin treatment either due to urinary

or respiratory tract infection were investigated. All the three patients received amikacin 1g/24 hours as initial therapy in

combination with defined doses of betalactam class of antibiotics. Plasma samples for amikacin levels determination were

collected in all cases just before the 2nd dose (trough) and half an hour after the application of the 2nd infusion (peak). The

plasma drug levels measured by fluorescent polarization immunoassay (FPIA) method were confronted with the serum

creatinine concentration based eGFR according to chronic kidney disease(CKD) equation versus cys-C concentration derived

GFR. Finally the dose adjustment has been done using the marker, which better corresponds with the observed drug

concentrations. Serum creatinine based eGFR extremely overestimated the renal function so that it may mislead the drug

dosage, while cystatin-C based estimation of GFR was with close agreement to the observed drug level. Due to certain reasons

including chronic immobility and muscle mass reduction, the low value of serum creatinine may not reflect the true renal

function as we have previously observed only slight elevation of serum creatinine associated with potentially toxic drug

concentration in a paraplegic patient treated with reduced dose of amikacin. In contrast to serum creatinine, cystatin–C is

declared to be stable marker, because it is not dependent on muscle mass, age, gender, and physical activity. Unfortunately,

many clinical pharmacokinetic soft ware providers have not yet incorporated this valuable marker for routine application in

eGFR including for drug dose adjustment. Nevertheless recent works including our experience in a number of case observations

prove that cystatin-C may replace serum creatinine to predict the extent of renal dysfunction in SCI patients. According to our

series of cases, cystatin-C appears very reasonable marker of actual renal function to guide amikacin dose adjustment improving

therapeutic drug monitoring in the care for SCI patients. We are incredibly grateful to the staff nurses for extraordinary care to

our patients. Many thanks also go to the laboratory staff for careful handling of samples and valid analysis to assure the unbiased

reports.

Metabolite of indoleamine 2,3-dioxygenase (IDO) L-kynurenine inhibits fibrotic signaling pathway and

proliferation of TGF-β stimulated NIH/3T3 cells Vavrinec P.1, Deelman L.E.2, Henning R.H.2, Cepcova D.1, Sandovici M.2,3, Vavrincova-Yaghi D.1

1 Department of Pharmacology and Toxicology, Faculty of Pharmacy, Comenius University in Bratislava, Slovakia, 2 Department of Clinical Pharmacy and Pharmacology, University of Groningen, University Medical Center Groningen, The Netherlands, 3 Department of Rheumatology and Clinical Immunology, University of Groningen, University Medical Center Groningen, The Netherlands

Renal fibrosis is a key pathological feature of chronic kidney failure, including the chronic transplant dysfunction. TGF-

β mediated pathway influences fibrotic processes and its inhibition could have beneficial effects. Indoleamine 2,3-dioxygenase

(IDO) is the rate-limiting enzyme in tryptophan catabolism. Due to its immunosuppressive effects IDO has an impact on the

graft acceptance. IDO also decreases the level of graft fibrosis through a currently unknown mechanism. We hypothesized that

a tryptophan metabolite, L-kynurenine, the result of IDO activity could play a role in the development and progression of

fibrosis by affecting TGF-β mediated signaling pathways. The aim of this study was to investigate the effect of L-kynurenine

on pSMAD2/3, pJNK, α-SMA and pERK on TGF-β mediated signaling pathways in mouse embryonal fibroblasts as well as

the effects of L-kynurenine on fibroblasts proliferation. NIH/3T3 mouse embryonal fibroblasts were stimulated 48 hrs with

recombinant TGF-β (20 and 30 ng/ml) and subsequently treated with L-kynurenine (KYN; 3 and 10 µmol/l). Untreated cells

served as control. To assess SMAD2/3 phosphorylation we used SMAD binding element (SBE)-luciferase reporter vector,

producing luciferase in response to SMAD2/3 activation. The expression of pJNK, α-SMA and pERK was assessed by

Western blotting. As a next step, we performed proliferation assay to determine the effect of same doses of L-kynurenine on

the fibroblast proliferation. Treatment of NIH/3T3 cells with TGF-β (20 and 30 ng/ml) increased significantly the expression

of pSMAD2/3, pJNK and α-SMA. We observed that kynurenin in both doses significantly decreased expression of pSMAD2/3,

pJNK and α-SMA after the stimulation with TGF-β in dose of 20 ng/ml. The expression of pERK was significantly decreased

by TGF-β and treatment of KYN had no effect. Moreover we also observed that kynurenin treatment markedly inhibited

NIH/3T3 proliferation. Based on these results we conclude that IDO metabolite L-kynurenine has promising potential in

treatment of renal fibrosis. However, further studies are required to elucidate its full effect on the TGF-β signaling. Hereby we

show for first time that L-kynurenine influences the TGF-β mediated signaling pathway and cell proliferation. This work was

supported by grants VEGA 1/0223/15 and VEGA 1/0667/14.

14

Dendritic cells transduced with indoleamine 2,3-dioxygenase suppress T cell proliferation via kynurenine

pathway Vavrincova-Yaghi D.1, Deelman L.E.2, Henning R.H.2, Gardlik R.3, Sandovici M.2, Vavrinec P.1

1 Department of Pharmacology and Toxicology, Faculty of Pharmacy, Comenius University in Bratislava, Slovakia, 2 Department of Clinical Pharmacy and Pharmacology, University of Groningen, University Medical Center Groningen, The Netherlands, 3 Institute of Molecular Biomedicine, Faculty of Medicine, Comenius University in Bratislava, Slovakia, 4 Department of Rheumatology and Clinical Immunology, University of Groningen, University Medical Center Groningen, The Netherlands

To steer the interaction between antigen presenting cells and T cells towards graft-specific tolerance represents an

appealing tool in the field of transplant allograft rejection. Indoleamine 2,3-dioxygenase (IDO) is one among the most

promising candidates in achieving the tolerogenic phenotype of antigen presenting cells. In this study, we investigated the

feasibility of enhancing the expression of IDO in dendritic cells (DC) both using an RGD modified adenoviral vector carrying

IDO gene and treatment with IDO inducer interferon-γ (IFN- γ). Moreover, we ivestigated the effect of DC treated with IDO

metabolite L-kynurenine (KYN). We analyzed the phenotype of adenoviral-transduced, IDO overexpressing DC, and both

IFN-γ and KYN treated DC. Moreover, we investigated the effects of DC on the proliferation and the phenotype of the T cells

in vitro. DC were obtained from bone marrow of Brown Norway rats. Nine days after harvesting, DC were transduced with an

RGD modified adenovirus carrying the gene for human IDO (RGD-AdTIDO) or the gene for luciferase (RGD-AdTL).

Moreover, cells were treated with IFN-γ (100 µmol/l) and KYN (10 µmol/l). Untransduced and untreated DC served as control.

At day 11, DC were used as stimulators in a mixed leukocyte reaction (MLR) with naïve Lewis rat lymphocytes as responders.

T cells isolated from Brown Norway served as syngeneic control. FACS and real-time PCR were performed to assess the

phenotype of the dendritic cells and T-cells, respectively. FACS analyses of DC transfected with adenovirus, treated with IFN-

γ and KYN has shown CD11c+, CD86+ and MHCII+ phenotype, respectively. Moreover, DC overexpressing IDO suppressed

the proliferation of the naïve T cells, as well as DC treated with KYN. This was associated with up-regulation of foxp3, IL-10

and IL-13 gene expression on one hand and on other hand with up-regulation of IL-2 and IL-17 gene expression. However,

treatment with IFN-γ did not influence the T cell proliferation. We observed that both IDO-overexpressing and KYN treated

DC inhibited the proliferation of the naïve T cells in vitro. Moreover, these T cells exhibited tolerogenic and anti-inflammatory

signature, as well as a pro-inflammatory cytokine profile. Whether the anti- or the pro-inflammatory effects predominate in

vivo remains to be established. Dendritic cells overexpressing IDO and/or treated with L-kynurenine can be a promising tool

in the therapy of transplant rejection. This work was supported by grants VEGA 1/0667/14 and VEGA 1/0223/15.

Differences in the vascular reactivity of diabetic and spontaneously hypertensive rats figured by computer-

based modeling Vojtko R., Petrová M., Dobiaš L., Kristová V.

Department of Pharmacology and Clinical Pharmacology, Faculty of Medicine, Comenius University, Bratislava

Current recording of vascular reactivity of perfused arterial segments usually offers two basic methods for quantifying

of numeric outputs: classical descriptive evaluation and analysis using computer-based modeling. Our aim was to evaluate both

of them in models of rat diabetes and spontaneous hypertension and to assess methodological benefits. Segments of rat renal

arteries were subjected to series of contractions induced by seven successively increasing bolus doses of noradrenaline and in

state of precontraction were exposed to final relaxation induced by single bolus dose of acetylcholine. Data extraction continued

by digital model analysis using methods Levy and Monte Carlo. We found out a clear reduction of relaxatory responses in the

diabetic group compared to controls. In contrast with nonsignicant differences between groups assessed by descriptive

evaluation, computational analysis revealed several significant differences of model parameters, especially in sensitivity and

AIC in the diabetic group vs. controls. Evaluation of contractile responses by the descriptive methods did not unveil significant

differences between groups at any used dose of noradrenaline. Computer modeling enables a much deeper insight into the

mechanisms of vascular reactivity alterations, resulting in the finding of changed characteristic parameters of contraction

profiles. Software design proposed for measuring, recording and computational modeling of perfused artery contractile

responses meets the expectations of accuracy and high reproducibility of data extraction. Therefore it seems to be a new

approach and promising improvement over recent routine methods. Financial support by the grant VEGA SR nr. 1/0886/14

and VEGA SR nr. 1/0186/16 is gratefully acknowledged.

15

POSTERY – PREZENTOVÁNY 16.9.2015

HYPERBILIRUBINAEMIA DECREASES PHYSIOLOGICAL MARKERS IN THE ADJUVANT-

INDUCED ARTHRITIS

Bauerová K., Dráfi F., Kuncírová V., Poništ S., Mihalová D., *Babál P.,*Sýkora T.

Institute of Experimental Pharmacology and Toxicology, Slovak Academy of Sciences, Dúbravská cesta 9, Bratislava, Slovak Republic;

katarina.bauerova@savba.sk *Institute of Pathological anatomy, FM CU and UHB in Bratislava, Špitálska 24, Bratislava, Slovak Republic.

Key words: bilirubin, hyperbilirubinaemia, autoimmune diseases, adjuvant-induced arthritis, rheumatoid arthritis

There is a clinical evidence that higher serum level of bilirubin (BIL) may be a protective factor for autoimmune diseases.

We examine BIL in adjuvant-induced arthritis (AIA) where oxidative stress, inflammation and inadequate immune response

are present. Male Lewis rats were randomised into groups: CO – control, AIA – untreated adjuvant-induced arthritis, CO-BIL

– control administrated BIL (200 mg/kg b.w. daily i.p. during 28 days), AIA-BIL – adjuvant-induced arthritis administrated

BIL (as described above). Change of hind paw volume in AIA-BIL group in comparison to AIA group was significantly

decreased after BIL administration. In CO and AIA groups we found almost untraceable levels of BIL. In CO-BIL group and

in the AIA-BIL group hyperbilirubinaemia was observed. BIL administration significantly decreased the level of CRP in AIA-

BIL group in comparison to AIA group. The values of white and red blood cells, haemoglobin and haematocrit were

significantly decreased in AIA-BIL after bilirubin administration. Liver, spleen and thymus, weighted less in AIA than in AIA-

BIL. Histological findings showed decreased or even absent damage in hind paw joint of AIA-BIL animals. We showed a

immunomodulatory effect of BIL on the AIA development which may also have a novel pharmacological and toxicological

impact. VEGA 2/0045/11 and VEGA 2/0044/15.

Porovnání protektivních účinků 6-gingerolu a kurkuminu v oxidačním poškození jater arsenem

Bludovská M., Kovaříková P., Potočová I., Soukupová V., Kotyzová D., Kmoníčková E.

Ústav farmakologie a toxikologie, LF UK v Plzni

Klíčová slova: 6-gingerol, kurkumin, oxidační stres, arsen

Arsenité sloučeniny jsou známé svými toxickými účinky. Důležitou roli zejména v hepatotoxickém působení hraje

oxidační stres. Přírodní fenoly izolované z rostlin čeledi Zingiberaceae, 6-gingerol (6-G) a kurkumin (CUR) jsou studovány

pro své antioxidační účinky. Cílem práce bylo zjistit možné protektivní účinky těchto dvou rostlinných fenolů v oxidačním

poškození jater arsenem (As3+) v pokusu in vivo. V pokusu na myších samcích (ICR, 32±3g) byly podány ekvimolární dávky

6-gingerolu (30 mg/kg těl.hm.) nebo kurkuminu (40 mg/kg těl.hm.), perorálně, 1x denně, celkem 2 dávky. Následně byl podán

NaAsO2 (7 mg/kg), intraperitoneálně, 1x, kontrolním a premedikovaným zvířatům. V jaterní tkáni byly stanoveny následující

markery oxidačního stavu: peroxidace lipidů (LP), redukovaný glutathion (GSH), aktivity antioxidačních enzymů glutathion

peroxidázy (GPx), katalázy (CAT) a glutathion reduktázy (GR). V jaterní tkáni způsobila intoxikace As3+ oxidační stres

vyjádřený signifikantním zvýšením (p <0.01) hladiny LP (138% kontrolní hladiny), deplecí GSH (72% kontr.) a snížením

aktivity CAT (78% kontr.). Premedikace CUR snížila negativní vliv As3+ na LP a GSH; premedikace 6-G kompletně zabránila

navýšení LP a depleci GSH. Snížení aktivity CAT po As3+ nebylo premedikací 6-G ani CUR ovlivněno. Deplece GSH u

intoxikovaných zvířat byla doprovázena zvýšenou aktivitou GR; u premedikovaných zvířat byla aktivita GR na kontrolní

hladině. Oba rostlinné fenoly působily protektivně proti As3+ indukovanému oxidační poškození jater. Ve srovnání s

kurkuminem vykazoval 6-gingerol v tomto experimentálním uspořádání výrazně lepší antioxidační účinky. Práce byla finančně

podpořena specifickým vysokoškolským výzkumem LF UK v Plzni, SVV 02674/2015.

THE INFLUENCE OF SEX ON OXIDATIVE STRESS AND MARKERS OF LIVER DAMAGE

INDUCED BY SUBCHRONIC ADMINISTRATION OF THIOACETAMIDE IN MALE AND FEMALE

WISTAR RATS

Bludovská M.a, Mistrová E.b, Kotyzová D.a, Jarkovská D.b, Chottová-Dvořáková M.b

a Department of Pharmacology and Toxicology, b Department of Physiology and Biomedical Centre, Charles University in Prague, Faculty of Medicine in Pilsen, Czech Republic

Key words: thioacetamide, liver, rat, lipid peroxidation, kidney, oxidative stress

Thioacetamide (TAA) is frequently used in experimental models of drug-induced acute and chronic liver injury.

Generation of reactive oxygen species plays an important role in the pathogenesis of TAA toxicity. TAA-induced liver damage

in rats has been often used for the evaluation of hepatoprotective and antioxidant effects of various substances.The aim of the

study was to evaluate the influence of gender on the oxidative stress in the liver and kidneys and markers of liver damage in

serum induced by subchronic administration of TAA in Wistar rats. TAA was administered over a period of 12 weeks

(intraperitoneally 200 mg/kg body weight, three times per week). Animals were sacrificed 8 weeks after the treatment

discontinuation. Lipid peroxidation (LP), reduced glutathione, activities of glutathione peroxidase, glutathione reductase and

catalase were estimated in liver and kidney homogenates. Activities of ALT, AST and GLDH were measured in the serum.

16

Very significant differences were found in some of the markers of oxidative stress. A significantly higher increase in LP was

found in male rats in the liver (by 45% vs. 8% in female rats; p<0.001) and in the kidneys (by 44% vs. 10% in female rats;

p<0.01). The decrease in glutathione peroxidase activity in the liver was significantly deeper in male rats (by 38% vs. 17% in

female rats; p<0.001). The activity of liver catalase was decreased in female rats only. Significant increase in GLDH activity

was seen in both sexes, the increase of AST activity was significant only in male rats and the increase in ALT activity was not

significant. Male Wistar rats showed higher susceptibility to oxidative stress induced by subchronic administration of TAA.

The response to TAA-induced oxidative liver damage in rats is influenced by gender and this fact might affect the evaluation

of antioxidant properties of various therapeutic agents. Support: CZ.1.05/2.1.00/03.0076 from European Regional

Development Fund and Charles University Research Fund (P36).

Effect of sulodexide on vascular responses and liver mitochondrial function in diabetic rats

DOBIAŠ L.1, PETROVÁ M.1, VOJTKO R.1, ULIČNÁ O.2 , VANČOVÁ O.2, KRISTOVÁ V.1

1 Department of Pharmacology and Clinical Pharmacology, Faculty of Medicine, Comenius University, Bratislava, Slovak Republic, 2 Pharmacobiochemical Laboratory of Third Department of Internal Medicine, Comenius University, Bratislava, Slovak Republic

Key words: Femoral artery, diabetes, sulodexide, oxidative phosphorylation

This study investigates the effects of long-term treatment with sulodexide (SLX) on norepinephrine(NE)-induced

contractions, acetylcholine(Ach)-induced relaxations, acute cyclooxygenase blockade by diclofenac (DIC) in isolated femoral

arteries (FA) and the parameters of oxidative phosporylation in liver mitochondria. 15-weeks old Wistar rats were divided into

four groups: control (C; injected with saline solution), treated control (C+SLX), diabetic (DM) and treated diabetic (DM+SLX).

Diabetes was induced with a single i.v. dose of streptozotocin (STZ) 45 mg.kg-1. SLX was administered i.p., at dose 100 IU.kg-

1 daily for 5 weeks. Vascular responses of isolated femoral arteries were measured using Mulvany-Halpern myograph.

Respiratory function of the mitochondria was determined using voltamperometric method on oxygraph Gilson. In diabetic rats

the NE dose-response curve was shifted to the left significantly and the amplitude of maximal response was elevated. DIC

pretreatment decreased the amplitudes of NE-induced contractions in all groups of rats. SLX treatment decreased sensitivity of

FA to NE and caused higher relaxatory responses to Ach in C and DM rats. Oxygen consumption rate in state 3 [QO2(S3)],

state 4 [QO2(S4)], oxidative phosphorylation rate (OPR) and respiratory control ratio (RCR) were decreased in the

mitochondria of DM rats. Mitochondria of C rats were not affected with SLX treatment. Administration of SLX in DM was

associated with increase of RCR, while other parameters were not affected. There are growing body of evidence that SLX

possess antithrombotic, antiinflammatory and endothelial-protective effects. Treatment with glycosaminoglycans recovers the

damaged endothelial glycocalyx, which appears as an initial step in the pathology of vascular complications during diabetes

mellitus. Our findings suggest that SLX treatment might be associated with vasculoprotective effects during diabetes and

improvement of mitochondrial function. The experimental work was supported by the research Grant UK/330/2014, Grant

VEGA 1/0886/14 and VEGA1/0058/15.

Can the Bioactive Compounds of Crocus sativus L. Influence the Metabolic Activity of Selected CYP

Enzymes in Rat?

Dovrtělová G.1,2, Nosková K.1,2, Juřica J.1,2, Turjap M.1,2, Zendulka O.1,2

1 CEITEC (Central European Institute of Technology) Masaryk University, Brno, Czech Republic 2 Department of Pharmacology, Faculty of Medicine, Masaryk University, Brno, Czech Republic

Key words: crocin, safranal, CYP, rat liver microsomes

Safranal and crocin are biological active compounds of Crocus sativus L. (saffron) which is a potentially valuable

alternative in the treatment of depression. The aim of our study was to determine the effect of systemic administration of

safranal and crocin on the metabolic activity of CYP2C11, CYP3A, CYP2B and CYP2A and the total protein content in rat

liver microsomes (RLM). The experiments were carried out on male Wistar albino rats. Each substance was administered in

three different concentrations. Liver microsomal fraction was then isolated from liver tissue of each animal and the total protein

content and total CYP content were determined. The activities of the CYP2C11, CYP3A, CYP2B and CYP2A enzymes were

assessed based on the rate of testosterone biotransformation in RLM with NADPH generating system. Metabolic activity was

expressed as metabolite concentration/min/mg of total protein in RLM. In the Experiment I, animals were administered

intragastrically with safranal dissolved in a mixture of propylene glycol and 5% glucose (1:1) at the doses of 4, 20 and 100

mg/kg/day for 10 consecutive days. The control group was administered with the vehicle in appropriate volume. In the

Experiment II, animals were treated intraperitoneally with crocin dissolved in saline at the doses of 4, 20 and 100 mg/kg/day

for 9 consecutive days. The control group was administered with saline (1 ml/kg). Safranal significantly increased the metabolic

activity of CYP3A, CYP2B, CYP2A enzymes only at the highest dose, although dose-dependent increase in the enzyme activity

was observed for most monitored enzymes. Crocin significantly increased the metabolic activity of all tested CYP enzymes,

again at the highest dose. Results revealed that safranal significantly increased the total protein content (128 % of the control

group content) but crocin did significantly decrease it (46 % of the control group content). Safranal and crocin influence the

metabolic activity of CYP enzymes, which are the major metabolic enzyme system for exogenous and endogenous compounds.

In case of use of these substances in clinical practice it would be necessary to take into account the risk of interactions with

substances metabolized exclusively by the same CYP enzyme. This risk becomes more serious as food supplements containing

saffron extracts are already available. To the best of our knowledge, no studies exist up to date dealing with the effects of

saffron or its compounds on CYP enzymes. Our results demonstrate the ability of systemic administration of safranal and crocin

17

the ability to change the total protein content in RLM and to increase the metabolic activity of several CYP enzymes, thereby

to increase the risk of interactions with co-administered substances metabolised by the same pathway. This work was supported

by projects CZ.1.05/1.1.00/02.0068 and MUNI/A/0885/2013.

Probiotické kmene ako súčasť výživových doplnkov používaných v lekárenskej praxi v Slovenskej

republike v rokoch 2013 - 2014

Fedorová M.1, Pirník Z.2, Marcinčáková D.3, Pistl J.4, Rychnavská D.1

1 Katedra lekárenstva a sociálnej farmácie, Univerzita veterinárskeho lekárstva a farmácie v Košiciach, Slovenská republika, 2 Ústav humánnej a klinickej farmakológie, Univerzita veterinárskeho lekárstva a farmácie v Košiciach, Slovenská republika, 3 Ústav farmakológie, Univerzita veterinárskeho lekárstva a farmácie v Košiciach, Slovenská republika, 4 Ústav mikrobiológie a gnotobiológie, Univerzita veterinárskeho lekárstva a farmácie v Košiciach, Slovenská republika

Klíčová slova: probiotické kmene, výživové doplnky, exopolysacharidy

Probiotiká sú definované Svetovou zdravotníckou organizáciou ako živé mikroorganizmy, ktoré ak sú podávané v

primeranom množstve, udeľujú zdravotné výhody hostiteľovi. Medzi probiotiká radíme baktérie mliečneho kvasenia,

Escherichia coli, ako aj kmene kvasiniek Saccharomyces boulardii a Saccharomyces cerevisiae. Probiotiká, s ktorými sa

farmaceuti pri poskytovaní lekárenskej starostlivosti vo verejných lekárňach na Slovensku stretávajú, sú poväčšine výživové

doplnky (VD) radené medzi potraviny. Cieľom tejto práce bolo zanalyzovať spektrum používaných probiotických kmeňov vo

VD dostupných v lekárenskej praxi v období rokov 2013 - 14 a zhodnotiť ich vo vzťahu k aktuálnym poznatkom v odbornej

literatúre. Informácie o dostupných výživových doplnkoch s obsahom probiotických kmeňov boli zbierané v rámci 5-mesačnej

lekárenskej praxe. Vo VD sa celkovo vyskytovalo 28 probiotických kmeňov mikroorganizmov, ktoré sa vyskytovali v rámci 8

rodov (Bacillus, Bifidobacterium, Enterococcus, Lactobacillus, Lactococcus, Pediococcus, Saccharoromyces, Streptococcus).

Až 77% VD (n=88) malo viaczložkové zastúpenie probiotických kmeňov. 44% prípravkov obsahovalo prebiotiká (s obsahom

polysacharidov), 32% prídavok vitamínov, rastlinných extraktov, resp. minerálov. Najčastejšie sa odporúčalo užívanie pri

obnove črevnej mikroflóry, pri podpore činnosti imunitného systému a ako súčasť antibiotickej terapie. Posúdenie relevantnosti

použitia jednotlivých probiotických kmeňov v odporúčaných indikáciách vo väčšine prípadov zlyhávalo na nešpecifikovaní

zbierkového čísla daného probiotického kmeňa vo VD. Z klinicky relevantných faktorov nebola uvádzaná schopnosť produkcie

exopolysacharidov, ktoré majú zdokumentovanú antioxidačnú aktivitu, imunomodulačné a antibakteriálne účinky, ako ani

schopnosť produkcie vitamínu K2. Očakávaný benefit užívania probiotík vo VD môže byť preto u pacientov z hľadiska vyššie

uvedených dôvodov minimálne kontroverzný. Táto práca bola podporená projektom VEGA: 1/0379/13.

Nárast srdcovej expresie endotelovej NO syntázy v metabolickom syndróme indukovanom dlhodobou

konzumáciou kolového nápoja u Wistar potkanov

Galková K., Malíková E., Kráľová E., Vavrinec P., Čavojský T., Křenek P., Klimas J.

Katedra farmakológie a toxikológie, Farmaceutická fakulta Univerzity Komenského, Odbojárov 10, 832 32 Bratislava, Slovenská republika

Klíčová slova: endotelová syntáza oxidu dusnatého, metabolický syndróm, kolový nápoj, potkan

V prípade metabolického syndrómu, doposiaľ nebol etablovaný experimentálny model s využitím kolového nápoja u

potkanov. Naším cieľom bolo popísať vplyv dlhodobej konzumácie colového nápoja na expresiu kardiálnych proteínov NO

signálnej kaskády. Samce potkanov Wistar dostali štandardnú stravu, pričom jedna skupina potkanov mala neobmedzený

prístup ku komerčnému kolovému nápoju (CC, n=12). Kontrolné zvieratá (CON, n=7) pili pitnú vodu. Merali sme prírastky na

váhe, hladiny triglyceridov (TG) a cholesterolu (CHOL) štandardným glukomerom v kvapkách kapilárnej krvi. Stanovenie

glukózovej intolerancie sme vykonali konvenčne, pomocou orálneho glukózového tolerančného testu (oGTT).

Hemodynamické parametre srdcová frekvencia (SF), systolický a diastolický krvný tlak (STK a DTK) boli merané tail-cuff

metódou. Expresia proteínov bola stanovená metódou Western blotting v ľavej komore. Zaznamenali sme významne zvýšenú

telesnú hmotnosť u CC potkanov (541±12 g, p <0,01) v porovnaní s kontrolami (443±23 g). Súčasne sme namerali zvýšený

STK (CC: 135±3 mmHg vs. CON: 120±4 mmHg, p <0,01) a nezmenený DTK (CC: 86±1 mmHg vs. CON: 85±1 mmHg; NS)

a taktiež sa signifikantne zvýšila SF (CC: 383±9 t/min. vs. CON: 312±15 t/min., p <0,01). Signifikantný rozdiel postprandiálnej

glykémie medzi skupinami bol prítomný po 60 minútach (CC: 9,3±0,5 mmol/l vs. CON: 6,6±0,6 mmol/l, p <0,01) a pretrval

aj po 90 minútach (CC: 8,3±0,4 mmol/l vs. CON: 6,6±0,5 mmol/l; P <0,01). Hladina TG nebola zmenená (CC: 5,2±0,4 mmol/l

vs. CON: 5± 0,5mmol/l; NS). Rovnako, hladiny CHOL neboli zmenené (CC: 3,7 ± 0,4 mmol/l vs. CON: 4,0 ± 0,4 mmol/l;

NS). Zístili sme signifikantné zvýšenie srdcovej expresie endotelovej NO syntázy (eNOS) (CC: 142±26% vs. CON: 100±26%,

p <0,01), pričom expresie jej modulátorov (heat shock protein; hsp90 a kaveolínu-1; CAV-1) boli nezmenené. Šesťmesačná

konzumácia kolového nápoja môže viesť k rozvoju kardiovaskulárnych (zvýšený STK, SF), molekulárnych (nárast expresie

eNOS) a rovnako i metabolických prejavov (porucha glukózovej tolerancie) u Wistar potkanov. Naše výsledky ukazujú tento

model ako spoľahlivý experimentálny model metabolického syndrómu u potkanov pre budúce uplatnenie vo farmakologických

experimentoch. Grantová podpora: APVV-0887-11, VEGA1 / 0564/13

18

VZTAH PLAZMATICKÝCH HLADIN LEPTINU A INSULINU A RŮZNÝMI FÁZEMI ZÁVISLOSTI

NA MORFINU V MODELU U POTKANŮ.

Havlíčková T., Jeřábek P., Lacinová Z., Potměšil P., Šustková-Fišerová M.

Ústav farmakologie 3. LFUK v Praze

V posledních letech vzrůstá počet prací, jež zkoumají vztahy mezi hormony ovlivňujícími apetit (včetně anorexigenního

leptinu a insulinu), poruchami příjmu potravy a drogovými závislostmi s nadějí, že studium povede k nalezení nového

léčebného přístupu k závislostem. Tyto vztahy u opioidní závislosti byly zatím zkoumány minimálně. Cílem naší studie bylo

monitorovat změny plazmatických hladin leptinu a insulinu v různých fázích morfinové závislosti v potkaním modelu, protože

případné nalezené korelace by doložily účast těchto hormonů v mechanismech morfinové závislosti, a tak podpořily představu

možného využití těchto mechanismů při vývoji nových léčiv pro terapii opioidních závislostí. U „opioidní“ skupiny (11 zvířat)

jsme podávali opakovaně morfin, jedenkrát denně ve zvyšující se dávce (10, 20, 20, 40, 40 mg/kg s.c.), pak následovala

abstinence provázená v prvních 3 dnech projevy somatických abstinenčních příznaků. Desátý až dvanáctý den abstinence jsme

podali „challenge“ dávku morfinu. „Kontrolní“ skupině (6 zvířat) jsme aplikovali opakovaně fyziologický roztok a

(akutní/„challenge“) dávku morfinu až v úplném závěru. Vzorky krve jsme odebírali z ocasní žíly opakovaně celkem pětkrát.

Leptin a insulin jsme následně stanovovali v plasmě metodou multi-ELISA (ve spolupráci s VFN). Oproti kontrolní skupině i

vlastním bazálním hodnotám byly hladiny insulinu i leptinu v závěru denních aplikací morfinu a během abstinenčních příznaků

významně nižší. V závěru dlouhodobější abstinence se hladiny leptinu vrátily k bazálním hodnotám a insulin je dokonce

významně přesáhl. „Challenge“ dávka morfinu (i akutní dávka morfinu u kontrolní skupiny) navodila u obou peptidů pokles

hladin. Nalezené významné změny hladin insulinu a leptinu v průběhu modelové morfinové závislosti, doložily účast obou

peptidů v mechanismech opioidní závislosti a podporují tak další výzkum možného terapeutického využití těchto mechanismů

u opioidních závislostí. Studie byla podpořena PRVOUK 34 a IGA NT 13687-3.

Dynamika změny plazmatické hladiny leptinu při chronické aplikaci risperidonu u laboratorního potkana

Horská K.1, Kotolová H.1, Karpíšek M.1,2, Suchý P.1

1 Ústav humánní farmakologie a toxikologie, Farmaceutická fakulta, Veterinární a Farmaceutická Univerzita, Palackého 1/3, Brno 612 42 2 BioVendor – Laboratorní medicína a.s, Karásek 1, Brno 621 00

Klíčová slova: leptin, risperidon

Cílem studie je posouzení vlivu intramuskulárně aplikovaného SDA atypického antipsychotika risperidonu (Risperdal

Consta®) u laboratorního potkana na rozvoj metabolických komplikací včetně rané alterace produkce relevantních adipokinů

– biomarkerů a potenciálních prediktorů metabolického syndromu, významně se podílejících na metabolických regulacích a

energetické homeostáze. Experiment byl proveden na laboratorních potkanech kmene Sprague-Dawley (Harlan) samičího

pohlaví, stejného staří (devět týdnů) s hmotností v rozmezí 200-250 g. Léčené skupiny byly aplikovány intramuskulárně

risperidonem (Risperdal Consta®) ve 14-ti denním intervalu. Placebová skupina byla aplikována intramuskulárně

fyziologickým roztokem. Experiment byl ukončován po 4, 6 a 10 týdnech. Biochemické parametry (leptin, IL – 6, IL – 1,

PAI, glukagon, glukagonlike peptid) byly stanoveny spektrofotometricky na Bio-Plex® readeru za použití setů firmy Bio-Rad.

V našem experimentu jsme prokázali vliv depotně podávaného risperidonu laboratornímu potkanovi na změnu hladiny leptinu,

IL – 6, IL – 1, glukagonu, glukakonlike peptidu v chronickém podávání v 6 a 10 týdnu léčby. Hladina PAI nebyla léčbou

ovlivněna. Leptin patří mezi endokrinně aktivní látky produkované tukovou tkání a hraje významnou roli v regulaci tělesné

hmotnosti, příjmu potravy i energetického výdeje. V našem pokusu jsme zaznamenali statisticky významně zvýšenou hladinu

leptinu u skupiny léčené risperidonem(40mg a 20 mg) 6 a 10 týdnů oproti skupině kontrolní. Náš experiment prokázal

statisticky zvýšené hodnoty IL – 6 a IL-1 u skupiny léčené risperidonem(40mg a 20 mg) 6 týdnů oproti skupině kontrolní.

Antipsychotiky indukovaný zánětlivý stav může přispívat k prozánětlivému stavu, který nacházíme u schizofrenních pacientů,

ale i k rozvoji inzulinové rezistence a kardiovaskulárních onemocnění. Odpověď na otázku jakým mechanizmem

antipsychotika indukují zvýšení tělesné hmotnosti a rozvoj kardiometabolických komplikací zatím není plně objasněna. Jednou

ze zkoumaných možností je zda AAP mohou narušit sekreci nebo signalizaci leptinu a dalších relevantních adipokinů.

Výsledky tohoto experimentů naznačují, že risperidon může ovlivněním sekrece relavantních adipokinů a hormonů akcelerovat

rozvoj metabolického syndromu.

New findings in the etiopathogenesis of tinnitus: prothrombogenic activity measured by levels of

thromboxane correlates with hearing disorders and tinnitus

Chrbolka P., Paluch Z., Hill M., Adámek T., Alušík Š.

Thomayerova nemocnice Praha

Key words: tinnitus – hearing impairment – vascular anomalies –– Meniere’s disease – vestibular schwannoma – multiple

sclerosis – 11-dTxB2

Aim of the study: The aim of the work is question, if patients with primary tinnitus have higher prothrombogenic activity

measured by determining the level of 11-dTxB2 and other coagulation parameters. Tromboxanes are a potent platelet agonists

and vasoconstrictors, which is thought to be of functional importance in the precipitation of certain vascular occlusive diseases.

Methods: The study group includes patients without evidence of organic causes of tinnitus, without cause of non-vascular

hearing impairment. Laboratory exams includes CBC, biochemistry, coagulation activity and 11-dTxB2. To exclude pathology

19

in cerebellopontine area was use CT scan or MRI imaging, together was make X-ray of cervical vertebra. For the purpose of

this study, whole blood samples obtained were screened for 11-dTxB concentrations using commercial kits. Results: The main

indicator of prothrombogenic activity was monitoring levels of 11-dTxB2 compared with the control group. We see increased

levels of prothrombogenic indicators, mainly levels of 11-dTxB2. Initial consideration of the possible use of drugs affecting

haemostasis and thus help patients suffering from hearing loss and tinnitus is by our exploration confirmed. It turns out that the

question of the relationship between hearing impairment, tinnitus and perfusion of sensory organs are very narrow and

interdependent. This hypothesis is supported by our results, which speaks of a higher tendency to clotting precisely in these

patients. Influence of atherosclerotic changes together with decrease cardiac output, arrhythmia, heart failure, hypertensive

encephalopathy, rheological properties of blood, often potentiated by dehydration in the elderly. All of these relationships to

each other gives the impression that increased thrombogenic activity may be associated with impaired hearing and tinnitus.

These considerations relationship is very important because it can pronounce the possibility of pharmacological effect on blood

clotting and slow down the progression of hearing loss or even improve the current state of the mitigation tinnitus. In the

literature are described protective effects of non-steroidal anti-inflammatory drugs (NSAIDs) on cochlear injury. They are

usually used as painkillers and antipyretics. Classic NSAIDs inhibits actitivy of cyclooxygenase. Various protective effects of

NSAIDs on cochlear injury have been reported in animal studies. Further studies on the arachidonic cascade in the cochlea and

its inhibitors (or modulators) will shed light on the understanding of the generation mechanisms of various cochlear injuries.

Conclusion: From the study it can be concluded that there is a relationship between primary tinnitus and increased levels of

prothrombogenic indicators, mainly levels of 11-dTxB2.

Dual role of neutrophil-derived oxidants in inflammation and its implication in the efficacy of anti-

inflammatory therapy

Jančinová V., Pažoureková S., Lucová M., Perečko T., Nosáľ R., Drábiková K.

Institute of Experimental Pharmacology and Toxicology, Slovak Academy of Sciences, Bratislava

Key words: neutrophils, reactive oxygen species, hydroxychloroquine, NADPH oxidase, protein kinase C, intracellular

calcium

Neutrophilic polymorphonuclear leukocytes liberate reactive oxygen species (ROS), operative not only in the elimination

of invading pathogens but also in tissue damage and in the development of persisting inflammation. From this perspective,

pharmacological intervention capable to diminish the release of oxidants from neutrophils represents a prospective way to

support the therapy of chronic inflammatory diseases. On the other hand, recent findings suggest that ROS formed inside

neutrophils can act as suppressors of inflammation. The protective role was confirmed by hyper-inflammatory responses found

in patients whose neutrophils displayed severely decreased intracellular ROS formation. The therapy of chronic inflammation

should thus preferentially decrease external oxidants, keeping ROS formed inside neutrophils untouched. The present study

examines whether this applies to hydroxychloroquine - a drug widely used in patients with rheumatoid arthritis. Neutrophils

were isolated from blood of healthy male donors who had not received any medication for at least seven days. Extra- and

intracellular formation of oxidants were evaluated on the basis of isoluminol- and luminol-enhanced chemiluminescence. The

phosphorylation of regulatory proteins (p40phox, PKCα, PKCβII and PKCδ) was assessed by western blotting, using

phosphospecific antibodies and luminescence detection. Mobilisation of intracellular calcium was recorded by flow cytometry

in Fluo-4 AM loaded neutrophils. Hydroxychloroquine decreased the concentration of extracellular oxidants, reduced calcium

mobilisation and inhibited the phosphorylation of Ca2+-dependent protein kinase C isoforms PKCα and PKCβII, which regulate

activation of NADPH oxidase on the plasma membrane. On the other hand, no reduction was observed in the formation of

reactive oxygen species inside neutrophils or in the phosphorylation of p40phox and PKCδ, two proteins directing the NADPH

oxidase assembly to intracellular membranes. Reactive oxygen species produced by neutrophils can exert pro- or anti-

inflammatory effects, with respect to their extra- or intracellular location. The optimal therapy should thus preferentially

decrease external radicals which may increase the risk of tissue damage and lead to permanent inflammation. On the other

hand, oxidants arising inside neutrophils should not be affected as they are involved in intracellular signalling and can suppress

inflammation. Hydroxychloroquine met these criteria and the interference with neutrophil-derived oxidants may represent a

new mechanism potentially involved in the anti-inflammatory activity of this drug. The study was supported by grants APVV-

0052-10 and VEGA 2/0010/13

In vitro a in vivo hodnocení kardioprotektivních účinků dusitanů a dusičnanů vůči antracyklinové

kardiotoxicitě

Jansová H.1, Lenčová O.2, Jirkovský E., Vostatková L.1, Jirkovská A., Pokorná Z.2 , Šimůnek T.1, Štěrba M.2

1 Farmaceutická fakulta v Hradci Králové, Univerzita Karlova v Praze, Česká Republika 2 Lékařská fakulta v Hradci Králové, Univerzita Karlova v Praze, Česká Republika

Klíčová slova: kardiotoxicita; ANT; dusitany/dusičnany

Antracykliny (ANT) jsou velmi účinná a široce užívaná protinádorová léčiva. Jejich užití v klinické praxi je

komplikováno jejich kardiotoxicitou. Kardioprotektivní účinek anorganických dusitanů/dusičnanů byl již dříve popsán u

ischemicko-reperfuzního poškození a v současnosti je zde v klinickém hodnocení. Stejně tak byla popsána i kardioprotekce u

akutní toxicity navozené ANT. Jejich použití u klinicky podstatně významnější chronické ANT kardiotoxicity ale zůstává stále

nejasné. V našich experimentech byly použité neproliferující H9c2 myoblasty a neonatální izolované kardiomyocyty ze srdce

potkana (NVCM). Ke stanovení jejich viability byla použita metoda MTT a stanovení LDH. Pro stanovení změny

20

mitochondriálního membránového potenciálu byla použita fluorescenční sonda JC1. Apoptotická signalizace byla stanovena

pomocí měření aktivit příslušných kaspas. V in vivo pokusech byl použit zavedený model chronické ANT kardiotoxicity u

králíků (DAU; 3 mg/kg týdně po dobu 10 týdnů). Pro určení genové exprese vybraných parametrů byla použita kvantitativní

RT-PCR metoda. Naše in vitro pokusy ukázaly, že dusitan sodný je schopný signifikantně chránit mitochondrie před jejich

depolarizací navozenou daunorubicinem (DAU; 1,2µM). Stejně tak dokážou snižovat apoptotickou signalizaci navozenou

DAU. Tato látka ale nebyla schopna signifikantně ochránit buňky před celkovou ztrátou viability způsobenou DAU. In vivo

pokusy ukázaly, že dusičnan sodný (1g/l p.o.) a nižší dávka dusitanu sodného (0,15 mg/kg i.v.) významně neovlivňují DAU

navozenou kardiotoxicitu. Naopak dusitan sodný ve vyšší dávce (5 mg/kg i.v.) byl schopný signifikantně chránit myokard před

ztrátou mitochondrií, poruchou mitochondriální biogeneze a také byl schopen významně zmírnit patologické změny v

proteinech regulujících homeostázu vápníku. Ačkoliv tato intervence zabránila předčasnému úhynu zvířat, evidentně

nedokázala účinně předejít vzniku srdeční dysfunkce, remodelaci myokardu a uvolňování srdečního troponinu T do cirkulace.

Vyšší dávky dusitanu sodného pozitivně modulují vybrané molekulární aspekty chronické ANT kardiotoxicity (především ty

spojené s mitochondriálním poškozením a apoptózou), ale kardioprotektivní potenciál této látky je evidentně velmi omezený.

Tato studie byla financována projektem IGA číslo NT/13457-4-2012.

Je chelatační metabolit (ADR-925) zodpovědný za kardioprotektivní účinky dexrazoxanu proti

antracyklinové kardiotoxicitě?

Jirkovský E.1, Lenčová O.1, Pokorná Z.1, Jirkovská A.2, Jansová H.2, Kovaříková P.2, Šimůnek T.2, Geršl V.1, Štěrba M.1

1 Lékařská fakulta v Hradci Králové a 2 Farmaceutická fakulta v Hradci Králové, Univerzita Karlova v Praze, Hradec Králové.

Klíčová slova: dexrazoxan, kardioprotekce, antracyklinová kardiotoxicita

Dexrazoxan (DEX) je jediným léčivem s jasně prokázaným účinkem proti chronické antracyklinové (ANT)

kardiotoxicitě v experimentech i klinické praxi. Ačkoli v literatuře chybí přímé důkazy, převládá názor, že DEX je proléčivo

účinkující kardioprotektivně prostřednictvím svého chelatačního metabolitu ADR-925. Cílem této studie je objasnění role

ADR-925 v kardioprotektivním účinku DEX. DEX byl podáván králíkům v kardioprotektivním schématu (60 mg/kg, i.p.);

ADR-925 byl podáván ve stejné dávce 30min infuzí a v další skupině navíc subkutánně ve 150. min (60 mg/kg). Koncentrace

ADR-925 v plazmě a myokardu byly stanoveny pomocí validované HPLC/MS metody. Kardioprotektivní účinky byly následně

studovány při podání metabolitu králíkům 45 min před každou dávkou ANT na zavedeném modelu (DAU; 3 mg/kg/týdně po

10 týdnů). Exogenní podávání ADR-925 je schopno navodit signifikantní expozici myokardu s koncentracemi výrazně vyššími

než po podání DEX po dobu 6-12 hodin v plazmě a 6-9 hodin v myokardu. Toto zjištění umožnilo přímé hodnocení

kardioprotektivního účinku domnělého aktivního metabolitu. Výsledky kardioprotektivní studie pak jasně ukázaly, že podání

metabolitu v uvedených schématech není schopné navodit významnou ochranu myokardu před kardiotoxicitou. K tomuto

závěru vedlo vyšetření funkčních, morfologických a molekulárních parametrů (např. index kontraktility dP/dtmax byl snížen

oproti kontrole o 46% a 39% v kombinačních skupinách s ADR-925 a o 41% v DAU skupině, p <0,05). Naše výsledky jasně

naznačují, že chelatačně aktivní metabolit ADR-925 není zodpovědný za účinnou ochranu myokardu proti chronické ANT

kardiotoxicitě navozenou DEX. Kardioprotektivní účinek je tedy patrně spojen s parentní látkou DEX a jinými mechanismy.

Podpořeno GAČR 13-15008S a PRVOUK 37/05.

Selektívna blokáda napäťovo riadeného sodíkového kanála Nav 1.8 a frekvencia kmitania cílií v dýchacích

cestách

Jošková M.1, Šutovská M.1, Koniar D.2, Hargaš L.2, Ďurdík P.3, Hrianka M.2, Bánovčin P.3, Kocmálová M.1, Pappová L.1 a

Fraňová S.1

1 BioMed Martin, Divízia- Respirológia, Ústav farmakológie, Jesseniova lekárska fakulta UK Martin, 2 Katedra mechatroniky a elektroniky, Žilinská Univerzita, Žilina, 3 Klinika detí a dorastu, JLF UK a UNM Martin

Klíčová slova: sodíkové kanály, frekvencia kmitania cílií

V dýchacích cestách bola potvrdená prítomnosť 3 izoforiem napäťovo riadených sodíkových kanálov (Nav): Nav 1.7,

Nav 1.8 a Nav 1.9. Cieľom našej štúdie bolo farmakologicky overiť hypotézu modulačného vplyvu Nav 1.8 na pohyb riasiniek

v dýchacích cestách experimentálnych zvierat. Ster z epitelu trachey zdravých a ovalbumínom-senzibilizovaných morčiat bol

odobratý „brushingovou metodikou“ v in vitro podmienkach. Vysoko potentný a selektívny antagonista Nav 1.8, A803467 (10-

7 mol.l-1, 10-6 mol.l-1, 10-5 mol.l-1), bol pridávaný priamo k biologickému materiálu. Kmitavý pohyb riasiniek, sledovaných v

svetelnom mikroskope (Kvant model IM1C), bol zaznamenávaný vysokorýchlostnou kamerou (Basler A504kc). Sledovaným

parametrom bola frekvencia kmitania riasiniek (CBF) analyzovaná pomocou softvéru Ciliary analysis. Za fyziologických

podmienok lokálne podanie selektívneho antagonistu Nav 1.8 kanálov viedlo k signifikantnému cilioinhibičnému účinku len

pri jeho nízkych koncentráciách (10-7 mol.l-1). V podmienkach ovalbumínom indukovaného alergického zápalu dýchacích

ciest spomalenie CBF po podaní A803467 nebolo štatisticky významné. Napäťovo riadené sodíkové kanály Nav 1.8

nezohrávajú rozhodujúcu úlohu v regulácii pohybu riasiniek v dýchacích cestách experimentálnych zvierat. Práca vznikla s

podporou projektov VEGA 1/0165/14; MZ 2012/35-UK MA-12; APVV-0305-12, Dobudovanie centra experimentálnej a

klinickej respirológie, CEKR II a BioMed Martin (ITMS 26220220187).

21

A single dose of amitriptyline and citaloprame influences myocardial electrical activity and hemodynamic

parameters in rats

Kralova E., Buckova L., Pobiecka A., Vicen M., Gulac P., Stankovicova T.

Comenius University in Bratislava, Faculty of Pharmacy, Department of Pharmacology and Toxicology

Key words: Antidepressants, ischemic – reperfusion injury, heart, rat , QT interval

Treatment of patients with psychiatric diagnoses is often complicated with accompanying cardiovascular diseases. It is

known that long administration of amitriptyline (AMT) and citalopram (CIT) increases the incidence of myocardial infarction

and risk of death in people with coronary artery diseases. The aime of this study was to analyze if a single dose of AMT and

CIT can influence selected electrical and hemodynamic parameters in spontaneously beating isolated rat heart under conditions

of ischemia-reperfusion injury. Experiments were performed on rat hearts from 3-months old Wistar rats. AMI (n=5) and CIT

(n=6) were administered once at the dose 30mg/kg s.c. Control animals were treated with vehiculum s.c.(n=5). After 24 hours

hearts were isolated and perfused according to Langendorff method. After 20 min of stabilization, the hearts were subjected to

a 30-minute period of global no-flow ischemia, followed by a 45-minute reperfusion period. During all experiment

hemodynamic, ECG parameters and dysrhythmias were recorded. In AMT group compared to control and CIT group increased

the myocardial contractility and the higher coronary flow were showed in the beginning of stabilization. In the end of

stabilization the myocardial contraction and coronary flow were decreased in all groups compared to begin of stabilization, the

most in AMT group. During ischemia, the reduction in the force of contraction was deepened in all groups, the weakest

contraction had hearts pretreated with AMT. During reperfusion, the force of contraction and coronary flow increased in all

three groups, but did not reached values from stabilization. One single dose of AMT and CIT increased heart rate and prolonged

the duration of QT and QTc animal compared to control animals in the beginning of stabilization. In contrast to the begining

of stabilization, the heart rate was slowed and the duration of the QT interval was extended in the end of the stabilization, the

most in the CIT group. During ischemia the heart frequency was weakened and duration of QT and QTC was prolonged in all

groups compared to stabilization. During reperfusion, hearts modified with AMT and CIT had increased heart rate and

prolonged duration of QT and QTc interval compared to control. During reperfusion in the groups premedicated with AMT

and CIT dysrhythmias passed from simple forms to more serious forms like ventricular tachycardia (VT) and ventricular

fibrillations (VF) compared to control rats. We recorded more episodes of VT, VF and cardiac arrests in group pretreated with

CIT compared to control and AMT group. We can concluded that the single dose of AMT and CIT caused slight changes in

hemodynamics and electrical activity of heart in rats. This work was supported by VEGA grant 1/1342/12.

Intramyokardiálna autológna transplantácia mononukleárnych derivátov z kostnej drene pacientom s

chronickou ischemickou chorobou srdca s postihnutím koronárnych artérii podstupujúci aorto-koronárne

premostenie.

Kyselovič J., Gažová A., Musil P., Hulman M.

Farmaceutická fakulta Univerzity Komenského, Katedra farmakológie a toxikológie Bratislava, Slovenská republika Lekárska fakulta

Univerzity Komenského, Ústav farmakológie a klinickej farmakológie, Bratislava, Slovenská republika Oddelenie srdcovej chirurgie, Klinika kardiochirurgie, Národný ústav srdcovo-cievnych chorôb, Bratislava, Slovenská republika

Klíčová slova: klinická štúdia, kmeňové bunky, CABG

Prebiehajúci výskum s kmeňovými bunkami jednoznačne poukazuje na benefičný efekt znižovania morbidity a mortality

u pacientov podstupujúcich autológnu transplantáciu kmeňovými bunkami nielen v rámci kardiovaskulárneho systému. Od

marca 2013 prebieha v 5 krajinách EU (Veľká Británia, Dánsko, Belgicko, Španielsko a Taliansko) najrozsiahlejšia klinická

štúdia bunkovej terapie u pacientov s akútnym infarktom myokardu. Dvojročná nekomerčná štúdia si dala za cieľ uskutočniť

na súbore 3 000 pacientov reinfúziu bunkami kostnej drene. V tejto štúdii sa má overiť efekt intrakoronárnej reinfúzie

mononukleárnymi autológnymi bunkami kostnej dreni na celkovú mortalitu pacientov po infarkte myokardu. Primárnym

cieľom našej klinickej štúdie je hodnotiť efekt intrakoronárnej reinfúzie mononukleárnymi autológnymi bunkami kostnej drene

na celkovú mortalitu pacientov s diagnózou chronická ischemická choroba srdca s postihnutím koronárnych artérii, ktorý

podstupujú aorto-koronárne premostenie (CABG – Coronary Artery Bypass Graft). Sekundárnym cieľom je sledovanie

parametrov (systolická a diastolická funkcia myokardu, hladina NTproBNP, záťažová kapacita, max. spotreba kyslíka na

vrchole záťaže, lézie myokardu, ischémia myokardu, poruchy rytmu), globálnych a regionálnych zmien a výskyt akýchkoľvek

vedľajších a nežiaducich účinkov. Cieľovou populáciou sú pacienti s chronickou ischemickou chorobou srdca s postihnutím

koronárnych artérii podstupujúci aorto-koronárne premostenie. Klinická štúdia má dve podštúdie - bunková terapia s

autológnymi mononukleárnymi derivátmi z kostnej drene získanými ako čerstvý separát z kostnej drene priamo na operačnej

sále, ktoré sa pred aplikáciou izolujú pomocou centrifugačnej gradientovej separácie a terapia s autológnymi progenitorovými

bunkami CD 133+ získané ako čerstvý separát z kostnej drene priamo na operačnej sále certifikovaným postupom

CliniMACS® CD133 Selection. Randomizácia pacientov prebieha na základe hospitalizácie a musia prejsť nezávislým

klinickým indikačný seminárom z hľadiska indikácie revaskularizácie. Vhodní jednotlivci po podpísaní informovaného súhlasu

sú randomizovaní do jednotlivých liečebných ramien. Priebeh klinickej štúdie je nasledovný. V deň operácie sa uskutočňuje

autológny odber kostnej drene v krátkej lokálnej anestéze z lopaty bedrovej kosti (odber cca 200 ml), následne sa bunky

separujú pomocou centrifugačnej gradientovej centrifugácie alebo certifikovaným postupom CliniMACS® CD133 Selection.

Počas uskutočnenia operačného výkonu CABG sa aplikuje 5ml roztoku s BMSC alebo CD133+ podľa intramyokardiálnej

mapy infarktového ložiska z MRI. Na základe už uskutočnených klinických a experimentálnych štúdii predpokladáme výrazný

22

pozitívny manifest regeneračnej schopnosti srdcového svalu u pacientov s injektovanými kmeňovými bunkami na základe

randomizácie. Projekt je podporovaný grantami: VEGA 1/905/14, VEGA 1/0949/15, APVV -14-0416

DYSFUNKCIA PRAVEJ KOMORY JE SPREVÁDZANÁ ZMENAMI HSP90 A CAV-1 PRI

MONOKROTALÍNOM INDUKOVANEJ PĽÚCNEJ HYPERTENZII

Malíková E., Galková K., Vavrinec P., Vavrincová D., Křenek P., Klimas J.

Katedra Farmakológie a Toxikológie, Farmaceutická fakulta, Univerzita Komenského, Bratislava, Slovensko

Klíčová slova: pľúcna hypertenzia, Hsp90, kaveolín-1, monokrotalín

Komponent NO signálnej kaskády heat shock proteín 90 (Hsp90) hrá úlohu v kompenzačných mechanizmoch zlyhania

srdca spolu s kaveolínom-1 (CAV-1). Pľúcna artériová hypertenzia (PAH) indukovaná u potkanov monokrotalínom je dobre

známym experimentálnym modelom pre štúdium zlyhania pravej komory. Predpokladáme, že hladiny Hsp90 a CAV-1 môžu

byť zmenené v pravej komore pri monokrotalínom vyvolanej PAH. Skupine (n=13) samcov Wistar potkanov bol podaný

monokrotalín (MCT; 60 mg/kg, s.c.) a 7 kontrolných potkanov (CON) dostalo vehikulum. Separátna skupina 20 (MCT) a 10

(CON) bola použitá na meranie hemodynamickej funkcie pravej komory použitím pravokomorovej katéterizácie. Vitálne

funkcie boli merané pomocou MouseOx prístroja. Potkany boli utratené po 4 týždňoch alebo pri prejavoch dyspnoe, letargie a

výrazného poklesu hmotnosti. MCT potkany preukázali pokles telesnej hmotnosti vzhľadom ku kontrolám (MCT: 294±9 g vs.

CON: 328±6; P<0,01). Pravokomorový tlak signifikantne narástol (MCT: 50,65±6,28 vs. CON: 21,52±2,49; P<0.01).

Hmotnosť pravej komory bola signifikantne zvýšená (MCT: 0,29±0,02 vs. CON: 0,17±0,01; P<0,05), kým hmotnosť ľavej

komory ostala nezmenená (MCT: 0,69±0,03 vs. CON: 0,70±0,05). Expresia Hsp90 v pravej komore MCT potkanov bola

signifikantne zvýšená (MCT: 234±44 vs. CON: 100±7; P<0,05), kým v ľavej komore ostala nezmenená (MCT: 126±14 vs.

CON: 100±3). Expresia kaveolínu-1 v pravej komore MCT skupiny bola znížená (MCT: 66±14 vs. CON: 100±9), ako aj v

ľavej komore (MCT: 67±11 vs. CON: 100±23). Expresia pTyr14CAV-1 bola signifikantne znížená v pravej komore (MCT:

48±13 vs. CON: 100±14; P<0,05), kým v ľavej komore bola nezmenená (MCT: 80±24 vs. CON: 100±22). Zvýšená hladina

Hsp90 spolu so zníženou expresiou CAV-1 izomérov môže hrať dôležitú úlohu v hypertrofovanej a dysfunkčnej pravej komore

v modeli monokrotalínom indukovanej pľúcnej hypertenzie. Táto práca bola podporená nasledovnými grantmi: APVV-0887-

11, VEGA 1/0981/12, VEGA 1/0564/13 and FaF UK/5/2015.

THE INFLUENCE OF CAROTENOID LYCOPENE ON THE METABOLIC ACTIVITY OF CYP2A,

CY3A, CYP2B, AND CYP2C1 IN RAT

Nosková K., Zendulka O., Dovrtělová G., Turjap M., Juřica J.

Department of Pharmacology, Faculty of Medicine, Masaryk University, Brno, Czech Republic, Experimental and Applied

Neuropsychopharmacology Group, CEITEC, Masaryk University, Brno, Czech Republic

Key words: lycopene, cytochrome P450, RLM, metabolic activity

Carotenoids have generally been shown to have modulatory effect on the cytochrome P450 (CYP) enzymes. The aim of

this study was to reveal the influence of lycopene on the metabolic activity of selected CYP enzymes. Lycopene was

administered intragastrically to male Wistar albino rats for 10 consecutive days at the doses of 4; 20; or 100 mg/kg. The control

group was administered with vehicle (5% glucose + propylene glycol 1:1) in an appropriate volume (1 ml/kg).Then, rat liver

microsomal fraction was isolated with differential centrifugation. The total protein content and CYP2A, 3A, 2B, and 2C11

metabolic activities were assessed. CYP metabolic activities were evaluated as CYP-specific hydroxylation of testosterone in

positions 2α, 16α (CYP2C11), 2β+6β (CYP3A), 16β (CYP2B) and 7α (CYP2A) according to the approach of Daniel et al. (1)

The metabolic activity was expressed as pmol/ml/min/mg protein. Data were analysed using non-parametric statistical methods. Neither total protein levels, nor CYP levels in RLM were affected by any of treatment regimes. The lowest dose of lycopene

did not influence the metabolic activities of observed CYP enzymes. Doses 20 and 100 mg/kg increased testosterone 16β, 7α

and 2α hydroxylation. 2β hydroxylation was increased only by dose 100 mg/kg. Up to date, lycopene is reported to inhibit

CYP2E1 metabolic activity. In our experiment, mid- and highest dose of lycopene increased metabolic activity of CYP2A and

CYP2B, compared to control group, while the activity of CYP3A was affected only by the highest level of lycopene in terms

of testosterone 2β hydroxylation, but not 6β hydroxylation. Similarly, CYP2C activity was affected only in terms of 2α

hydroxylation, but not 16α hydroxylation. The change of CYP activity by lycopene can lead to clinically relevant interactions

with co-administered medicines. The present results suggest that lycopene does not influence the total CYP content in rat liver

microsomes, but depending on the dose, lycopene may increase CYP2A, CYP3A, CYP2B and CYP2C11 metabolic activity.

The work was supported by projects CZ.1.05/1.1.00/02.0068 and MUNI/A/0886/2013.

1.Wojcikowski J, Haduch A, Daniel WA. Effect of antidepressant drugs on cytochrome P450 2C11 (CYP2C11) in rat liver.

Pharmacol Rep. 2013 Oct;65(5):1247–55.

23

Vliv proteasomového inhibitoru bortezomibu na rozvoj chronické antracyklinové kardiotoxicity in vivo

Pokorná Z.1, Jirkovský E.1, Lenčová O.1, Adamcová M.1, Mazurová Y.1, Šimůnek T.2, Geršl V.1, Štěrba M.1

1 Lékařská fakulta v Hradci Králové, Univerzita Karlova v Praze, Hradec Králové 2 Farmaceutická fakulta v Hradci Králové, Univerzita Karlova v Praze, Hradec Králové.

Klíčová slova: antracyklinová kardiotoxicita, inhibitor proteazomu bortezomib

Antracyklinová (ANT) kardiotoxicita je spojována s poruchou funkce ubikvitin proteazomového systému, ale jeho

skutečný význam pro rozvoj kardiotoxicity je nejasný. Inhibitory proteazomu (např. bortezomib) se užívají k léčbě

hematologických malignit a jsou také spojovány s určitým rizikem kardiotoxicity. Kombinace ANT a inhibitorů proteazomu

se zdá býti účinnou při léčbě rezistentních forem hematologických malignit, nicméně kardiovaskulární bezpečnost této

kombinace je prozatím nejistá. Cílem této práce je studium vlivu inhibitoru proteazomu bortezomibu na rozvoj chronické ANT

kardiotoxicity u králíka. Chronická ANT kardiotoxicita byla navozena u králíka pomocí daunorubicinu (DAU 3 mg/kg ≈ 50

mg/m2 i.v., 1 x týdně po 10 týdnů, n=11). Bortezomib byl podáván i.p. 1 h před každou dávkou DAU. Pilotní experimenty

ukázaly na potřebu redukce klinicky užívaných dávek bortezomibu v monoterapii (1.0-1.2 mg/m2) při kombinaci s DAU na

dávku 0,75 mg/m2, z důvodu omezení časnějších extrakardiálních komplikací. Další skupinám byl podáván samotný

bortezomib (0,75 mg/m2 i.p.) a fyziologický roztok. Opakované podání samotného bortezomibu bylo dobře tolerováno a nebylo

spojeno s rozvojem systolické srdeční dysfunkce a významných morfologických změn v myokardu. Kombinace bortezomibu

a DAU vedla paradoxně k o něco nižší mortalitě než ve skupině se samotným DAU (11% vs. 27%). Invazivní vyšetření

systolické srdeční funkce ale odhalilo u obou skupin obdobný signifikantní pokles indexu kontraktility dP/dtmax (o 51% vs

46% v porovnání s kontrolou, p<0.05) a významný rozdíl mezi skupinami nebyl nalezen i při stanovení cTnT a histologickém

vyšetření. Výsledky naznačují, že reversibilní inhibice proteazomu pomocí bortezomibu výrazně nezvyšuje chronickou ANT

kardiotoxicitu u králíka. Podpořeno grantem IGA NT/13457-4-2012.

Positive effects of different drug formulation of silybin in the treatment of metabolic syndrome

Poruba M.1, Matušková Z.1, Kazdová L.2, Oliyarnik O.2, Malínská H.2, Večeřa R.1

1 Department of Pharmacology, Faculty of Medicine and Dentistry, Palacký University Olomouc, Czech Republic 2 Center of Experimental Medicine, Institute for Clinical and Experimental Medicine, Prague, Czech Republic

Key words: HHTg rats, hyperglycemia, hyperlipidemia, metabolic syndrome, silybin

Metabolic syndrome (MS) is a state associated with a high risk of cardiovascular morbidity and mortality. Patients

suffering from MS usually have hyperlipidemia and impaired metabolism of glucose. It seems that natural products derived

from polyphenolic compounds are capable to improve these pathologic states. Our previous studies confirm positive effect of

silymarin - milk thistle extract. Probably the most important polyphenol isolated from silymarin is silybin, widely used for its

hepatoprotective, antioxidative and anti-amanita poisoning properties. Although the positive properties of silymarin and silybin

are well established, poor bioavailability limits their therapeutic potential. Advanced drug formulations may improve the

bioavailability of these compounds. In this study, we tried to evaluate the hypolipidemic and hypoglycemic properties of silybin

in different drug formulation (standardized extract of silybin, micronized silybin, and silybin in form of phytosomes) on

hereditary hypertriglyceridemic (HHTg) male rats. HHTg rats are accepted animal model for metabolic syndrome study. A

total of 28 male HHTg rats were divided into four groups of seven animals. Diets were follows: Control group were fed on

standard laboratory diet, SSD group were fed on standard laboratory diet with standardized silybin, MSD group were fed on

standard laboratory diet with addition of micronized silybin and PSD group of animals were fed on standard laboratory diet

containing silybin in form of phytosomes. Four weeks treatment of standardized silybin and silybin in form of phytosomes

significantly decreased level of triglycerides in HHTg rats. In comparison with previous study with silymarin, silybin did not

affect the total cholesterol level, but significantly increased the level of anti-atherogenic HDL cholesterol in all groups of

animals. There was observed a significant decrease of glucose and insulin levels in rats fed by standardized extract of silybin

and micronized silybin. Our results suggest that silybin is a very important part of silymarin (milk thistle extract) and is

responsible for many positive properties of silymarin. These are the first published results about the effects of silybin on the

increase in protective HDL cholesterol level and decrease in high blood glucose level. Our results suggest that silybin alone

may be beneficial for the treatment of hyperlipidemia and hyperglycemia in states associated with metabolic syndrome. Very

important is an information that silybin is a natural product with minimal toxicity on organism. This study was supported by

grant P303/13-10813S from the Czech Science Foundation, Czech Republic and IGA UPOL LF_2015_004.

24

Drug efficacy and safety in asphyxiated newborns treated with whole-body hypothermia.

Posch L.1, Pokorná P.1, 2, 3, Bašková M.1, Slanař O.2

1 Department of Pediatrics - PICU/NICU, General University Hospital, 1st Faculty of Medicine Charles University in Prague, Ke Karlovu 2,

128 02 Prague 2, Czech Republic 2 Department of Pharmacology, First Faculty of Medicine, Charles University in Prague and General University Hospital in Prague,

Albertov 4, 128 00 Prague 2, Czech Republic; 3 Intensive Care and Department of Pediatric Surgery, Erasmus MC - Sophia Childrens Hospital, Rotterdam, the Netherlands, Wytemaweg

80, 3015 CN - Rotterdam, the Netherlands

Key words: hypoxic-ischemic encephalopathy, COMFORT-B score, newborn

Whole-body hypothermia (HT) in asphyxiated newborns is known to result in a significant improved outcome at 18

months of age. However data reporting efficacy and safety of drug therapy under HT are not available. Phenobarbital (PHE),

midazolam and sufentanil are frequently used therefore we aimed to investigate the effects of neuroprotective drugs. 80

newborns (gestational age ≥36 weeks) treated with HT (33-34°C) for hypoxic-ischemic encephalopathy (HIE) were included

in a prospective observational study. Variables of drug efficacy and safety were determined during HT, rewarming (RW) and

normothermia (NT): HIE using aEEG (CFM 6000), COMFORT-B score, respiratory (respiratory rate, ventilatory support,

oxygen saturation), circulatory (heart rate, blood pressure), metabolic hepatic, renal, and coagulation parameters. All newborns

received phenobarbital; midazolam (83%); sufentanil (96%). 1460 individual Comfort-B scores were determined (1026 during

HT and 433 during RW/NT): 61% of all measurements were <11 (63.5% under HT; 54.7% during RW/NT). Prolonged seizures

occurred in 20% of newborns treated with PHE monotherapy and co-medication was needed.23% did not reach normal aEEG

pattern, refractory bradycardia (<80/min) occurred in only two cases. Therapy with phenobarbital was safe and comparable

with literature however newborns were considered to be oversedated during HT/RW. Prof Monique van Dijk, Erwin Ista and

Prof Dick Tibboel.

Zmeny kašľového reflexu po aplikácii arabinogalaktánu

Sivová V.1, Nosáľová G.1, Fraňová S.1, Ray B.2

1 Ústav farmakológie Jesseniovej lekárskej fakulty v Martine, Univerzita Komenského v Bratislave, Biomed Malá Hora 4A, 036 01 Martin,

Slovensko. 2 Natural Products Laboratory, Department of Chemistry, The University of Burdwan, Golapbag, Burdwan, West Bengal 713 104, India

Klíčová slova: Kašeľ, polysacharid, špecifický odpor dýchacích ciest, antitusická aktivita, Nyctanthes

Polysacharidy izolované z liečivých rastlín majú mnoho pozitívnych účinkov na ľudské telo. Z našich doterajších

výsledkov vieme, že niektoré z nich sú schopné ovplyvniť aj obranné reflexy dýchacích ciest. Cieľom práce bolo sledovanie

takéhoto ovplyvnenia u polysacharidu z Nyctanthes arbor-tristis.V práci sme použili zdravé bdelé morčatá u ktorých sme v

bodypletyzmografe vyvolali kašeľ inhaláciou kyseliny citrónovej (c=0,3 mol/l). U morčiat sme zaznamenávali kašľovú

odpoveď a hodnoty špecifického odporu dýchacích ciest v norme a 30, 60, 120 a 300 min po podaní polysacharidu v dávke 25

mg.kg-1 a 50 mg.kg-1 hmotnosti zvieraťa. Štatisticky významné potlačenie kašľového reflexu sme pozorovali už 30 minút po

podaní nižšej dávky polysacharidu, pričom supresia kašľa zostala signifikantná až do konca experimentu. Zvýšenie dávky

polysacharidu na 50 mg.kg-1 viedlo z zvýšeniu jeho antitusickej aktivity, ktorá dokonca prevýšila aktivitu kodeín fosfátu v

dávke 10 mg.kg-1. Perorálne podanie polysacharidu neovplyvnilo významne špecifický odpor dýchacích ciest. Skúmaný

arabinogalaktánový polysacharid prejavil dávkovo závislú schopnosť potlačiť kašľový reflex, pričom sa na tomto účinku

pravdepodobne nepodieľala bronchodilatácia. Predpokladáme, že v antitusickej aktivite polysacharidov sa podieľajú najmä ich

fyzikálno-chemické vlastnosti. Polysacharidy predstavujú v terapii kašľa veľmi perspektívnu skupinu látok, nakoľko mnohé z

nich majú významnú antitusickú aktivitu avšak bez prítomnosti závažných nežiadúcich účinkov. grant UK/24/2014, grant MZ

č. 2012/35-UKMA-12, grant APVV-0305-12, BioMed Martin ITMS 26220220187

Monitoring the length of telomeres in the process of heart failure.

Sprušanský O.1, Gažová A.2, Musil P.1, Hulman M.3, Goncalvesová E.3, Kyselovič J.1

1 Faculty of Pharmacy, Comenius University in Bratislava, Department of Pharmacology and Toxicology 2 Faculty of Medicine Comenius University Bratislava, Institute of Pharmacology and Clinical Pharmacology 3 National Institute of Cardiovascular Diseases, Bratislava

Keywords: Telomere. Heart failure. Polymerase chain reaction

Telomeres are specialized structures at the ends of eukaryotic chromosomes that contribute to the maintenance of genome

integrity by preventing chromosomal rearrangements and fusions. At the same time they allow for full replication of

chromosome ends , linear DNA molecules, without loss of information. Telomeric DNA in humans consists of a sequence of

ds repeats 5'-TTAGGG-3 'and the single stranded 3' overhang at the end. Telomere length can be an important indicator of

certain biological processes. Significant lengthening of telomeres occurs during a process of carcinogenesis especially in the

case of ALT tumor types. This lenghtening is related to the process of immortalization of cells. On the contrary, in the process

of cell aging telomere shortening eventually leads to senescence and apoptosis. Recently, telomeric dysfunction was indicated

as an important factor in the pathogenesis of hypertension, atherosclerosis, and heart failure. The aim of our work was to

determine the relative length of the telomere repeats by quantitative PCR in samples from explanted hearts. We examined the

25

length of telomeres in the cells from the aorta, left ventricle, right ventricle and right atrium. Using qPCR method we

investigated the relative telomere length, while the absolute length was determined by Southen blot analysis In order to

normalize the results, we used two independent endogenous controls (albumin, beta2microglobulin), Normalization to both

controls gave almost identical results. An interesting finding is, as it appears, that some patients showed in sample of the left

ventricle the highest content of telomeric repeats, and thus the longest telomeres. This work was supported by a grant VEGA

1/0905/14

Účinok pyridoindolového antioxidantu SMe1EC2 na funkčný stav pyramidálnych buniek CA1 vrstvy

hipokampu potkana redukovaný vplyvom trimetylcínu

Stará V., Gáspárová Z.

Ústav experimentálnej farmakológie a toxikológie, SAV, Bratislava, Slovenská republika

Key words: trimethyltin, SMe1EC2, oxidative stress, synaptic transmission, neurodegeneration

Neurodegeneratívne ochorenia predstavujú závažný zdravotný a sociálny problém. Pacienti s Alzheimerovou chorobou

(AD) sú dezorientovaní, majú výrazne zhoršenú pamäť a celkovú kvalitu života. Hipokampus je jednou z prvých štruktúr

mozgu, kde sa u pacientov s AD začína prejavovať poškodenie. Napriek celosvetovému úsiliu vedcov a lekárov sú v terapii

AD za čiastočne účinné považované iba štyri látky (donepezil, rivastigmín, galantamín a memantín). Liečba je zameraná na

spomalenie progresu ochorenia. Cieľom našej práce bolo testovať účinok perspektívneho pyridoindolového derivátu, látky

SMe1EC2 na synaptický prenos v hipokampe potkana, ktorý bol zoslabený vplyvom trimetylcínu (TMT). TMT je modelová

neurotoxická látka poškodzujúca najmä hipokampus. Následkom pôsobenia TMT dochádza v hipokampe zvierat ku strate

neurónov a aktivácii glie. In vitro štúdie poukazujú na TMT sprostredkovaný oxidačný stres sprevádzaný znížením hladín

endogénnych antioxidačných enzýmov. Poškodenie membránovej dvojvrstvy vedie k destabilizácii membránových štruktúr a

následnej apoptóze, čo má za následok zhoršenie priebehu synaptickej transmisie v hipokampe. V tejto práci sme sa zamerali

na testovanie neuroprotektívneho účinku pyridoindolu SMe1EC2 (0,1 a 1 µmol.l-1) aplikovaného formou predinkubácie rezov

hipokampu v rôznych časových intervaloch a sledovali sme jeho vplyv na TMT (30 µmol.l-1) sprostredkované poškodenie

synaptického prenosu. Priebeh nervového prenosu na CA3-CA1 synapse v hipokampe sme merali extracelulárnym snímaním

akútnych rezov vo vrstve stratum pyramidale. Na základe našich výsledkov môžeme konštatovať, že sme pozorovali od dĺžky

predinkubácie závislý protektívny účinok SMe1EC2, ktorý by mohol súvisieť s jeho známymi antilipoperoxidačnými

vlastnosťami a pravdepodobne takto sprostredkovanou ochranou lipidickej dvojvrstvy nervových buniek. Táto práca vznikla

za podpory grantu VEGA 2/0054/15 a ITMS 26240220040.

Vzťah farmakodynamických a farmakokinetických vlastností teofylínu v experimentálne navodenom

modeli alergickej astmy

Urbanová A., Kertys M., Šimeková M., Mokrý J.

Ústav farmakológie Jesseniova lekárska fakulta v Martine, Univerzita Komenského v Bratislave

Klíčová slova: astma, teofylín,

Cieľom tejto štúdie bolo porovnať pôsobenie jednorázovo a dlhodobo podávaného teofylínu v rôznych dávkach na

reaktivitu dýchacích ciest v modeli experimentálne navodeného alergického zápalu u morčiat a zistiť, či dosiahnuté plazmatické

hladiny sú relevantné k takémuto pôsobeniu. Dospelé morčatá boli rozdelené do šiestich skupín. Zvieratá v piatich skupinách

boli senzibilizované ovalbumínom. Posledná skupina bola ponechaná bez senzibilizácie a bez liečby ako kontrola. Jedna

senzibilizovaná skupina bola ponechaná bez liečby. Zvieratám v dvoch skupinách sa podával teofylín intraperitoneálne v dávke

20,0 mg/kg buď jednorázovo alebo opakovane raz denne počas 7 dní. V ďalších dvoch skupinách sa teofylín aplikoval podobne,

avšak v dávke 50,0 mg/kg. Špecifický odpor DC ako marker in vivo reaktivity DC sa hodnotil použitím celotelového

dvojkomorového pletyzmografu po aplikácii histamínu. In vitro reaktivita bola hodnotená prostredníctvom orgánových

kúpeľov na prúžkoch tracheálneho a pľúcneho tkaniva po aplikácii kumulatívnych dávok bronchokonstriktorov (histamín a

acetylcholín). Plazmatické hladiny boli merané v liečených skupinách použitím HPLC metodiky. Aplikáciou teofylínu v

dávkach 20,0 mg/kg ako aj 50,0 mg/kg sa dosiahli u morčiat odporúčané hladiny pre jeho farmakoterapeutické použitie.

Pozorovali sme významné potlačenie reaktivity DC in vivo, kde najvýznamnejšie poklesy boli zistené po sedemdňovej aplikácii

obidvoch dávok. V porovnaní s nameranou hodnotou in vivo reaktivity DC u neliečenej senzibilizovanej skupiny bol rozdiel

významný na 21. deň aj po sedemdňovej aplikácii v dávke 20,0 mg/kg. V podmienkach in vitro bola pozorovaná tendencia k

poklesu reaktivity DC, pričom hladina významnosti bola dosiahnutá najmä pri nižších koncentráciách histamínu a acetylcholínu

a po aplikácii vyšších dávok teofylínu. Plazmatická hladina teofylínu po jednorázovom a dlhodobom podaní sa pri rovnakej

aplikovanej dávke významne nelíšila. Teofylín spôsobuje pokles in vivo aj in vitro reaktivity DC u morčiat s ovalbumínom

navodenou hyperreaktivitou DC. Vyššia účinnosť pri dlhodobejšom podávaní naznačuje prevahu protizápalového pôsobenia

teofylínu, ktoré sa môže nepriamo podieľať na jeho bronchodilatačnom pôsobení. APVV-0305-12, Grant MZ 2012/35-UKMA-

12, VEGA 1/0260/14, UK/131/2015

26

Ovlivnění cytochromu P450 2E1 silymarinem – porovnání dvou modelů metabolického syndromu

Večeřa R.1*, Poruba M.1, Kazdová L.2, Škop V.2, Matušková Z.1

1 Ústav farmakologie, Lékařská fakulta Univerzity Palackého v Olomouci 2 Centrum experimentální medicíny IKEM, Praha

Klíčová slova: silymarin, CYP2E1, HHTg potkan

Cytochrom P450 2E1 (CYP2E1) metabolizuje v játrech řadu látek na toxické metabolity. Cílem naší práce bylo zjistit,

jak ovlivňuje silymarin aktivitu cytochromu P450 2E1 u dvou modelů potkana s metabolickým syndromem. Silymarin byl

podáván ve třech různých lékových formách po dobu 4 týdnů. Následně byla stanovena aktivita CYP2E1 v játrech. HHTg a

OVX potkani byli rozděleni do 4 skupin (n ≥ 5) a krmeni 4 týdny (ad libitum) experimentálními dietami. První (kontrolní)

skupině byla podávána standardní laboratorní dieta – STD, druhá skupina byla krmena STD obohacenou o 1% (w/w)

standardizovaného silymarinu – SSD, třetí skupina byla na STD obohacené o 1% (w/w) mikronizovaného silymarinu – MSD.

Čtvrtá skupina potkanů byla krmena STD s přídavkem 1% (w/w) silymarinu ve formě fytozomů - PSD. Po exsanguinaci byly

odebrány vzorky jater pro izolaci mikrozomů. Odborná etická komise MŠMT ČR schválila projekt pokusů pro práci s těmito

experimentálními zvířaty. Aktivita CYP2E1 byla sledována inkubací jaterních mikrozomů s chlorzoxazonem. Vzniklý

metabolit byl měřen metodou HPLC. Všechna data byla statisticky vyhodnocena jednofaktorovou analýzou rozptylu a

Studentovým t-testem na hladině významnosti 0,05. Na rozdíl od OVX samic došlo u HHTg potkanů ke statisticky

významnému poklesu aktivity CYP2E1 po podávání všech tří lékových forem silymarinu. Nejvýznamnější byl pokles po

podání silymarinu ve formě fytozomů, který má dle literatury lepší biodostupnost než ostatní námi studované formy silymarinu.

Vzhledem k tomu, že zvýšená aktivita CYP2E1 je dávána do souvislosti se vznikem řady závažných patologických stavů, je

její studium v jaterní tkáni zajímavé a cenné. Přeměna paracetamolu na toxický NAPQI se děje cestou cytochromů P450.

Zásadní roli, mimo CYP1A2, 2A6 nebo 3A4, zde hraje právě CYP2E1. U HHTg potkana silymarin ve všech svých lékových

formách statisticky významně snížil aktivitu cytochromu P450 2E1 v jaterní tkáni. K nejvýraznějšímu poklesu došlo po

podávání silymarinu ve formě fytozomů, který má dle studií lepší biodostupnost než ostatní námi studované formy silymarinu.

Jeho zvýšená hladina v cirkulaci a následně pak i jeho koncentrace v jaterní tkáni patrně vede ke zvýšení jeho inhibičního

působení na aktivitu CYP2E1. Tato zjištění jsou v souladu s literaturou, která uvádí, že silymarin má spíše inhibiční vliv na

expresi cytochromů P450. Na druhé straně u druhého modelu metabolického syndromu (postmenopauzální OVX samice) jsme

nenalezli významnější ovlivnění CYP2E1 silymarinem. Regulace cytochromů P450 může být ovlivněna hormonálně. Někteří

autoři uvádějí, že by silymarin mohl svým vlastním estrogenovým působením modulovat cytochromy P450. Jeho estrogenová

aktivita by také mohla zlepšit stav vyvolaný ovariektomií. V našich předešlých studiích (data ještě nebyla publikována) jsme

zjistili, že silymarin neovlivňuje expresi a aktivitu CYP2E1 u zdravých potkanů. Naše zjištění ukazují, že silymarin ve všech

námi sledovaných lékových formách snížil aktivitu CYP2E1 u hereditárně hypetriglyceridemického potkana. Tyto výsledky

naznačují, že silymarin může mít touto cestou určitý potenciál ke snížení hladiny vznikajících toxických metabolitů řady látek

a léčiv. To může následně zabránit nebo alespoň zmírnit možné toxické poškození jaterních buněk způsobené indukčním

efektem etanolu na aktivitu cytochromu P450 2E1. Interpretace námi získaných závěrů a jejich využití u člověka (pacienta)

není jednoduchá. Je třeba dalších experimentů, které detailněji objasní působení tohoto extraktu na expresi a aktivitu

cytochromů P450 u jedince s patologickým stavem organismu. Autoři děkují za finanční podporu grantu GAČR 13-10813S a

grantu IGA UPOL LF_2015_004.

Sirtuin 1 modulation in rat model of acetaminophen-induced hepatotoxicity

Wojnarová L., Kutinová Canová N., Farghali H.

Institute of Pharmacology, First Faculty of Medicine in Prague

Key words: SIRT1, resveratrol, acetaminophen, CAY10591, liver

Sirtuin 1 (SIRT1) is involved in important biological processes such as energy metabolism and regulatory functions of

the cell cycle, apoptosis, and inflammation. Our previous studies have shown hepatoprotective effect of polyphenolic

compound resveratrol, which is also an activator of SIRT1. Therefore, the aim of our present study was to clarify the role of

SIRT1 in process of hepatoprotection in animal model of drug-induced liver damage. Male Wistar rats were used for both in

vivo and in vitro studies. Hepatotoxicity was induced by single dose of acetaminophen (APAP). Some rats and hepatocytes

were treated by resveratrol or synthetic selective activator of sirtuin 1 (CAY10591). The degree of hepatotoxicity, the activity

and expression of the SIRT1 were determined by biochemical, histological and molecular-biological assesments of gained

samples (plasma, liver tissue, culture media and hepatocytes). Resveratrol and CAY attenuated APAP-induced hepatotoxicity

in vivo and in vitro. Moreover, both drugs enhanced APAP-reduced SIRT1 activity. Our results show that modulation of the

SIRT1 activity plays a role in hepatoprotection. Synthetic activators of SIRT1 would help in understanding the role of SIRT 1

and are therefore a major boost towards the search for specific treatment of liver disease. PRVOUK-P25/LF1/2 a GAUK

916314

27

Boldine attenuates cholestasis associated with nonalcoholic fatty liver disease in hereditary

hypertriglyceridemic rats fed by high sucrose diet

Zagorova M.1, Prasnicka A.1, Kadova Z.1, Dolezelova E.3, Kazdova L.4, Cermanova J.1, Rozkydalova L.1,5, Hajkova J.1,

Mokrý J.2, Micuda S.1

1 Department of Pharmacology, 2 Department of Histology and Embryology, Charles University in Prague, Faculty of Medicine in Hradec Kralove; 3 Department of Biological and Medical Sciences, 4 Centre for Experimental Medicine, Institute for Clinical and Experimental Medicine, Prague, Czech Republic. 5 Department of Pharmacology, Charles University in Prague, Faculty of Pharmacy in Hradec Kralove;

Key words: Boldine, high sucrose diet, hereditary hypertriglyceridemic rats, NAFLD

The aim of the current study was to clarify the effect of high sucrose diet (HSD) on bile production in rats with

spontaneous hypertriglyceridemia (HHTg). Potentially positive effects were studied for boldine, a natural choleretic agent.

Administration of HSD to HHTg rats over 6 weeks led to increased glucose levels and further rise of triglyceride levels in

serum accompanied with fatty liver infiltration without presence of inflammation. HSD reduced bile formation as a

consequence of decreased secretion of bile acids and glutathione, the main osmotic constituents of bile. Molecular mechanism

behind this cholestatic effect was down-regulation of rate limiting transporters for bile acids and glutathione at apical

membranes of hepatocytes, Bsep and Mrp2, respectively. Moreover, gene expression of transporters for other constituents of

bile, Abcg5/8 for cholesterol, Abcb4 for phospholipids, and Slco1a4 for xenobiotics, was also down-regulated by HSD.

Administration of boldine reduced triglyceride concentrations in serum, and partially attenuated cholestatic effect of HSD by

up-regulation of both Bsep and Mrp2, respectively, and by increase in hepatic disposition of glutathione in reduced form. This

study demonstrates mechanisms of impaired bile production and biliary secretion of some endobiotics during fatty liver disease

induced by HSD in sensitive HHTg rats. Altered function of responsible transporters suggests also potential for changes in

kinetics of xenobiotics including drugs, which may complicate pharmacotherapy in subjects with high intake of sucrose, and

with fatty liver disease. Majority of these alterations may be alleviated by long term administration of boldine. This study was

supported by grants from the Grant Agency of Charles University Prvouk P37/05, and SVV-2015-260179.

28

POSTERY – PREZENTOVÁNY 17.9.2015

Vliv anthokyanů a proanthokyanidinů na metabolismus warfarinu v primárních kulturách lidských

hepatocytů

Anzenbacherová E.1, Tománková V.1, Jourová L.1, Kosina P.1, Anzenbacher P.2

1 Ústav lékařské chemie a biochemie a 2 Ústav farmakologie, Lékařská fakulta Univerzity Palackého v Olomouci, Hněvotínská 3, 775 15 Olomouc

Klíčová slova: cytochromy P450, lékové interakce

Anthokyany a proanthokyanidiny jsou součástí mnoha barevných bobulovitých plodů. Významné množství

proanthokyanidinů se vyskytuje např. v brusinkách, anthokyanidin pelargonidin je obsažen v jahodách. Pozitivní efekt těchto

látek na lidský organismus je způsoben jejich antioxidačními vlastnostmi. Vzhledem k možné současné konzumaci většího

množství anthokyanů a léčiv je třeba se zabývat také možnostmi lékových interakcí těchto látek, zejména na úrovni

metabolismu. V naší práci jsme se zaměřili na studium vlivu pelargonidinu a prokyanidinu A2 na metabolismus warfarinu za

účasti cytochromů P450. Primární kultury lidských hepatocytů byly připraveny podle protokolu, který byl schválen Etickou

komisí LF UPOL. Hepatocyty byly inkubovány 24 hodin s pelargonidinem a s prokyanidinem A2. Jako kontrola byl použit

DMSO (0,1%). Po inkubaci byl k hepatocytům přidán warfarin a po dvou hodinách byly s využitím HPLC kvantifikovány

vzniklé metabolity. Exprese proteinů byla stanovena metodou Western blottingu. Byly stanoveny tři metabolity warfarinu: 7-

hydroxywarfarin, 6-hydroxywarfarin a 4´-hydroxywarfarin. Za vznik 7-hydroxywarfarinu je zodpovědný hlavně cytochrom

P450 2C9, ale také formy cytochromu P450 1A1/2 a 2C8. Na vzniku 6-hydroxywarfarinu se podílí zejména cytochrom P450

1A1/2 (a také 2C19), 4 ´-hydroxywarfarin vniká za účasti cytochromů P450 2C9/18. Po inkubaci s pelargonidinem byla

pozorována zvýšená hladina 6-hydroxywarfarinu, menší zvýšení bylo nalezeno také i u dalších metabolitů. Po inkubaci s

prokyanidinem A2 byly pozorovány zvýšené hladiny 7-hydroxywarfarinu a 6-hydroxywarfarinu naopak pokleslo množství 4

´-hydroxywarfarinu. Vzrůst hladiny 7- a 6- hydroxyderivátů warfarinu lze vysvětlit indukcí cytochromu P450 1A1/2, která je

zřejmě důsledkem aktivace receptoru AhR pozorované v naší dřívější práci (Kameníčková et al. Toxicol Lett. 2013). K

hodnocení možnosti lékových interakcí těchto přírodních látek s warfarinem bude v další práci třeba zjistit závislost uvedených

změn v metabolismu warfarinu na množství antokyanidinu. Autoři děkují Ministerstvu zemědělství ČR za podporu projektu

QJ1510206.

New methodological approaches for distinguishing direct and indirect Constitutive androstane receptor

(CAR) activators

Carazo A., Pávek P.

Farmaceutická fakulta v Hradci Králové, Univerzita Karlova

Key words: CAR, gene regulation, flavonoids, nuclear receptors

The constitutive androstane receptor (CAR, NR1I3) is the critical transcriptional regulator of key xenobiotic-

metabolizing enzymes such as cytochrome P450 CYP3A4, CYP2C9 and specially, CYP2B6. CAR can be activate either via

direct ligand binding to CAR ligand binding domain (LBD) or indirectly via inhibition of EGFR signaling with subsequent

dephosphorylation of CAR at Thr38. The CITCO 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-

dichlorobenzyl)oxime) is the prototype direct CAR ligand and phenobarbital is the classic indirect CAR activator. In the work,

we introduce methods that can demonstrate direct interaction with the CAR ligand binding pocket. Further, we studied whether

the tested compounds exert their activity through the EGFR signaling, phenobarbital-like activation. We tested the flavonoids

chrysin, balcalein and galangin, which have been reported to be activators of CAR and interacting compounds with EGFR

signaling. Employing a TR-FRET coactivator assay with recombinant human CAR and gene reporter-based CAR assembly

assay, a consistent activation of CAR with flavonoids and phenobarbital was not observed. It was determined, however, that

galangin, chrysin, and baicalin may repress EGFR-Tyr1068 autophosphorylation after EGF treatment. These data suggest that

flavonoids chrysin, galangin and balcalein are not direct human CAR agonists and that they may interfere with EGFR signaling.

This study thus demonstrates the application of new approaches for the testing of the direct CAR interaction of both natural

and synthetic ligands. This research has been supported by the Czech Scientific Agency GACR303/12/G163 and SVV project.

29

GALECTIN-1-EXPOSED FIBROBLASTS PRODUCE BIOLOGICALLY ACTIVE SUBSTRATUM

WHICH FAVORS GROWTH OF ENDOTHELIAL CELLS IN VITRO

Gál P.1,2,3,4, Varinská L.1, Peržeľová V.1, Dvořánková B.3, André S.4, Kaltner H.4, Gabius H-J.4, Smetana K. Jr.3, Mojžiš J.1,

Mirossay L.1

1 Department of Pharmacology, Faculty of Medicine, PJ Šarárik University, Košice, Slovak Republic 2 Department for Biomedical Research, East-Slovak Institute of Cardiovascular Diseases, Košice, Slovak Republic 3 Institute of Anatomy, 1st Faculty of Medicine, Charles University, Prague, Czech Republic 4 Institute of Physiological Chemistry, Faculty of Veterinary Medicine, Ludwig-Maximilians-University Munich, Munich, Germany

Key words: galectin-1, fibroblasts, endothelial cells

The tumor microenvironment is formed by both malignant and non-malignant cells as well as by extracellular matrix

(ECM) glycoproteins and various types of soluble mediators. Stromal cells located in the tumor are primarily considered as

sources of promalignant factors. Toward this end, we here address the issue of testing whether ECM affects vessel growth,

considering the impact of a potent effector for conversion of fibroblasts to myofibroblasts and ECM production, i.e. the

adhesion/growth-regulatory galectin-1 (Gal-1). This endogenous lectin, known for triggering diverse cellular responses such

as growth modulation, invasion or motility and production of vascular endothelial growth factor-C, is here studied for its impact

on the qualities of ECM to sustain proliferation of human umbilical vein endothelial cells (HUVECs). Fibroblasts had been

cultured for 10 days with the lectin, followed by removing cellular constituents after an osmotic shock. Freshly isolated

HUVECs were placed on the ECM. In parallel, HUVECs were seeded on untreated and gelatin-coated surfaces as controls. A

positive control for growth of HUVECs culture using medium supplemented with vascular endothelial growth factor completed

the test panel. Cells were kept in contact to the substratum for two days and then processed for immunocytochemistry. HUVECs

seeded on fibroblast-generated ECM presented a comparatively high degree of proliferation. Furthermore, contact to substratum

produced by tumor-associated fibroblasts led to generation a meshwork especially rich in fibronectin. Galetctin-1 is apparently

capable to trigger ECM production favorable for growth of HUVECs, prompting further work on characterizing structural

features of the ECM and in situ correlation of lectin presence, ECM constitution and neo-angiogenesis. This study was

supported in part by the Grant Agency of Ministry of Education, Science, Research, and Sport of the Slovak Republic (VEGA

1/0299/13, VEGA 1/0404/15, and VEGA 1/0048/15) and the Agency for Science and Research under the contract no. APVV-

0408-12 and APVV-14-0731.

Vyhodnocování genové exprese neuropeptidů z imunofluorescenčních řezů

Hudáková J., Mrázová M., Skorkovský T., Hynie S., Klenerová V.

Laboratoř neurofarmakologie ÚLBLD 1. LFUK, Albertov 4, Praha 2, ČR

Klíčová slova: Imunofluorescenční vizualizace neuropeptidů, obrazová analýza, ImageJ sofware, program NIS-Elements

Pro vyhodnocení imunofluorescenčních studií se používá obrazová analýza zpracování obrazových dat z kamery

mikroskopu a jejich transformace a kvantifikace pomocí matematických metod. Cílem práce je navrhnout a implementovat

algoritmy pro analýzu obrazů tkání, využitelné pro kvantitativní hodnocení neuropeptidů. Použili jsme software ImageJ a NIS-

Elements. Výchozím materiálem byly obrazy řezů tkání ve formátu .jpeg pořízené fluorescenčním mikroskopem Leica

DM5000 B se systémem pro digitalizaci snímaného obrazu Leica DFC420 C. U kvantifikace jednotlivých snímků jsme použili

k úpravě binárního obrazu kromě prahování a klasifikátoru objektů i funkce matematické morfologie. Implementovali jsme

software ImageJ a NIS-Elements. Ve srovnání obou metod jsme zjistili, že NIS-Elements vykazuje řadu výhod oproti ImageJ,

umožňuje citlivější a přesnější kvantifikaci, lehčí odstranění vad snímků, a především snížení subjektivní chyby. Vyhodnotili

jsme vztah záznamu obrazů a jejich analýzy pro správný výsledek. Kvalitu digitálního záznamu obrazu ovlivňuje řada faktorů

- chyba samotné metodiky, např. zhášení fluorescenčního signálu, chyba převodu analogového signálu na digitální parametry,

a především individuální chyba pozorovatele. Implementovali jsme metody programu NIS-Elements a ImageJ a získali jsme

srovnatelné výsledky. NIS-Elements poskytuje rozsáhlejší spektrum funkcí ke zpracování obrazových dat než software ImageJ.

Podporováno granty PRVOUK 25/LF1/2 a SVV 260149.

Účinek stresu na expresi mRNA CRH receptorů R1 a R2 v hypofýze kontrolních a CRH knockoutovaných

myší

Hynie S. a Klenerová V.

Laboratoř neurofarmakologie ÚLBLD 1. LFUK, Albertov 4, Praha 2, ČR

Klíčová slova: Kortikotrofní hormon, CRH receptory, hypofýza, CRH knockoutované myši, stres

Aktivace HPA osy je mírou stresové odpovědi, která je spuštěna CRH z hypotalamu, s následným účinkem CRH na

sekreci ACTH v adenohypofýze. CRH působí prostřednictvím dvou receptorů CRHR1 a CRHR2. Receptor CRHR1 má v

hypofýze primární neuroendokrinní úlohu, zatímco CRHR2 je zapojen v doladění stresové odpovědi a jeho funkce není plně

objasněna. Cílem bylo studium exprese mRNA obou receptorů v hypofýze za fyziologických podmínek a po stresu, u

kontrolních a CRH knockoutovaných myší. Použili jsme kontrolní (wild type wt +/+) a CRH knockoutované (CRH-KO -/-)

myší samce. Aplikovali jsme akutní a opakovaný imobilizační stres v intervalech 30 a 120 min. Exprese mRNA byla stanovena

metodou Real Time qPCR a relativní exprese byla počítána pomocí 2-ΔΔCT metody. Nalezli jsme rozdíl v expresi mRNA

CRH obou receptorů po akutním i opakovaném imobilizačním stresu, zejména u CRH-KO myší. CRH-KO myši vykazovaly

30

signifikantně vyšší expresi mRNA CRHR2. V této studii jsme rozšířili naše předchozí nálezy týkající se CRH receptorových

subtypů v adenohypofýze po působení dvou odlišných akutních stresorů, s převahou emoční resp. fyzické komponenty. Použití

CRH knockoutovaných myší umožnilo sledovat úlohu CRH v expresi CRH receptorových subtypů. Naše výsledky svědčí o

možném zapojení obou CRH receptorů v regulaci stresové odpovědi. Podporováno granty PRVOUK 25/LF1/2 a SVV 260149.

Pregnane X receptor (PXR) down-regulates organic cation transporter 1 (OCT1) in primary hepatocytes

Hyrsova L.1, Smutny T.1, Carazo A.1, 1Stefan Moravcik S.1, Mandikova J.1, Trejtnar F.1, Gerbal-Chaloin S.2, Pavek P.1*

1 Department of Pharmacology and Toxicology, Faculty of Pharmacy in Hradec Kralove, Charles University in Prague, Heyrovskeho 1203,

Hradec Kralove, CZ-500 05, Czech Republic, 2 Hepatic Differentiation of Stem Cells and Biotherapy of Liver Diseases, INSERM U1040, Institut de Recherche en Biothérapie, Hôpital

St. Eloi, 80 Avenue Augustin Fliche, FR-34090 Montpellier, France

Key words: OCT1, PXR, down-regulation, squelching of coactivators, primary human hepatocytes

OCT1 is a polyspecific carrier delivering numerous organic cationic drugs into the hepatocytes. Its hepatic expression is

controlled mainly by HNF4α. PXR nuclear receptor mediates induction of principal xenobiotic metabolizing enzymes and drug

transporters. In current study we elucidate regulation of OCT1 transporter by PXR. The effect of PXR activation on the OCT1

expression was examined in several validated hepatocyte cellular models including primary human hepatocytes, differentiated

HepaRG cells, and in two cell lines with overexpressed PXR (HepG2, HuH-7). Several gene reporter experiments were

performed to assess the activity of pOCT1 1.8 kb-luc reporter construct after activation of PXR in HepG2, HuH-7 and HeLa

cells. The influence of PXR on the function of OCT1 was determinated in accumulation studies with the use of [3H]MPP+ in

HepaRG cell line. The level of OCT1 mRNA was decreased after 24 hours treatment with PXR ligands (10 µM rifampicin or

hyperforin 1 µM) in 13 out of 15 primary human hepatocytes and in three model cell lines – HepaRG, HepG2 and HuH-7.

Moreover the function of OCT1 transporter in accumulation study in HepaRG cells was significantly reduced after 24 hours

treatment of 10 µM rifampicin compared to 0,1% DMSO treated cells. PXR activation caused decrease of pOCT1 1.8kb-luc

reporter construct activity in HepG2 cells. This phenomenon was observed also in HuH-7 (low level of HNF4α) and in non-

hepatic HNF4α-deficient cell line HeLa after co-transfection these cells with HNF4α. Co-transfection of SRC-1 coactivator

abrogates PXR-mediated suppression of pOCT1 reporter construct in HepG2 cells in dose-dependent manner. Mutated or

truncated pOCT1 1.8 kb-luc reporter construct (mutations have been introduced into both DR-2 motifs of the HNF4α response

element and/or E-box) were used to confirm mechanism of this regulation. We observed that the PXR-mediated suppressive

effect was partly alleviated, but not abrogated, in pOCT1 1.8 kb-luc construct with mutated HNF4α responsive element and in

pOCT1 1.8 kb-luc construct with mutated E-box in comparison with wild-type construct. In the HNF4α/E-box double mutant,

we observed no suppressive effect of PXR and rifampicin treatment on OCT1 promoter construct activation. We also performed

chromatin immunoprecipitation experiments with probes designed to amplify both HNF4α RE and E-box elements using qPCR.

It was detected lower amount of DNA fragments immunoprecipitated with anti-FLAG SRC-1 antibody after rifampicin

treatment in both cases. In the current study, we examined the effect of PXR activation on the OCT1 expression in several

validated hepatocyte cellular models including primary human hepatocytes and differentiated HepaRG cells. All our data

indicate that OCT1 hepatic expression is down-regulated by PXR agonists at the transcriptional level. In addition, we propose

the mechanism of the down-regulation and postulate that PXR reduces OCT1 transactivation by squelching of SCR-1

coactivator from HNF4α and USF factors controlling high constitutive hepatic expression of OCT1 gene. This conclusion is

supported by results of experiments involving the over-expression of SRC-1 coactivator or various PXR mutants, silencing of

PXR and HNF4α in HepaRG cells, mutagenesis of the HNF4α responsive DR-2 and E-box response elements, and chromatin

immunoprecipitation assay. OCT1 transporter is down-regulated by PXR via squelching of SRC-1 coactivator. This conclusion

is supported by results of gene reporter experiments employing the mutated or truncated OCT1 gene promoter constructs and

chromatin immunoprecipitation study. This work was funded by the Czech Scientific Agency (GACR 303/12/G163 to P.P.)

and by SVV project 260 186.

Comparison of cytotoxicity in selected β-cyclodextrin derivatives

Janoušek J., Mandíková J., Trejtnar F.

Charles University in Prague, Faculty of Pharmacy in Hradec Kralove, Department of Pharmacology and Toxicology

Key words: cyclodextrin, cytotoxicity, MTS, drug carriers

Cyclodextrins (CD) are cyclic oligosaccharides with a broad range of pharmaceutical applications including use as

anticancer drug carriers. They exhibit an intrinsic cytotoxicity potency. However, experimental data on cytotoxicity of CDs are

insufficient. The aim of the work was to determine intrinsic cytotoxicity of selected β-CD derivatives face-to-face in one

laboratory using the same in vitro model. Methyl-β-cyclodextrin, heptakis(2,6-di-O-methyl)-β-cyclodextrin and 2

hydroxypropyl-β-cyclodextrin were tested. The method based on determination of reduction capacity of cells using tetrazolium

dye MTS was used. Standard human hepatic cell line Hep G2 was used as in vitro model. The parameter IC50 was calculated

to evaluate cytotoxicity of the tested CDs. The results showed a significant cytotoxicity of all tested CDs. The cytotoxicity in

Hep G2 cells may be ranged according to IC50 values in the following order: heptakis(2,6-di-O-methyl)-β-cyclodextrin <

methyl-β-cyclodextrin < 2-hydroxypropyl-β-cyclodextrin. This order matches with hypothesis based upon calculated

lipophilicity clog P. In summary, the performed in vitro study provided a valid data on cytotoxicity in basic β CD derivatives.

The results of the study will be used in comparative studies on newly synthetized β-CD derivatives and for selection of

appropriate concentrations in further pharmacological studies. The study was supported by Charles University in Prague (SVV

260185 and PRVOUK P40).

31

Epigenome and differentiation affecting drugs change expression of nucleoside transporters in placental

BeWo and JEG-3 cell lines

Jirásková L., Červený L., Štaud F.

Department of Pharmacology and Toxicology, Charles University in Prague, Faculty of Pharmacy in Hradec Králové

Key words: nucleoside transporters, BeWo cells, JEG-3 cells, epigenome and differentiation affecting drugs

Nucleoside transporters are proteins that control transport of substances containing nucleoside in their structure across cell

membranes. Two major families of nucleoside transport systems have been established: the equilibrative nucleoside

transporters (ENTs; SLC29A) which are involved in facilitated diffusion on one hand and the secondary active concentrative

nucleoside transporters (CNTs; SLC28A) on the other hand. These transporters are localized in all of important cell membranes

including the placental barrier. The goal of the project was to analyze effect of epigenome and differentiation affecting drugs

(butyrate sodium, valproic acid sodium salt, forskolin and 5-azacytidine) on gene expression of ENTs and CNTs in established

human in vitro model of placental barrier – BeWo and JEG-3 cell lines. BeWo and JEG-3 cell lines were cultivated in growth

medium containing 10% FBS at 37°C in humidified atmoshere of 5% CO2/air. Cell was treated with particular agent (1mM

butyrate sodium, 2mM valproic acid sodium salt, 100µM forskolin and 2,5µM 5-azacytidine) for 48 or 72 hours. Quantify

changes in gene expression of nucleoside transporters was analyzed by qRT-PCR using the comparative CT method. Statistical

analysis was performed by the use of one-way ANOVA followed by Dunnett‘s post hoc test. In BeWo cells valproic acid and

forskolin significantly increased levels of expression of CNT2. Exposure to 5-azacytidine reduced expression of CNT2 and

CNT3. In JEG-3 cell line expression of CNT2 and CNT3 was upregulated by butyrate sodium, forskolin and valproic acid,

respectively. Forskolin significantly decreased level of expression of CNT3 in these cells. In conclusion, our results

demonstrated that under in vitro condition placental expression can be changed by the activity of epigenome and/or

differentiation affecting agents. Interestingly, process of placentation of BeWo a JEG-3 cells widely affect CNTs that are

largely responsible for uptake of pyrimidine derived nucleobases. The exact mechanism of changes and reasons of cell line

dependent response to tested drugs should be elucidated. Even though both BeWo and JEG-3 are cells lines derived of human

placental choriocarcinoma our results describe different effect of tested compounds of nucleoside transporters. Particularly in

the case in forskolin were observed different results in the expression of transporters CNT2 and CNT3. These differences may

be caused by the absence some regulation factors in JEG-3 cells which may affect response on the tested component. Lacking

these important factors can cause different differentiation than BeWo cells. Additional analysis have to be carried out to confirm

the changes observed at the protein/function level and comparison of nucleoside transporter expression in the placental cell

lines and directly in the placenta will be also performed. We would like to grafully acknowledge the fund of Charles University

in Prague SVV/2015/260-185 for the financial support.

Nucleoside transporters does not affect transport of zidovudine across the placenta

Karbanová S., Neumanová Z., Červený L., Štaud F.

Department of pharmacology and toxicology, Faculty of Pharmacy in Hradec Králové, Charles University in Prague, Czech Republic

Key words: placenta, zidovudine, nucleoside transporters, HIV, AIDS, pregnancy, drug transporters

Zidovudine (AZT) is a nucleoside reverse transcriptase inhibitor frequently used for prevention of mother-to-child

transmission of HIV. It readily crosses the placenta reaching therapeutic concentrations in fetal circulation. The aim of our

study was to elucidate whether the nucleoside transporters (NTs) contribute to transplacental transport of AZV. In this study

we used in vitro accumulation assay in placenta derived BeWo and JEG 3 cell lines and in situ dually perfused rat term placenta

model to assess role of NTs on transplacental passage on organ level. NBMPR, a model inhibitor of ENTs, was used at low

and high concentrations to discriminate between ENT1 and ENT2 activities; ENT1 is sensitive to NBMPR at 100 nM

concentration, ENT2 is blocked only at high (100 µM) concentration. Moreover, uridine (5 mM) was used as non-selective

inhibitor of both ENTs and CNTs. Thymidine, a model substrate of NTs, was used as a positive control for in vitro experiments.

Thymidine showed a significant sensitivity to uridine and both concentrations of NBMPR confirming functioning ENT1, ENT2

and CNTs in both cell lines. Conversely, AZT uptake by the cell lines used was not affected by presence of both inhibitors.

Then we performed dually perfused rat term placenta model, where any statistically significant differences between maternal-

to-fetal and fetal-to-maternal clearances of AZT were observed. BeWo, JEG-3 cells and the rat placenta express predominantly

ENT1, ENT2, CNT2, and CNT3 and importantly, ENT2, CNT3 recognize AZT as substrate. Both experimental setups used,

however, did not show involvement of NTs in AZT uptake. Lack of significance might be related to higher lipophilicity of

AZT or potentially lower expression of ENT2 and CNT3 in cell lines used or rat placenta. In summary we demonstrated that

NTs did not mediate AZT transplacental transfer. Our data thus propose that AZT overcome placental barrier only by

mechanism of passive diffusion or is transported by other AZT specific transporter as suggested in several published papers.

The study was kindly supported by SVV/2015/260-185 and GAUK/324215/C/2015.

32

In vitro analysis of antiproliferative effects of newly synthesized nitro-substituted hydroxynaphthanilides

with a focus on cell cycle of tumor cell lines

Kauerová T.1, Goněc T.2, Kos J.2, Jampílek J.2, Kollár P.1

1 Department of Human Pharmacology and Toxicology, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences Brno, Palackého Třída 1-3, 612 42 Brno, Czech Republic

2 Department of Chemical Drugs, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences Brno, Palackého Třída 1-3,

612 42 Brno, Czech Republic

Key words: antiproliferative effect, cell cycle, apoptosis, hydroxynaphthanilides, flow cytometry

Ring-substituted hydroxynaphthanilides are known as multitarget agents with antibacterial effect. Recent studies

identified them also as potential anti-inflammatory as well as anticancer agents. The aim of this study was to evaluate the effect

of newly synthesized nitro-substituted hydroxynaphthanilides on viability and proliferation of different human cancer cell lines

and to describe their effect on cell cycle progression. Our analyses were realized on THP-1 monocytic leukaemia cell line and

MCF-7 breast carcinoma cell line. Cell viability was evaluated by dye exclusion test and antiproliferative effect was determined

by the colorimetric WST-1 assay. Cell cycle analysis and detection of apoptosis using Annexin V-FITC assay was performed

by flow cytometry. After 24-hour incubation, two nitro-substituted derivatives showed inhibition of cell proliferation of both

cancer cell lines in a dose-dependent manner. Cell cycle analysis showed that 24-hour incubation with both

hydroxynaphthanilides JK-24 and CP-25 induced a concentration-dependent accumulation in G1 phase together with inhibition

of cell transition from G1 to the synthetic phase of the cell cycle. The percentage of cells in G2/M phase remained unaffected.

Significant increase of sub-G1 peak was observed only after treatment of THP-1 cells with compound JK-24 (5 µM). Further

detection of apoptosis using staining of THP-1 cells with Annexin V-FITC and PI did not demonstrate that the apoptosis would

be the prevailing type of cell death induced by JK-24 compound. Our results demonstrated that both tested compounds

effectively inhibited the transition of cancer cells from G1 to S phase of the cycle in non-toxic concentrations. Still, detailed

analyses on a molecular level are necessary to reveal mechanisms of their antiproliferative effect. Based on the results it can

be concluded that two nitro-substituted hydroxynaphthanilides JK-24 and CP-25 are suitable for further studies of their

mechanism of action as potential anticancer agents.

Nová chiméra oxytocinu využitelná pro detekci oxytocinového receptoru

Klenerová V. a Hynie S.

Laboratoř neurofarmakologie ÚLBLD, 1. LF UK, Albertov 4, Praha 2, ČR

Klíčová slova: oxytocin, oxytocinový receptor, chiméra, OT-Bodipy

Oxytocin (OT) je hormon s periferními i centrálními regulačními účinky. OT působí prostřednictvím jednoho typu

oxytocinového receptoru (OTR). Exprese OTR se určuje pomocí imunofluorescenčních metod s využitím specifických

protilátek. Tyto metody jsou velmi náročné nejen technicky, časově, ale i finančně. Cílem práce je syntéza chimér oxytocinu

pro fluorescenční vizualizaci OTR ve tkáních. Pro studium OTR byla jako referenční tkáň použita děloha potkana, s vysokou

denzitou OTR. Pro značení OTR byly použity dvě metody: značení OTR s využitím imunofluorescenčních specifických

protilátek, a značení pomocí námi syntetizované chiméry OT-Bodipy, kde je OT konjugovaný se zeleně emitujícím

fluorescenčním barvivem, tzv. „fluoroforem Bodipy“ (alternativní metoda). Prokázali jsme navázání chiméry na OTR a

porovnali jsme tuto vazbu s vazbou s imunofluorescenčním značením. Nalezli jsme obdobný vzorec značení pro obě použité

metody, svědčící, že jak specifická antiOT-protilátka, tak OT-chiméra značí stejný cíl – oxytocinový receptor. Vazebná místa

Bodipy s navázaným OT odpovídají vazebným místům protilátky proti OTR. Ověřili jsme tak validitu námi navržené

alternativní metody určení OTR. Nová chiméra oxytocinu spojeného s fluoroforem Bodipy pro vizualizaci oxytocinových

receptorů umožňuje rychlý screening k průkazu OTR bez použití protilátek. Rozšiřuje metody současného výzkumu

mechanismů působení OT na molekulární a buněčné úrovni, s cílem terapeutického využití těchto poznatků. Podporováno

granty PRVOUK 25/LF1/2 a SVV 260149.

Immunomodulatory effects of lipoteichoic acid (LTA) in isolated rat hepatocytes.

Kmoníčková E., Kostecká P., Dusilová A., Zídek Z.

Institute of Experimental Medicine, Department of Pharmacology, Academy of Sciences of the Czech Republic, v.v.i., Vídeňská 1083, 142

20, Prague.

Key words: rat hepatocytes, lipoteichoic acid, nitric oxide, IL-6, TNF-α

In the liver, not only immune cells, but also the parenchymal cells play an important role in the immune response to a

wide range of liver problems. The effect of lipopolysaccharide on pro-inflammatory pathways is well known, but the effects of

gram-positive bacterial components in the liver are not studied properly. In the present study we investigate the effects of

lipoteichoic acid on NO production, cytokine secretion and viability in isolated rat hepatocytes. Primary rat hepatocytes were

isolated using the standard two phase collagenase perfusion method. Freshly isolated hepatocytes were seeded (0.66×10(6)

cells/ml) into collagen-coated cell culture plates in complete William's medium E and maintained at 37°C, 5% CO2 in

humidified incubator for 24 hours. Hepatocytes were stimulated by lipopolysaccharide (LPS, 1 µg/ml) or treated with

lipoteichoic acid (LTA- Bac. subtilis) in concentration range of 1-200 µg/ml. Supernatants of cells were analysed for NO (nitric

oxide) production (Griess reagent). Secretion of cytokines was determined in supernatants by ELISA kits (R&D Systems,

33

USA). Concentration of urea in samples was measured using customized diagnostic kit (BioVision, USA). We found dose-

dependent increase of NO production in primary rat hepatocyte culture stimulated by LTA. Significant changes in NO

expression started at 1µg/ml concentration of LTA. In the same samples, cytokine secretion was detected. Similarly to results

of NO, cultured rat hepatocytes treated with LTA enhanced secretion of IL-6 and TNF-α significantly. The effect of urea

synthesis was compared in LPS ant LTA treated samples. Only mild decrease in urea synthesis was found in the presence of 1

µg/ml or higher of both types of bacterial products. Immunomodulatory potential of LTA, i.e. NO production and pro-

inflammatory cytokine secretion, was newly documented in primary rat hepatocyte culture. The extent of significant changes

induced by LTA corresponded to the stimulation by LPS. A possibility that LTA is contaminated by LPS was excluded in other

experiments (Zidek et al., Nitric Oxide, 2010). Our results are in accordance with the findings that hepatocytes themselves

express Toll-like receptors. It should be further mentioned that bacterial translocation during increased intestinal permeability

is thought to progress the liver harm. The immune response of hepatocytes upon lipoteichoic acid, a product of Gram-positive

bacterial cell wall, is documented. These results can contribute to detail knowledge of interplay between immune and other

type hepatic cells or to can explain increased drug toxicity in liver observed in the presence of bacteria. Also utilization

probiotics in various liver diseases is an open question and is extensively studied. This work was supported by Czech Science

Foundation (GAČR) 303/12/0535.

Enantiospecific effects of amlodipine enantiomers on cytochrome P450 inhibition in vitro

Krasulová K.a, Anzenbacher P.a, Dvořák Z.b

a Department of Pharmacology, Faculty of Medicine and Dentistry, Palacky University, Olomouc, Czech Republic b Department of Cell Biology and Genetics, Faculty of Science, Palacky University, Olomouc, Czech Republic

Key words: enantiomery, amlodipine, cytochrome P450

Amlodipine, a dihydropyridine calcium channel antagonist, is one of the most commonly prescribed drugs for the

treatment of hypertension and ischemic heart disease. It belongs to the class of chiral drugs which undergo so called “chiral

switching”. A popular theme in drug development, an existing compound is changed for a pure enantiomeric chemical entity.

Amlodipine is a racemic mixture, composed of the pharmacologically active S(-)-enantiomer and 1000-fold less active R(+)-

enantiomer. In the most cases, calcium channel blockers are coadministered with other drugs. Therefore, the interactions

between them and other drugs should be well explained. Single enantiomers may act differently in the body as a consequence

of stereoselectivity of the interactions with three dimensional structures of proteins acting as enzymes, receptors or ion

channels. In the present study, we examined inhibitory effects of both amlodipine enantiomers as well as the racemic mixture

on main drug-metabolizing cytochromes P450 in human liver microsomes in vitro. All activities of individual CYP forms were

determined according to established protocols [1]. The following microsomal CYP activities were assayed: CYP1A2 7-

ethoxyresorufin O-deethylation; CYP2A6 coumarin 7-hydroxylation; CYP2B6 7-ethoxy-4-(trifluoromethyl)coumarin 7-

deethylation; CYP2C9 diclofenac 4´-hydroxylation; CYP2D6 bufuralol 1´-hydroxylation; and CYP3A4 testosterone 6β-

hydroxylation, midazolam 1´-hydroxylation and CYP2E1 activity was assessed by chlorzoxazone 6-hydroxylation. It was

already reported that amlodipine may act as a weak inhibitor of CYP2B6 [2]. However, according to our results, amlodipine

and both its enantiomers are able to interact with more drug-metabolizing cytochromes P450 as CYP1A2 (IC50: R-amlodipine

67 µM, S- higher than 100 µM); CYP2B6 (IC50: R- 35 µM, S – amlodipine 65 µM) or CYP3A4 (IC50: R- 2,7 µM, S –

amlodipine 3,4 µM) with an enantiospecific manner. [1] Cytochrome P450 Protocols (Phillips IR, Shephard EA, Eds,), 2nd

Edition, Humana Press, Totowa, 2006. Financial support from Czech Science Agency GACR 13-01809S project and IGA

UPOL_LF_2015_004 is acknowledged.

Impact of newly developed carboxymethyl cellulose materials on the healing dynamics

Kružicová A., Suchý P., Klusáková J., Parák T., Chalupová M., Kuzmínová G., Sopuch T.*

Department of Human Pharmacology and Toxicology, *FaF VFU Brno; Holzbecher, Ltd

Key words: wound care, TNF alpha, TGF beta, VEGF

The aim of this study was to determine the impact of newly developed carboxymethyl cellulose materials on the healing

dynamics (production of cytokines and growth factors) on an acute wound model in rats, and to study the activity of matrix

metalloproteinases 2 and 9, compared with a standard commercially used dressing Aquacel®. Under general anesthesia, a

circular excision of all skin layers in the suprascapular region with 15 mm diameter was performed in male Wistar rats (180–

220g). The tested preparations and secondary dressing were applied on the wound; the control group was applied only the

secondary dressing. In total, 75 animals were divided into three groups of 15 animals. A third of animals from each group was

euthanized after 2, 7 and 14 days from the surgery. The wound size (diameter) was measured and a tissue sample was taken

from the wound for western blot and zymography and for histological examination. Using the western blot, levels of selected

cytokines and growth factors were determined semiquantitatively. The level of TNF-α was increased with a statistical

significance compared to the control after 2 and 7 days in the substances Aquacel®, Hcel®NaT and Hcel®CaHT. After 14

days, no statistically significant differences between the control and other groups were observed. The amount of TGF-α

significantly increased in the group Aquacel® after 2 days and in the group Hcel®NaT after 7 days. Furthermore, the levels of

VEGF, which is necessary for the formation and growth of new blood vessels in the wound, were determined in the test groups.

After 14 days an increase in the VEGF level was observed in all the test groups except Hcel®HTB. We also observed an impact

of the tested substances on the MMP-2 and MMP-9 activity. The presented data suggest that the newly developed

34

carboxymethyl cellulose materials can affect the healing dynamics (production of TNF alpha, TGF beta1 and VEGF) on an

acute wound model in rats, compared with a control group. This work was supported by grants TA ČR (TA04010065).

Real-time monitoring of nephrotoxicity of selected antimycotics by using xCELLigence system

Marcinčáková D., Kšonžeková P., Falis M., Legáth J., Čonková E., Šnajderová Z.

Univerzita veterinárskeho lekárstva a farmácie v Košiciach, Komenského 73, 041 81 Košice, Slovensko

Key words: nephrotoxicity, antimycotics, xCELLigence system, metabolic activity

Drugs used in treatment of yeast infections are known for their nephrotoxic and hepatotoxic effects. The aim of this work

was to monitor the effect of selected antimycotics – fluconazole, amphotericin B and itraconazole in concetrations derived from

recommended daily dosage on model cell line – rabbit kidney cells (RK13). The response of cells after antimycotics exposure

was evaluated using real-time cell analyser xCELLigence system. The system monitored behaviour of treated cells (adherence,

proliferation, morphology changes) in real-time during the whole time of experiment (60 h). Changes were expressed as cell

index and recorded each hour into curves. To determine degree of cytotoxic effect in 24th h of experiment also the colorimetric

end-point analyse – MTT test was employed. This test is used for determination of metabolic activity in treated cells.

Nephrotoxic effcet of tested antimycotics was observed in all tested substances in all concentrations (P < 0,05) except

intraconazole in concentration (0,05 mg.l-1, 0,005 mg.l-1).

HEPATOTOXIC EFFECT OF SELECTED NUTRITIONAL SUBSTANCES AND ANABOLICS USED

TO ENHANCE SPORTS PERFORMANCES

Matejovič, A., Marcinčáková, D., Csank, T., Legath, J., Kyselovič, J.

Faculty of Pharmacy, Comenius University Bratislava, Slovakia University of Veterinary Medicine and Pharmacy, Department of

pharmacology and toxicology, Slovakia

Key words: BCCA; creatine; hepatotoxicity; MTT; nandrolone; RTCA; testosterone

This study investigated the effects of various concentrations of creatine, Branched Chain Amino Acids (BCAA)

preparations and the anabolics, testosterone and nandrolone (hormonal preparations), on hepatocytes. Real time cell analysis

(RTCA) was carried out during the action of the tested substances (24 h) and the changes in the state of cells expressed as cell

index (CI) values were recorded and the respective curves were constructed using an xCELLigence system. At the 24th hour,

we measured the viability of the cells by a colorimetric Methyl Thiazole Tetrazolium (MTT) test. The most pronounced

cytotoxic effect was recorded for nandrolone decanoate and the lowest for BCAA. Monitoring by the RTCA system showed

marked changes in CI at all tested concentrations of testosterone and nandrolone. Creatine and BCAA caused significant

differences only at higher concentrations, while differences at the remaining concentrations were insignificant in comparison

with untreated control cells. The MTT showed a significant effect of the test substances on the viability of cells at all

concentrations of testosterone, nandrolone, creatine and at 3 concentrations of BCAA.

The metabolism of natural compounds from silymarin by Escherichia coli Nissle 1917

Matušková Z.1, Anzenbacherová E.2, Kolář M.3, Papoušková B.4, Tlaskalová-Hogenová H.5, Anzenbacher P.1

1 Department of Pharmacology, 2 Department of Medical Chemistry and Biochemistry, and 3 Department of Microbiology, Faculty of Medicine and Dentistry, Palacky University Olomouc, Czech Republic, 4 Department of Analytical Chemistry, Faculty of Sciences, Palacky University Olomouc, Czech Republic, and 5 Institute of Microbiology, Academy of Sciences, Prague, Czech Republic

Key words: intestinal microflora, probiotics, Silybum marianum

Intestinal microflora has an important role in protection of organism against penetration of xenobiotics or pathogens into

bloodstream. The positive effect on the health of organism and on the stimulation its immunity is typically mediated by

probiotic bacteria which are naturally present in the intestine or can by administered with food or with dietary supplements.

Their application can help in treatment of different diseases. The aim of our study was to find whether probiotic strain of E.

coli Nissle 1917 (EcN) has an effect on natural compounds from silymarin, extract of Silybum marianum. Silymarin is used

especially as a supportive therapy of liver diseases. The question is, if probiotic bacteria (EcN in our study) can metabolize

these compounds and can cause a lower efficacy of the treatment or, on the other hand, improve therapy. Our preliminary study

was performed in vitro. The live bacterial suspension of EcN (108 CFU/ml) was incubated with silymarin solution (24 µg/10

µl) for 24 h. Also, three control samples (only bacterial suspension of EcN, the medium, or the medium with silymarin solution)

were incubated for 24 h. Then, all samples were centrifuged, processed and measured by LC/MS. Our results show that

probiotic bacteria EcN caused increased levels of taxifolin in comparison with control samples. Taxifolin is a silymarin

compound present there in very low concentrations. After incubation with EcN, its concentration has been increased by 63 %.

Our preliminary results suggest that bacterial strain EcN causes a cleavage of glycoside from taxifolin glycoside and therefore

causes a formation of free taxifolin compound. Financial support from Grant Agency of the Czech Republic grant

P303/12/G163 is gratefully acknowledged.

35

BLOOD-BASED GENOMIC SIGNATURE FOR AUTISM SPECTRUM DISORDERS

Nakládal D.1, Gažová A.2, Musil P.1, Kyselovič J.1, Castejon A.M.3

1 Farmaceutická Fakulta Univerzity Komenského Bratislava, Katedra Farmakológie a Toxikológie 2 Lekárska Fakulta Univerzity Komenského Bratislava, Ústav Farmakológie a Klinickej Farmakológie 3 College of Pharmacy, Nova Southeastern University

Key words: Autism Spectrum Disorders, ASD, autism, microarray, gene expression

Autism spectrum disorders (ASD) continue to follow their trend and increase in prevalence. Diagnosis of these

neurodevelopmental disorders is usually not possible before 18 months of age and some novel approaches to prediction have

been proposed. Currently, a standardized biological diagnostic test for ASD does not exist. Early diagnosis is a significant topic

in autism research, as it enables early intervention which is essential for a child’s development and the reduction of the impact

of disabling impairments. ASD etiology has a strong genetic background, and various gene expression signatures have already

been described. The aim of our project was to build upon current advances in autism research with reproducible research and

look for patterns of gene expression in data freely accessible at NCBI-GEO. Data was acquired at the College of Pharmacy of

Nova Southeastern Uni- versity. We used R, a programming language for statistics and visualization to process and analyze

two publicly available microarray datasets, which were generated using an identical platform and include probands diagnosed

according to the same standardized criteria. We test statistical properties for comparability and conduct reproducible re- search

by providing R code, attempting to reproduce findings from original papers and validating results between datasets. Two

evaluated microarray datasets GSE6575 and GSE25507 show sig- nificantly different patterns of value distribution in addition

to their different statistical properties, making them unsuitable for pooling despite attempts at adjusting for batch effects. We

managed to reproduce results from an original paper only in its associated dataset. Results from a differential expression

analysis indicated different signatures across datasets. Results from a hierarchical model analysis suggest a set of differen-

tially expressed genes, which are associated with immune system activity. Association of immune system disturbances with

ASD has been implicated before, hence the results of our analysis appear to be consistent with scientific evidence. Project was

financially supported by the National Scholarship Program of the Slovak Republic

Interactions of selected flavonoids with the transporters hOATP2B1 and hOATP1A2

Navrátilová L., Mandíková J., Trejtnar F.

Department of Pharmacology and Toxicology, Faculty of Pharmacy in Hradec Králové, Charles University in Prague, Czech Republic

Key words: drug transporters; flavonoids; OATP; intestinal absorption

Organic anion transporting polypeptides (OATPs) are the major intestinal transporters for the organic anion uptake. Some

dietary flavonoids have been shown to inhibit hOATP2B1 and/or hOATP1A2, human OATP subtypes involved in the drug

intestinal absorption. Therefore, the potential interactions of flavonoids with hOATPs should be considered as a factor

potentially affecting pharmacokinetics of relevant drugs. The aim of this study was to assess the potential inhibition of

hOATP2B1 and hOATP1A2 by natural compounds from the group of flavonoids (quercetin, myricetin, galangin, pinobanksin,

pinocembrin, chrysin, fisetin) in vitro. MDCK II cells transiently transfected with hOATP2B1 and HEK293 cells transiently

transfected with hOATP1A2 were used as experimental models. The appropriate cell line transfected with the empty vector

served as a control. Inhibitory studies measuring hOATP2B1- and hOATP1A2-mediated uptake of the typical OATP substrate,

radiolabelled [3H]-estrone 3 sulfate, were employed to determine the inhibitory potency of flavonoids. Quercetin, the known

OATP inhibitor, served as a comparator. All mentioned flavonoids showed the statistic significant inhibition of the hOATP2B1-

and hOATP1A2-mediated uptake. However, the found inhibitory potency towards the each transporter was significantly

different. For hOATP2B1 galangin, chrysin and fisetin were the most potent inhibitors. Myricetin and pinobanksin exhibited

IC50 more than one order of magnitude higher. The most potent hOATP1A2 inhibitors seem to be fisetin, pinocembrin and

galangin. According to the obtained results, these natural compounds could potentially affect in varying degrees uptake of the

drug substrates transported by human OATP2B1 and OATP1A2 (e.g. statins) into the enterocytes. So there is a possibility of

food-drug interactions in humans at the level of absorption. The study was supported by grant of the Czech Science Foundation

(GAČR) no. 303/12/G163 and Charles University in Prague (SVV 260185 and PRVOUK P40).

On the pharmacology of the oxidative burst of human neutrophils.

NOSÁĽ R., DRÁBIKOVÁ K., JANČINOVÁ V., MAČIČKOVÁ T., PEČIVOVÁ J., PEREČKO T.

Institute of Experimental Pharmacology and Toxicology SAS, Bratislava

Key words: human neutrophils, chemiluminescence, oxidative burst, brompheniramine, carvedilol, dithiaden, natural

polyphenols, apoptosis, proteinkinase C

The effect of 3 therapeutically used drugs and 5 polyphenolic compounds was compared on the mechanism of oxidative

burst in whole blood and isolated neutrophils at cellular and molecular level. The chemiluminescence(CL) method was applied

for determination of oxidative burst in whole human blood and isolated neutrophils at extra and intracellular level. Apoptosis

was determined in cell free system with luminometric determination of Z-DEVD- enzymatic aminoluciferine substrate in the

presence of tested compounds. Proteinkinase C activation.was evaluated by means of Wester blotting for isoenzymes alpha

and beta II on SDS polyacrylamide gels. Superoxide activity was determined using superoxide-dismutase inhibitable reduction

of cytochrome C and myeloperoxidase by measuring oxidation of o-dianizidine in the presence of peroxide, both measured

36

spectrophotometrically. The compounds investigated decreased the oxidative burst of whole blood in the rank order of potency:

N-feruloyl-serotonin (N-f-5HT) > curcumin (CUR) > quercetin (QUER) > arbutin (ARB) > resveratrol (RES) > dithiaden

(DIT) > carvedilol (CARV) > bromphenyramine (BPA). The generation of intracellular reactive oxygen species(ROS) in

isolated neutrophils was decreased in the same rank order, while CARV was ineffective. Scavenging of extracellular oxygen

radicals followed the rank order of potency: N-f-5HT>CUR>QUER>DIT. ARB and CARV had no effect. The ratio between

the percentage inhibition of extracellular versus intracellular CL was highest for CARV and DIT (1.0) and lowest for RES

(0.34), N-f-5HT(0.14) and QUER (0.13). All compounds tested increased the activity of caspase-3 in cell-free system indicating

a positive effect on apoptosis of neutrophils. Activation of protein kinase C was significantly decreased by DIT, CUR, QUER

and N-f-5HT. CARV, DIT, QUER and ARB reduced more significantly activated neutrophil myeloperoxidase release as

compared with the less pronounced effect on superoxide anion generation. Neutrophil leukocytes represent professional

phagocytes which participate essentially in oxidative burst and contribute to many pathological events. In this study we

investigated the effect of three therapeutically used drugs and five natural polyphenolic compounds on the mechanism of

oxidative burst of human neutrophils concerning they participation in the generation of reactive oxygen species. The rank order

potency for investigated compounds to inhibit whole blood CL was similar with the effect of tested drugs and polyphenolic

compounds on extracellular CL of isolated neutrophils indicating the whole blood CL is generated from neutrophils. The

decrease in intracellular CL of isolated neutrophils was significantly decreased with CUR, RES and N-f-5HT, but not with

ARB, BPA and CARV. All tested compounds decreased activation of proteinkinase C, one of the essential regulatory enzyme

in neutrophil NADPH oxidase activation transfering an electron from NADPH to molecular oxygen, generating superoxide

anion. CARV and ARB decresed superoxide generation in stimulated neutrophils and together with DIT and QUER activation

of neutrophil myeloperoxidase. The ratio between the inhibition of extracellular versus intracellular CL is indicative for positive

effect of tested compounds against oxidative burst of neutrophils demonstrating suppression of reactive oxygen species

extracellularly with minimum alteration of intracellular ROS necessary for neutrophil essential functions, particularly

phagocytosis. It is suggested that other regulatory mechanisms like proteinkinase C, might participate in the inhibition of

neutrophil activation with tested compounds. It concerns different mechanisms controling the assembly of the NADPH oxidase

and the interaction with regulatory role of calcium. Therapeutically used bromphenyramine, carvedilol and dithiaden and cyclic

polyphenolic compounds of natural origin altered oxidative burst of neutrophils in vitro in different ways. This resulted from

the inhibition of free oxygen radical production in whole blood and both at extra- and intracellular level of isolated neutrophils.

Compounds decreasing the amount of extracellular yet with minimum effect on intracellular ROS generation are promising for

further investigation in vivo. The presented results are indicative of pharmacological intervention with neutrophils in

pathological conditions. Of particular interest was the effect of natural compounds. The study was supported by the Slovak

Research and Development Agency [APVV-0052-10] and by the Scientific Grant Agency of the Slovak Republic [VEGA

2/0010/13 and VEGA 2/0044/15].

Susceptibility of selected antibiotics, used individually and in their combinations, to the most common

causative agents of nosocomial infections in cardiovascular surgery

Paprskářová A.1,2; Hrudíková R.3,4; Švadlák D.2; Zápotocký V.2,5; Velebný V.6; Suchý P.1

1 Department of Human Pharmacology and Toxicology, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences Brno,

Palackého tř. 1/3, 612 42 Brno, Czech Republic; 2 Contipro Pharma a.s., Dolní Dobrouč 401, 561 02 Dolní Dobrouč, Czech Republic; 3 Faculty of Chemistry, Brno University of Technology, Purkyňova 464/118, 612 00 Brno, Czech republic; 4 Contipro Biotech a.s., Dolní Dobrouč 401, 561 02 Dolní Dobrouč, Czech Republic; 5 Department of Medical Biophysics and Informatics, Third Faculty of Medicine, Charles University in Prague, Ruská 87, 100 00 Prague 10,

Czech Republic; 6 Contipro Group s.r.o., Dolní Dobrouč 401, 561 02 Dolní Dobrouč, Czech Republic

Key words: E-test, cefazolin, gentamicin, minocycline, fractional inhibitory index

The aim of this study was to determine the final effect of different combinations of antibiotics on selected microbial

species as the most common causative agents of nosocomial infections in cardiovascular surgery. Generally, interaction of two

antibiotics is a tricky proposition. We came across almost no results or very contradictory data in literature according to the

resulting effect of selected antibiotics working in couples. It is a general rule that interaction between bactericidal and

bacteriostatic drugs leeds to antagonistic effect. Anyway, there are some articles that deny this theory or emphasize that the

final effect depends on the concentration of substances. There is also no current assessment of the antibiotic‘s effect on the all

microbial tribes we used in the study. That was the main reason why to apply the E-test on our substances and why to carry out

this assessment. Minimum inhibitory concentration of individual antibiotics and their mutual combination were determined by

using commercially available E-test strips with a predefined concentration gradient of the antibiotic in the range of 0.016 – 256

µg/ml. The final effect was calculated using the fractional inhibitory index. We didn’t observe any case of antagonism. There

were signs of synergy, additive or indifference effects according to the selected microbial tribes and used combinations of

antibiotics. We will carry on with this testing, repeat the experiment and try other potential antibiotic combinations against

chosen microbial species.

37

Modulation of Neutrophil Activity by Equol

Pažoureková S.1, Lucová M.1, Nosáľ R.1, Drábiková K.1, Harmatha J.2, Jančinová V.1

1 Institute of Experimental Pharmacology and Toxicology, Slovak Academy of Sciences, Dúbravská cesta 9, 841 04 Bratislava, Slovak

Republic 2 Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic v.v.i., Flemingovo náměstí 2, 166 10 Praha,

Czech Republic

Key words: neutrophils, Equol, adjuvant arthritis, chemiluminescence, p40phox

Equol shows strong antioxidant properties. We investigated the effect of Equol on human neutrophils, extra- and

intracellular formation of oxidants and the phosphorylation of protein regulating NADPH. The whole human blood and isolated

neutrophils were used for chemiluminescence analysis. Chemiluminescence of human whole blood (250x diluted) enhanced

with luminol (250 µmol/l) was stimulated with phorbol myristate acetate (PMA, 0.05 µmol/l), opsonized zymosan (OpZ, 0.5

mg/ml), n-formyl-methionyl-leucyl-alanine (FMLP, 1 µmol/l), or Ca2+-ionophore A23187 (1 µmol/l). Chemiluminescence of

isolated human neutrophils (5 × 105/sample) was initiated by PMA (0.05 µmol/l). Oxidants released extracellularly were

determined in the system containing isoluminol (5 µmol/l) and horseradish peroxidase (HRP) (8 U/ml). Intracellular

chemiluminescence was enhanced with luminol (5 mol/l) in the presence of the extracellular scavengers superoxide dismutase

(100 U/ml) and catalase (2 000 U/ml). Phosphorylation of protein p40phox was analyzed by western blotting. The data showed

that chemiluminescence of neutrophils in whole blood was increased in all stimulants used in comparison with spontaneous

reaction. The highest effect was shown in the case of bypassing membrane stimulant PMA. The response was 66 times higher

than the response of the unstimulated sample. The inhibitory effect of Equol on the production of ROS was established mainly

in the case of FMLP (IC50 0.5 µM). This impact was detected on using Ca2+-ionophore A23187, PMA and OpZ (IC50 2.7 µM,

3.6 µM and 5.8 µM, resp.). In isolated neutrophils stimulated with PMA, extra- and intracellular chemiluminescence was

recorded separately. Equol decreased both the extracellular and intracellular chemiluminescence of neutrophils at the respective

mean effective concentrations 2.5 µM and 2.6 µM. The phosphorylation of p40phox was increased almost three times after

PMA stimulation. This increase was significantly modified by the treatment of neutrophils with Equol. In our study Equol

gradually decreased chemiluminescence response in concentration dependent manner. This could be caused by direct

antioxidant effect of Equol or by its regulation activity. We used different stimulants for better understanding of Equol of

molecular mechanism. Our analysis showed that the most evident effect was in the case of receptor initiated stimulant FMLP.

However receptor initiated mechanisms might not be the only possibility with regard to the pronounced inhibition of

chemiluminescence initiated with PMA and A23187. Equol decreased extracellular ROS production which is potentially

dangerous for host tissues. We also discovered a strong antioxidant effect of Equol on intracelullar ROS production. It is well

known, that the process of ROS production is iniciated by the activation of NADPH oxidase. Western blot analysis showed

that Equol affected the phosphorylation of p40phox - a component of NADPH oxidase, responsible for the assembly of

functional oxidase in intracellular membranes. The isoflavone slightly decreased the phosphorylation, which could also result

in decreased production of ROS. Equol appears to be a noticeable compound for affecting neutrophil activity, suggesting the

possibility to use it as a support therapy for diseases in which ROS are overproduced. Supported by grants APVV-0052-10,

VEGA 2/0010/13 and APVV-0315-07.

Efavirenz decreases renal excretion of lamivudine through inhibition of OCT1, OCT2 and MATE1 drug

transporters

Reznicek J., Ceckova M., Staud F.

Department of Pharmacology and Toxicology, Faculty of Pharmacy in Hradec Králové, Charles University in Prague, Czech Republic,

reznj7ba@faf.cuni.cz

Key words: OCT1, OCT2, MATE1, Efavirenz, Lamivudine, Drug transporter

Efavirenz (EFV) is a non-nucleoside reverse transcriptase inhibitor that constitutes an important part of combination

antiretroviral therapy (cART) in HIV positive patients. Efavirenz is often combined with other antiretrovirals, mainly

nucleoside reverse transcriptase inhibitors (NRTI), many of which are substrates of solute uptake carriers OCTs (SLC22A) and

efflux transporters MATEs (SLC47A). These membrane proteins are functionally expressed in kidneys and contribute to

vectorial transfer of many drugs across tubular cells from blood to urine. Drug-drug interactions (DDI) have been observed on

these trasnporters, leading to changed drug excretion. The aim of our study was to evaluate inhibitory potential of efavirenz

towards SLC uptake transporters OCT1 and OCT2 and to efflux transporter MATE1. Moreover, we aimed to evaluate possible

transporter-mediated DDI between efavirenz and NRTI lamivudine that is excreted into urine by means of OCTs and MATE

transporters. MDCK cell lines stably expressing human OCT1, OCT2 or MATE1 transporters were used for in vitro

accumulation and transport assays. ASP+ and MPP+ were used as model substrates of OCT1, OCT2 and MATE1. Effect of

efavirenz on pharmacokinetics of lamivudine was further evaluated in male Wistar rats in vivo. Employing in vitro approaches,

efavirenz (10 µM), was able to significantly reduce (P ≤ 0.01, one-way ANOVA) uptake of ASP+, into relevant MDCK-OCT1,

MDCK-OCT2 and MDCK-MATE1 cells and transport of MPP+ across cell monolayers. In Wistar rats, 10 µM efavirenz

significantly decreased (P ≤ 0.01, t-test) renal excretion of lamivudine by up to 90.7%, exceeding the effect of cimetidine as a

control inhibitor of OCT and MATE transporters. Our data suggest that efavirenz is an inhibitor of OCT1, OCT2 and MATE1

transporters, able to influence renal excretion of lamivudine in vivo. Further study will be needed to justify these findings in

clinical pharmacotherapy. The study was supported by the Grant Agency of the Charles University in Prague (GAUK

1148213/C/2013), Grant Agency of Czech Republic (303/13-31118P) and by SVV/2015/260-185.

38

M. pachydermatis isolates susceptibility to antifungal agents

Sihelská Z., Čonková E., Váczi P.

Univerzita veterinárskeho lekárstva a farmácie v Košiciach

Key words: M. pachydermatis, antifungals, susceptibility, disk diffusion method, dogs

The lipophilic yeasts Malassezia are part of the normal flora of the skin and mucosa in warm-blooded animals and human.

At the same time these can act as opportunistic organisms that under certain conditions can become pathogenic. Most often

they are a cause of otitis and dermatitis in animals and in humans it may result in various diseases of skin or fungaemia.

Currently only little information is known about the sensitivity of Malassezia isolates obtained from animals to antifungal

agents. Most of article is focused on those that are found in humans. Today, 14 species of Malassezia are known from which

M. pachydermatis is the most common species in dogs. Determination of the sensitivity of this yeast to the tested antifungals

would allow targeted therapy and thereby reduce the risk of resistance development. The aim of this study was to determine

the sensitivity of M. pachydermatis to antifungal agents. In this work were used 29 field isolates of M. pachydermatis obtained

from healthy dogs. These were tested by disk diffusion method to the antifungal activity of ketoconazole, clotrimazole,

itraconazole, fluconazole, voriconazole, and nystatin. Isolates tested were most susceptible to voriconazole (96.6 %) and

fluconazole (93.1 %). The high antifungal activity was achieved by ketoconazole and clotrimazole (89.7 %) and also by

itraconazole and nystatin (86.2 %). Three isolates of M. pachydermatis were resistant to three and one to four antifungal agents.

The results show a very good sensitivity of M. pachydermatis field isolates against antifungal agents which are commonly used

in clinical veterinary practice.

Immunosuppressive effect of 6-gingerol in rat macrophages.

Soukupová V.1, Potočová I.1, Bučková L.1, Bludovská M.1, Kotyzová D.1, Kovaříková P.1, Zídek Z.2, Kmoníčková E.1

1 Inst. of Pharmacology and Toxicology, Faculty of Medicine in Pilsen, Charles University in Prague, alej Svobody 76, 301 00 Pilsen; 2 Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Vídeňská 1083, 142 20, Prague.

Key words: 6-gingerol, nitric oxide, cytokines, rat macrophages

Ginger (Zingiber officinale) is widely used in diet and employed in Asian traditional medicine. Therapeutic potential is

given by the active principles such as gingerols or shogaols. Immunomodulatory properties of ginger constituents are described

in animal models and humans. Surprisingly, poor data are given for effects of an aromatic polyphenols, 6-gingerol and 10-

gingerol, in primary rat macrophages. Resident peritoneal cells isolated from Wistar rats were (2x106/ml) were cultured 24 h

in the presence of test compounds in RPMI-1640 medium. Supernatants of cells were analysed for NO (nitric oxide) production

(Griess reagent). Concentrations of cytokines (IL-1β, TNF-α) were determined by ELISA kits. Cytotoxicity of gingerols was

evaluated (0.5x106 cells/ml) by tetrazolium-based chromogenic WST-1 test. Compounds 6- and 10-gingerol were obtained

from Sigma-Aldrich. The 100 mM stock solutions were prepared in DMSO. We found that 6-gingerol in low concentration

(0.01-1 µM) sharply decreased nitric oxide production in rat peritoneal macrophages stimulated by lipopolysaccharide (LPS, 1

ng/ml). The effect is in contrast to 10-gingerol. This derivative did not change nitric oxide production in wide range of

concentrations (0.01-100 µM). Also, IL-1β and TNF-α secretion were significantly reduced by 6- gingerol, not by 10-gingerol.

The viability cells was not influenced by both gingerol derivatives, with an exception of the highest concentration of 10-

gingerol, which was found to decrease a viability of macrophages substantially. In this study, we found strong inhibitory effect

of 6-gingerol on NO biosynthesis in rat resident macrophages. Our results are in agreement with authors, who observe

concentration-dependent inhibition of NO production (IC50 ~ 2.0 µM) in other macrophage cell lines. We newly described the

inhibition of pro-inflammatory cytokines in LPS stimulated rat macrophages. Cytokine inhibition is related selectively to 6-

gingerol. In comparison to our previous experiments with curcumin, 6-gingerol seems to be more effective immunomodulatory

polyphenolic compound. Our results showed clear immunosuppressive effect of 6-gingerol but not 10-gingerol on primary rat

macrophages. These results can contribute to appropriate dietary supplement of ginger components and can serve for further

development of drug with potent immunomodulatory/immunosuppressive action. This work was supported by Charles

University/SVV 260 174 (2015).

Studium galaninu a určení subtypů jeho receptorů v kolokalizaci s dalšími neuropeptidy v hypofýze

potkana

Šída P., Hynie S., Klenerová V.

Laboratoř neurofarmakologie ÚLBLD, 1. LF UK, Albertov 4, Praha 2, ČR

Klíčová slova: galanin, galaninové receptory, kolokalizace, hypofýza, stres a anxieta

Multitalentový neuropeptid galanin je široce rozšířený neurotransmiter zapojený v řadě centrálních i periferních funkcí

jako inhibiční modulátor. Galanin působí prostřednictvím tří galaninových receptorových (GalR) subtypů patřících mezi G-

proteinové receptory. Cílem práce bylo studium GalR v hypofýze a určení jejich subtypů v kolokalizaci s dalšími neuropeptidy,

neboť takto komplexní studie nebyla dosud provedena. Exprese mRNA GalR subtypů a dalších neuropeptidů byla stanovena

metodou kvantitativního RT qPCR a exprese těchto proteinů byla určena pomocí immunofluorescence a metodou Western blot

v hypofýze potkana. Galanin a další neuropeptidy byly syntetizovány v naší laboratoři, což umožnilo experimenty, které by

byly jinak finančně nedostupné. Ve všech oddílech hypofýzy jsme jako první provedli komplexní stanovení exprese mRNA a

39

genovou expresi Gal a tří GalR subtypů, a jejich kolokalizaci s oxytocinem, vasopresinem a ACTH. Práce vycházela z našich

nálezů protistresových a anxiolytických účinků galaninu po systémovém podání, svědčící pro jeho centrální působení.

Dosavadní údaje o GalR subtypech v CNS jsou často kontroverzní. Pro možné terapeutické využití Gal u stresu a anxiety je

nutná znalost denzity a lokalizace GalR subtypů. Naše výsledky demonstrují různou distribuci a denzitu GalR subtypů v

jednotlivých oddílech hypofýzy a přispívají k pochopení regulačních mechanismů Gal ve vztahu k dalším neuropeptidům s

perspektivou terapeutického ovlivnění. Podporováno granty PRVOUK 25/LF1/2 a SVV 260149.

Interactions of cyclin-dependent kinase inhibitors ribociclib, AZD5438 and R547 with drug efflux

transporters ABCB1, ABCG2 and ABCC1

Šorf A., Číhalová D., Čečková M., Štaud F.

Department of Pharmacology and Toxicology, Charles University in Prague, Faculty of Pharmacy, Heyrovského 1203, Hradec Králové

Key words: ABC transporters, cancer, cyclin-dependent kinase inhibitors, multidrug resistence

Drug transporters from the ATP-binding cassette (ABC) family actively efflux wide range of substrates including drugs

out of the cells. Their overexpression in cancer cells leads to the development of multidrug resistance resulting in treatment

failure. The aim of this study was to characterize the possible interactions of novel anticancer cyclin-dependent kinase inhibitors

(CDKi) ribociclib, AZD5438 and R547 with ABC transporters ABCB1, ABCG2 and ABCC1. To evaluate whether any

transporter can be causative of resistance to CDKi, XTT proliferation assays were employed. We determined cytotoxic IC50

values for each CDKi in MDCKII cell lines expressing ABCB1, ABCG2 or ABCC1 transporters and in parental MDCKII

cells. Inhibitory properties of CDKi to studied transporters were evaluated by accumulation assays with relevant fluorescent

substrates. The cytotoxicity values indicated that ABCB1 significantly increased IC50 of ribociclib (P≤0.01, t-test), while

ABCG2 activity increased the IC50 of AZD5438 (P≤0.01, t-test). The accumulation assays showed that all studied CDKi were

able to increase fluorescence of Hoechst 33342 in MDCKII-ABCB1 and MDCKII-ABCG2 cells whereas only AZD5438 and

R547 affected daunorubicin accumulation in MDCKII-ABCC1 cells. We demonstrate that all three novel CDKi possess

inhibitory effect to ABCB1 and ABCG2, AZD5438 and R547 have also been revealed as inhibitors of ABCC1. Based on the

cytotoxicity data we conclude that ABCB1 can drive resistance to ribociclib, while ABCG2 expression decrease cytotoxicity

of AZD5438. The inhibitory properties of ribociclib, AZD5438 and R547 to ABCB1, ABCG2 and ABCC1 classifies these

compounds as modulators of multidrug resistance. A fact that ABCB1 and ABCG2 can be causative of resistance to ribociclib

or AZD5438, respectively, should be noted when introducing these substances to clinical area. The study was supported by

SVV/2015/260-185 and by GAUK 344315/C/2015.

Vplyv derivátov brasinínu na in vitro proliferáciu ľudských nádorových buniek

Tischlerová V., Kello M., Mojžiš J.

Ústav farmakológie, Univerzita Pavla Jozefa Šafárika v Košiciach, Lekárska fakulta

Klíčová slova: indolové fytoalexíny, brasinín, kolorektálny karcinóm, nádory

Cieľom práce bolo zistiť antiproliferatívny účinok brasinínu a jeho derivátov na ľudských nádorových bunkových líniách

v in vitro podmienkach. Antiproliferatívny účinok bol študovaný pomocou kolorimetrického MTT testu. Analýza bunkového

cyklu a farbenie anexínom V/PI boli realizované pomocou prietokovej cytometrie. Apoptóza bola tiež detekovaná DNA

fragmentáciou a kondenzáciou chromatínu. Zo siedmych testovaných zlúčenín najlepší účinok vykazoval homobrasinín (HB)

s IC50 = 8,0 µM na ľudskej nádorovej línii kolorektálneho karcinómu CaCo2, a následne bol zvolený na ďalšie štúdium. Analýza

bunkového cyklu pomocou prietokového cytometra preukázala HB indukovaný nárast v G2/M fáze spojený s porušenou

reguláciou expresie α-tubulínu, α1-tubulínu a β5-tubulínu. Tieto zistenia naznačujú možný inhibičný účinok HB na tvorbu

mikrotubulov. Súčasne so zastavením bunkového cyklu v G2/M fáze bol pozorovaný nárast populácie buniek s obsahom sub-

G1 DNA, ktorý je považovaný za marker apoptózy. Prítomnosť apoptózy bola tiež potvrdená značením anexínom V/PI, DNA

fragmentáciou a kondenzáciou chromatínu. Naše výsledky preukazujú, že HB indukuje apoptózu na CaCo2 bunkách a

opodstatňujú ďalšie in vivo štúdium protinádorového účinku HB. Práca bola podporená grantom VEGA 1/0322/14.

40

HPLC method for determination of sulforaphane in human liver cells treated with this compound in vitro

Vanduchova A.1, Tomankova V.2, Anzenbacherova E.2, Anzenbacher P.1

1 Department of Pharmacology and 2 Department of Medical Chemistry and Biochemistry, Faculty of Medicine and Dentistry, Palacky University Olomouc, Hnevotinska 3, 775

15 Olomouc, Czech Republic

Key words: HPLC, sulforaphane, human hepatic cells

In this work a simple and rapid method of determination of a biologically active compound of broccoli, sulforaphane in

human liver cells is described. Natural sulforaphane (SFR) is formed from glucoraphanin (stored form of SFR in cruciferous

vegetables such as broccoli, cabbage, cauliflower, kale) by action of enzyme myrosinase. SFR has anti-cancer and anti-

inflammatory properties and may protect against diabetes or cardiovascular disease and inhibits drug metabolizing enzymes,

cytochrome P450 3A4 and 2D6, in human liver microsomes in vitro. Analysis of sulforaphane treated with human hepatocytes

is based on cell disruption by sonication, methylene chloride extraction and reversed-phase high-performance liquid

chromatography method (HPLC). Conditions of analyses were as follows: column LiChroCART® RP-18 (25cm x 4mm, 5µm),

column temperature 30°C; isocratic elution of mobile phase 54% methanol/46% water with flow rate of 0,6ml/min. Under

these conditions, sulforaphane was detected by absorbance at 245nm with retention time 7 min. The results show only slight

penetration of sulforaphane into the human hepatic cells in vitro. Financial support from Grant Agency of Czech Republic

P303/12/G163 project and from IGA UPOL_LF_2015_004 is gratefully acknowledged.

Effect of galectin-8 on endothelial cell proliferation

Varinska L., Gal P., Kello M., Mojzis J.

Department of Pharmacology, Faculty of Medicine, PJ Šarárik University, Košice, Slovak Republic

Key words: galectins, VEGF, endothelial cells

Galectins are a family of sugar-binding proteins that are able to transfer biological signals with remarkable role in the

tumor biology. It has been shown that the tandem-repeat lectin galectin-8 plays an essential role in tumor angiogenesis by

improving endothelial cell migration and capillary tube formation. Interestingly by a more prominent way than the pro-

angiogenic growth factor - VEGF. Thus, new question arises, i.e. does galectin-8 improves endothelial cell proliferation? To

provide answer to that question the MTT and BrDu proliferation assays using HUVEC as a model was used. Different

concentrations of galectin-8 (3 - 0.004 µg/ml) were tested in a series of in vitro experiments. For galectin-8 detection in

HUVECs, western blot and flow cytometry analysis were used. Galectin-8 in HUVECs was detected only by flow cytometry.

It was foun that galectin-8 induced cell proliferation in combination with VEGF (25 ng/ml). Galectin-8 alone does not exhibit

any significant effects on cell proliferation. Therefore, further experiments revealing the mechanism of action as well as

potential roles of galectin-8 in selected steps of angiogenesis need to be answered. This study was supported in part by the

Grant Agency of Ministry of Education, Science, Research, and Sport of the Slovak Republic (VEGA 1/0299/13) and the

Agency for Science and Research under the contract no. APVV-0408-12.

41

Generální sponsor

Zlatí sponzoři

Stříbrní sponzoři

Sponzoři

Generální sponzor

Zlatí sponzoři

Stříbrní sponzoři

Sponzoři

EPHAR poster award