Jiri Lamka

Charles University in Prague, Praha, Praha, Czech Republic

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Publications (17)22.75 Total impact

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    ABSTRACT: The aim of present project was to find out if in vivo contact of Haemonchus contortus with benzimidazole an-thelmintic flubendazole (FLU) during treatment of its hosts (sheep) with low doses of FLU affects helminths' drug-metabolizing enzyme activities. Four groups of lambs, experimentally infected with H. contortus, were treated three-times orally with 0.0, 0.25, 0.50 or 1.00 mg per kg of body weight of FLU in three consecutive days. Twenty four hours after the last FLU dose, the nematodes were isolated, homogenized and subcellular fractions were prepared. In these subcellular fractions, biotransformation of FLU and the activities of carbonyl reducing enzymes and conjugation enzymes were as-sayed. The results showed that H. contortus enzymes were able to conjugate p-nitrophenol with glucose but not with glu-curonic acid. The exposure of H.contortus to FLU (the highest FLU dose) caused a significant increase in activities of FLU reductases, D,L-glyceraldehyde reductases and glutathion S-transferases.
    The Open Parasitology Journal 08/2010; 4(1):24-28. DOI:10.2174/1874421401004010024
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    ABSTRACT: The effect of flubendazole (FLU) therapy on in vitro FLU biotransformation and the activities of selected biotransformation enzymes were investigated in male and female lambs. Four experimental groups were used: control (untreated) ewes and rams and FLU-treated ewes and rams (orally, 15 mg/kg per day, for three consecutive days). Subcellular fractions were prepared from liver and intestinal mucosa 24 h after the final dosage was administered. Activities of cytochromes P450 (CYP), flavine monooxygenases (FMO), carbonyl reducing enzymes, UDP-glucuronosyl transferase (UGT) and glutathione S-transferase were tested. Significant gender differences were observed for FMO-mediated activity (2-fold higher in ram lambs) and UGT activity (up to 30% higher in ewe lambs), but no gender differences were observed in FLU metabolism. FLU-treatment of lambs moderately changed the activities of some CYPs, FMO, and UGT in liver microsomes. In vitro FLU reduction was not altered in the liver, but was slightly higher in the small intestine of FLU pre-treated lambs. This correlated with the higher carbonyl reductase activities measured in the gut mucosa of these animals.
    Pharmacological reports: PR 03/2010; 62(2):362-73. DOI:10.1016/S1734-1140(10)70276-0 · 2.17 Impact Factor
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    ABSTRACT: With further use of chemical agents in the control of parasitic infections, an increased number of drug resistance occurrences to antiparasitic drugs has been reported. Induction of enzymes responsible for detoxification of given drugs can contribute to drug resistance development in a parasitic organism. The identification of formed metabolites allows the characterization of the enzymes participating in biotransformation and possibly in drug resistance development. The objective of our work was to find and identify phase I and phase II metabolites of the anthelminthic drugs albendazole, flubendazole and mebendazole formed in ex vivo incubations by the parasitic helminth Dicrocoelium dendriticum, a parasite of ruminants and other grazing animals, using liquid chromatography/mass spectrometric (LC/MS) techniques. In the ex vivo study, approximately 50 living D. dendriticum adults were incubated in 5 mL RPMI-1640 medium in the presence of 10.0 micromol L(-1) benzimidazole drug (5% CO(2), 38 degrees C) for 24 h. The bodies of the parasite were then removed from the medium. After homogenization of parasites, both parasite homogenates and medium from the incubation were separately extracted using solid-phase extraction. The extracts were analyzed using LC/MS with electrospray ionization. The results showed that D. dendriticum enzymatic systems are capable of phase I oxidation and reduction as well as phase II conjugation reactions. Detected phase I metabolites comprised albendazole sulfoxide, reduced flubendazole and reduced mebendazole. As for phase II metabolites, methyl derivatives of both reduced flubendazole and reduced mebendazole were observed.
    Rapid Communications in Mass Spectrometry 07/2009; 23(17):2679-84. DOI:10.1002/rcm.4170 · 2.64 Impact Factor
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    ABSTRACT: Anthelminthics remain the only accessible means in the struggle against helminth parasites, which cause significant morbidity and mortality in man and farm animals. The treatment of helminthic infections has become problematic because of frequent drug resistance of helminth parasites. The development of drug resistance can be facilitated by the action of xenobiotic metabolizing enzymes (XMEs). In all organisms, XMEs serve as an efficient defense against the potential negative action of xenobiotics. The activities of XMEs determine both desired and undesired effects of drugs, and the knowledge of drug metabolism is necessary for safe, effective pharmacotherapy. While human and mammalian XMEs have been intensively studied for many years, XMEs of helminth parasites have undergone relatively little investigation, so far. However, many types of XMEs, including oxidases, reductases, hydrolases, transferases, and transporters, have been described in several helminth species. XMEs of helminth parasites may protect these organisms from the toxic effects of anthelminthics. In case of certain anthelminthics, metabolic deactivation was reported in helminth larvae and/or adults. Moreover, if a helminth is in the repeated contact with an anthelminthic, it defends itself against the chemical stress by the induction of biotransformation enzymes or transporters. This induction can represent an advantageous defense strategy of the parasites and may facilitate the drug-resistance development.
    Drug Metabolism Reviews 02/2009; 41(1):8-26. DOI:10.1080/03602530802602880 · 6.29 Impact Factor
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    ABSTRACT: Although benzimidazole anthelmintic flubendazole, methyl ester of [5-(4-fluorobenzoyl)-1H-benzimidazol-2-yl]carbamic acid, is extensively used in veterinary and human medicine for the treatment of gastrointestinal parasitic helminth infections, reliable data about its pharmacokinetics in various species have not been reported. Our previous work [M. Nobilis, Th. Jira, M. Lísa, M. Holcapek, B. Szotáková, J. Lamka, L.Skálová, J. Chromatogr. A 1149 (2007) 112-120] had described the stereospecificity of carbonyl reduction during phase I metabolic experiments in vitro. For in vivo pharmacokinetic studies, further improvement and optimization of bioanalytical HPLC method in terms of sensitivity and selectivity was necessary. Hence, a modified chiral bioanalytical HPLC method involving both UV photodiode-array and fluorescence detection for the determination of flubendazole, both enantiomers of reduced flubendazole and hydrolyzed flubendazole in the extracts from plasma samples was tested and validated. Albendazole was used as an internal standard. Sample preparation process involved a pH-dependent extraction of the analytes from the blood plasma into tert-butylmethyl ether. Chromatographic separations were performed on a Chiralcel OD-R 250 mm x 4.6mm column with mobile phase methanol-1M NaClO(4) (75:25, v/v) at the flow rate 0.5 ml min(-1). In quantitation, selective UV absorption maxima of 290 nm (for reduced flubendazole), 295 nm (for albendazole), 310 nm (for flubendazole) and 330 nm (for hydrolyzed flubendazole) were used in the UV photodiode-array detection, and lambda(exc.)/lambda(emis.)=228 nm/310 nm (for reduced flubendazole) and lambda(exc.)/lambda(emis.)=236 nm/346 nm (for albendazole) were set on the fluorescence detector. The fluorescence detection was approximately 10-times more sensitive than the UV detection. Each HPLC run lasted 27 min. The validated chiral HPLC-PDA-FL method was employed in the pharmacokinetic studies of flubendazole in sheep. The stereospecificity of the enzymatic carbonyl reduction of flubendazole was also observed in vivo. (+)-Reduced flubendazole was found to be the principal metabolite in ovine blood plasma and only low concentrations of hydrolyzed flubendazole, the parent flubendazole and (-)-reduced flubendazole were detected in this biomatrix.
    Journal of Chromatography B 01/2009; 876(1):89-96. DOI:10.1016/j.jchromb.2008.10.032 · 2.69 Impact Factor
  • The Open Veterinary Science Journal 03/2008; 2(1):23-32. DOI:10.2174/1874318800802010023
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    ABSTRACT: Flubendazole (FLU) is a widely administered benzimidazole anthelmintic indicated for the control of parasitic diseases in farm animals including pigs and pheasants. This study was designed to test the biotransformation of FLU in control animals and animals treated with FLU in recommended therapeutic doses. The activities of several pheasant and porcine hepatic and intestinal carbonyl reducing enzymes and their modulation by FLU were also studied. Twelve adult pheasant hens, approximately 1 year old, were divided into two groups and treated for 7 days with placebo or 6 mg of FLU/kg of body weight. Eight male hog weaners, approximately 3 month old, were divided into two groups and treated for 5 days with placebo or 1.57 mg of FLU/kg of body weight. Subcellular fractions, prepared from livers and small intestines of control and FLU treated animals, were incubated with FLU. In vitro formation of two main FLU metabolites, reduced FLU, and hydrolyzed FLU were analyzed using HPLC. While FLU was reduced significantly more intensively in FLU-treated pheasants than in control animals, no differences were observed in pigs. These results were confirmed by measuring the enzyme activities: carbonyl reducing enzyme activities were increased in pheasants treated by FLU, whereas FLU did not affect these enzymes in pigs.
    02/2008; 2(1):29-34. DOI:10.2174/187231208783478452
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    ABSTRACT: Cytochromes P450 (CYP) belong to the most important biotransformation enzymes, therefore, their inhibition may lead to serious pharmacological and toxicological consequences. Albendazole (ABZ) is a benzimidazole anthelmintic widely used in human and veterinary medicine. The effects of ABZ on CYP were investigated on the rat (Rattus norvergicus) and mouflon (Ovis musimon) hepatic microsomes. Besides ABZ, its two main metabolites (albendazole sulfoxide, ABZSO, and albendazole sulfone, ABZSOO) were tested to clarify which compound is responsible for the inhibitory effect. After preincubation of microsomes with the benzimidazoles (1, 5 and 25 microM), CYP activities, ethoxyresorufin O-deethylase (EROD) and benzyloxyresorufin O-dearylase activities were measured. The results showed that both ABZ and ABZSO, but not ABZSOO, exhibited significant potency to inhibit CYP activities measured in both tested species. Since ABZ as well as ABZSO are known inducers of EROD activity, our results clearly demonstrate that the drug can act as inducer and also as inhibitor of the same enzyme. In in vitro studies the CYP inhibition may mask the CYP induction. The extent of inhibition observed in mouflon was significantly higher than in rat. This finding emphasizes the importance of performance of inhibition studies in target animal species. Possible consequences of CYP inhibition should be taken into account during the anthelmintic therapy of mouflons with ABZ.
    Pharmacological reports: PR 01/2005; 57(1):97-106. · 2.17 Impact Factor
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    ABSTRACT: Our current knowledge about the biotransformation enzymes in wild ruminants is limited. The present study aimed to compare basic levels and specific activities of cytochrome P450 isoforms (CYP1A, 2A, 2B, 2C, 2D, 2E, 3A, 4A) in males of red deer ( Cervus elaphus), fallow deer ( Dama dama), roe deer ( Capreolus capreolus) and mouflon ( Ovis musimon). The proteins from the major cytochrome P450 (CYP) subfamilies were detected in all ruminant species by Western blotting, using polyclonal antibodies raised against rat or human CYP enzymes. The immunochemical data seem to suggest that humans and wild ruminants share some similar hepatic CYP enzymes corresponding to members of subfamilies 2 and 3; ruminant liver samples also contained two proteins cross-reacting with anti-rat CYP1A antibodies. High activities of CYP1A enzymes found in liver microsomes of male fallow deer and roe deer are indicative of increased susceptibility of these species towards promutagens that are metabolically activated by these CYPs. On the other hand, low activities of CYP1A-dependent alkoxyresorufin O-dealkylase activities were detected in male mouflons. Oxidative metabolism of testosterone was significantly higher in wild ruminants than the values previously reported from bulls. Androstene-3,17-dione and 6beta-hydroxytestosterone were the most important products of testosterone oxidation in liver microsomes of all the ruminant species under study. The highest CYP3A-dependent testosterone 6beta-hydroxylase activity was found in mouflons and fallow deer. A different pattern of CYP activities towards testosterone was found in roe deer, which showed high activities of testosterone 2beta-hydroxylase and lower production of androstene-3,17-dione. An increased activity of CYP4A-dependent laurate 12-hydroxylase found in roe deer and mouflons might indicate a higher metabolic turnover of fatty acids. The data on CYP activities indicated that high metabolic rates of steroids, fatty acids, and xenobiotics may occur in male wild ruminants. The highest hepatic activities specific for CYP3A, CYP2C, CYP2D, and CYP2E enzymes were found in mouflon, suggesting that this species has the highest biotransformation capacity.
    Archive für Toxikologie 11/2003; 77(10):555-60. DOI:10.1007/s00204-003-0477-4 · 5.08 Impact Factor
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    ABSTRACT: Knowledge of the biotransformation processes of veterinary drugs and food supplements in food-producing animals is increasingly important. Residual levels of parent compounds or their metabolites in food of animal origin may differ with the breed, breeding conditions, and gender of animals. The nonsteroidal antiinflammatory drug flobufen, 4-(2',4'-difluorobiphenyl-4-yl)-2-methyl-4-oxobutanoic acid (racemic or its individual enantiomers) was used as a model to evaluate differences in activity, stereoselectivity, and stereospecificity of reductases in primary cultures of hepatocytes from intact male or castrated male domestic pigs (Sus scrofa domestica) or male wild pig (Sus scrofa scrofa). Time-dependent consumption of flobufen enantiomers and formation of dihydroflobufen (DHF) diastereoisomers as their principal metabolites in hepatocytes were measured using chiral HPLC. Flobufen reduction in hepatocytes from all three experimental groups of animals was stereoselective ((+)-R-flobufen was predominantly metabolized) and stereospecific (2R;4S-DHF and 2S;4S-DHF diastereoisomers were preferentially formed). Flobufen reductases activity in male domestic pigs was 30 times higher compared to castrated pigs. Flobufen reductases activity was similar in domestic and wild pigs. The stereospecificity and stereoselectivity of DHF production did not significantly differ with breed or castration of animal. Chiral inversion of flobufen enantiomers was also studied and differences between castrated and intact male pigs were seen.
    Chirality 04/2003; 15(3):213-9. DOI:10.1002/chir.10182 · 1.72 Impact Factor
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    ABSTRACT: Lancet fluke (Dicrocoelium dendriticum), being causal organism of dicrocoeliosis known in animals and man, continuously attract attention of scientific community. Only detailed knowledge of the properties of this parasite, including his defence mechanisms, can lead to successful control of parasitosis in mentioned hosts. The only means so far generally accepted against dicrocoeliosis is the use of suitable anthelmintics, especially benzimidazole ones. Biotransformation enzymes can, to a certain extent, protect the parasitic worms against the effects of xenobiotics including anthelmintics. The present study was designed to evaluate drug metabolizing enzymes activities in lancet fluke subcellular fractions. Activities of oxidation enzymes, carbonyl-reducing enzymes and conjugation enzymes were tested in subcellular fractions of D. dendriticum homogenate. Several enzyme activities corresponding to main isoforms of cytochrome P450 were measured in microsomes-like fraction but no activity was detected. On the other hand, activity of catalase (using europium (III)–tetracycline–hydrogen peroxide system) and peroxidase (assayed by measuring the H2O2 dependent oxidation of o-phenylendiamine) was proven in subcellular fractions. From conjugation enzyme activities, UDP-glucuronosyl transferase activity towards p‑nitrophenol in microsomes-like fraction and glutathione-S-transferase (GST) activity towards 1‑chloro-2,4-dinitrobenzene in cytosol-like fraction were assayed. The apparent kinetic parameters for GST reaction were determined: V´max = 1.64±0.25µM.min-1, K´m = 2.57±0.38 mM. Activities of carbonyl-reducing enzymes were tested using metyrapone, acenaphthenol, daunorubicin, and oracin. The results proved the ability of D. dendriticum enzymes to reduce the carbonyl group of xenobiotics. In lancet fluke, many model substrates were metabolized and the specific activities of some biotransformation enzymes were similar to the activities found in mammal host organisms. These results document the ability of D. dendriticum to effectively metabolize different xenobiotics and by this way be protected against the toxic effect of xenobiotics. This project was supported by the Grant Agency of Czech Republic, Grant No. 524/07/0611.
    10th European Regional International society for the study of xenobiotics Meeting;
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    ABSTRACT: Dicrocoeliosis, a helminthosis caused by lancet fluke (Dicrocoelium dendriticum), is at present considered a world-widely significant but little investigated parasitosis of farm, domestic and wild animals, and a relatively rare parasitosis of man. Lancet fluke particularly parasitizes in biliary duct, gallbladder and at the outlet of pancreas of its host, small ruminants. The only means so far generally accepted against fluke is pharmacotherapy and pharmacoprophylaxis practised in endangered or attacked breeds with the use of suitable anthelmintics (preference is given to benzimidazole anthelmintics). The aim of this project was to study the metabolism of benzimidazole anthelmintic albendazole ([5-(propylthio)-1H-benzimidazol-2-yl] methylcarbamate, ABZ) in lancet fluke subcellular fractions and to characterise the responsible enzymes. The results showed that ABZ is metabolised via sulphoxidation in lancet fluke in vitro. The highest velocity of ABZ sulphoxide (ABZSO) formation was found in mitochondria-like fraction, lower in microsomes-like fraction and none ABZSO arise in cytosole-like fraction. Several model inhibitors of biotransformation oxidation enzymes were used to characterise the enzymes responsible for sulphoxidation of ABZ. Salicylhydroxamic acid, mercaptosuccinic acid, octylamine, methimazole (MET) and alpha-naphthyl thiourea (ANTU) were used in incubations of ABZ with fluke's subcellular fractions. MET and ANTU, typical inhibitors of flavine monooxygenases (FMO) in mammals, significantly inhibited the ABZSO formation in lancet fluke. The concentration dependent inhibition was observed both in mitochondria-like and microsomes-like fractions. Values of IC50 were calculated. Based on inhibition study, FMO seemed to be responsible for ABZSO formation, although no activity of thiobenzamide-S-oxidase (typical reaction catalysed by FMO in mammals) was detected in lancet fluke. This contradiction indicate that lancet fluke's FMO does not use thiobenzamide as a substrate or MET and ANTU inhibit other oxidase than FMO. The results demonstrate considerable differences in biotransformation enzymes properties in mammals and helminths. This project was supported by Czech Science Foundation, grant No. 524/07/0611.
    11th European Regional International society for the study of xenobiotics Meeting;
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    ABSTRACT: In our previous papers [1,2], the in vitro and in vivo phase I metabolites of anthelmintic flubendazole were studied in pig, pheasant and sheep. In all mentioned species, (+)-reduced flubendazole was identified as the principal metabolite in various biomatrices, whereas hydrolyzed flubendazole and (-)-reduced flubendazole were found in only low concentrations. The reduction of flubendazole carbonyl group and hydrolysis of its ethyl carbamoyl moiety form the prepositions for the conjugation reactions leading to polar phase II metabolites. To complete the information about the fate of flubendazole in various species, the conjugates of phase I flubendazole metabolites were searched in urine of sheep and rats and faeces of rats.Thirty mg of flubendazole per kg of body weight were administered to animals and the excretes were collected during 24 hours. Comparative HPLC-PDA-FLUOR analyses of the samples of urine and homogenized faeces and of the same biomatrices after their enzymatic treatment using beta-glucuronidase afforded the qualitative and quantitative information about the phase II flubendazole metabolites. References : [1] Nobilis M., Jira T., Lsa M., Holcapek M., Szotkov B., Lamka J., Sklov L.: Achiral and chiral high-performance liquid chromatographic determination of flubendazole and its metabolites in biomatrices using UV photodiode-array and mass spectrometric detection. J. Chromatogr. A 1149 (2007) 112-120 [2] Nobilis M., Vybralov Z., Krzov V., Kubcek V., Soukupov M., Lamka J., Szotkov B., Sklov L.: Sensitive chiral high-performance liquid chromatographic determination of anthelmintic flubendazole and its phase I metabolites in blood plasma using UV photodiode-array and fluorescence detection. J. Chromatogr. B 876 (2008) 89-96 This project was supported by the Ministry of Education of the Czech Republic (project No.MSM 0021620822)
    11th European Regional International society for the study of xenobiotics Meeting;
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    ABSTRACT: The aim of this study was to evaluate the age- and sex-differences in pharmacokinetic of anthelmintic drug flubendazole (FLU; methyl-[5-(4-fluorbenzoyl)-1H-benzimidazole-2-y1]-carbamate) in sheep. Three male and three female lambs (three-months-old) were used for the first pharmacokinetic study and five month later the same animals (as matured rams and ewes) were used for repetition of pharmacokinetic FLU study. During whole experiments, all animals were healthy, parasitologically negative, without any pharmacological treatment and bred under the same conditions. In both pharmacokinetic studies, single dose of FLU (30 mg per kg of body weight) in oral suspension were administered. Blood samples were taken before administration of FLU and than in 0.5, 1, 2, 4, 6, 8, 10, 12, 24, 36, 48, 60 and 72 hour intervals after FLU administration. Blood samples were centrifuged and plasma was frozen until the extraction and analysis. FLU and its metabolites were extracted into tert-butylmethyl ether and analysed by HPLC with albendazole as internal standard and mixture of methanol and sodium perchlorate (3:1) as mobile phase. The main detected metabolite was reduced flubendazole (FLU-R, compound with reduced keto group) and only traces of other metabolite (hydrolysed flubendazole) were found. Two enantiomers of FLU-R were detected on the chromatogram: (‑)‑enantiomer at retention time 14.2 min and (+)-enantiomer at 17.0 min. Considerable differences between lambs and mature sheep in plasmatic FLU-R concentrations were observed. In lambs, the Cmax value was achieved at 10 hours after FLU administration (300 pmol of (+)‑FLU-R and 14 pmol of (-)-FLU-R per ml of male plasma and 210 pmol of (+)-FLU-R and 7 pmol of (-)-FLU-R per ml of female plasma) while in mature animals Cmax value was achieved at 36 hours after FLU administration (590 pmol of (+)-FLU-R and 22 pmol of (-)-FLU-R per ml of male plasma and 440 pmol of (+)-FLU-R and 24 pmol of (-)-FLU-R per ml of female plasma). In mature sheep high concentration of both isomers of FLU-R remained in plasma up to 72 h after FLU administration of experiment. These findings document that lambs in comparison to mature animals absorb and metabolise FLU much faster but in lower range. This project was supported by Grant Agency of Czech Republic (Grant No 524/06/1345)
    10th European Regional International society for the study of xenobiotics Meeting;
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    ABSTRACT: Flubendazole, methyl ester of [5-(4-fluorobenzoyl)-1H-benzimidazol-2-yl]carbamic acid, belongs to the group of benzimidazole anthelmintics, which are widely used in veterinary medicine. The phase I flubendazole biotransformation includes the hydrolysis of the carbamoyl methyl moiety accompanied by a decarboxylation (desmethylcarboxy-flubendazole) and a carbonyl reduction of flubendazole (reduced flubendazole). Flubendazole is a prochiral drug, hence two enantiomers of reduced flubendazole are formed during non-specific carbonyl reduction. For the metabolic studies, a chiral HPLC method enabling the separation of desmethylcarboxyflubendazole, both enantiomers of reduced flubendazole and parent flubendazole was developed. The sample preparation step involves pH-dependent liquid-liquid extraction of flubendazole and its phase I metabolites into t-butylmethyl ether. Chromatographic analyses were performed on a Chiralcel OD-R 250 x 4.6 mm column (Daicel Chemical Industries, Ltd). A mixture of acetonitrile 1 M aqueous NaClO4 (4 : 6, v/v) served as an isocratic mobile phase. The column effluent was monitored using a photodiode-array detector (scan or single wavelength at 300 nm). The whole analysis lasted 30 min at a flow rate of 0.5 ml/min. Extracts from liver and intestinal cytosole of pigs and pheasants after the incubation with flubendazole and coenzyme NADPH or NADH were analyzed. Enantioselectivity in the enzymatic carbonyl reduction of flubendazole was observed. While synthetic racemic mixture of reduced flubendazole was separated to equimolar amounts of both enantiomers, only one enantiomer was detected in the extracts from all incubates. This work was supported by Grant Agency of Czech Republic (524/06/1345) and by Grant Agency of Charles University (108/2005/C).
    9th European Regional International society for the study of xenobiotics Meeting;
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    ABSTRACT: Activities of biotransformation enzymes represent the major factor which determines efficacy of pharmacotherapy and behavior of drugs in organism. Present knowledge of biotransformation enzymes and their modulation in avian farm species, including pheasants, is quite insufficient, in spite of frequent use of veterinary drugs in pheasant semidomestic breeds. Mebendazole represents benzimidazole anthelmintic which was for a long period indicated for antiparasitic treatment and prophylaxis of pheasant parasitic diseases. This benzimidazole was well effective especially against main species of pararasitic nematodes. Mebendazole is declared to be safe without adverse effects at therapeutic levels (www.emea.eu.int). Activities of pheasant hepatic and intestinal carbonyl reducing enzymes and their modulation by anthelmintic mebendazole were investigated. Biotransformation of mebendazole in subcellular fractions prepared from livers and small intestines of control and mebendazole treated birds was investigated as well. Twelve adult pheasant hens, approx. two years old, were randomly divided into two groups. Birds in the first group were treated 7 days with therapeutic dose of mebendazole (6 mg/kg). Pheasants of the second group representing controls were administered with a placebo. After the termination of experimental schedule, the whole livers and small intestines were removed; the gut content washed out. Intestinal mucosa and liver tissue were homogenized and subcellular fractions were isolated. Microsomes and cytosol were incubated with mebendazole in various concentrations and NADPH. After incubation, extracts were analyzed using an HPLC. Cytosolic reductases activities were determined using metyrapone, daunorubicin and acenaphthenol as model substrates. Activities of pheasant carbonyl reducing enzymes were slightly but significantly increased in animals treated by mebendazole both in liver and in small intestine. These results were confirmed by incubation of mebendazole with subcellular fractions. Differences of data obtained from the treated and the control birds were quantified using One-way ANOVA. Summed up, the repeated administration of mebendazole to pheasant caused only slight modulation of tested biotransformation enzymes. Mebendazole seems to be relatively safe for pheasant therapy from the view of an enzymatic modulation. This project was supported by Grant Agency of Charles University, Grant No 108/2005/C.
    9th European Regional International society for the study of xenobiotics Meeting;
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    ABSTRACT: Benzimidazole anthelmintics are widely used in veterinary medicine for prophylaxis and treatment of different endoparasitic diseases. Flubendazole, methyl ester of [5-(4-fluorobenzoyl)-1H-benzimidazol-2-yl]carbamic acid, has been mainly indicated for antiparasitic control in pig and poultry breeds. Before potential use of this drug in ruminant species, more informations about its biotransformation are necessary. A bioanalytical HPLC method was developed, validated and employed for flubendazole in vitro biotransformation studies in domestic sheep (Ovis aries) and wild sheep - mouflon (Ovis musimon). Microsomal and cytosolic subcellular fractions were prepared from liver of adult young rams (castrated or non-castrated) and adult mouflons (males or females). Flubendazole (in various concentrations) was incubated with NADPH and ovine microsomes or cytosol under aerobic conditions. The sample preparation step prior HPLC analyses involves pH-dependent liquid-liquid extraction of flubendazole and its phase I metabolites into ethyl acetate. Chromatographic analyses were performed on a LiChroCART 250 x 4 mm column containing LiChrospher 60 RPselectB, 5m (Merck, Darmstadt, Germany) with a precolumn (4 x 4 mm). A mixture of acetonitrile 0.025M phosphate buffer pH=3 (3:7, v/v) served as an isocratic mobile phase. The column effluent was monitored using a photodiode-array detector (scan or single wavelength at  = 246 nm). The whole analysis lasted 22 min at a flow rate of 1 ml.min-1. Two phase I flubendazole metabolites were found in the extracts: desmethylcarboxy flubendazole (tR = 5.2 min) and flubendazole with reduced carbonyl group (tR = 6.0 min). The chemical structures of these metabolites were confirmed using pure standards. With the aim to study the sex-differences in flubendazole biotransformation, the data obtained in castrated vs. noncastrated rams and male vs. female mouflons were compared. Comparing the data obtained in sheep and mouflon the differences in flubendazole biotransformation between domestic and wild ruminants were evaluated. The project was supported by Grant Agency of Czech Republic, Grant No 524/06/1345
    9th European Regional International society for the study of xenobiotics Meeting;

Publication Stats

87 Citations
22.75 Total Impact Points

Institutions

  • 2003–2010
    • Charles University in Prague
      • Faculty of Pharmacy in Hradec Králové
      Praha, Praha, Czech Republic
    • Veterinary Research Institute, Brno
      • Immunology
      Brünn, South Moravian, Czech Republic
  • 2009
    • Academy of Sciences of the Czech Republic
      • Ústav experimentální biofarmacie
      Praha, Hlavni mesto Praha, Czech Republic
  • 2005–2008
    • University of Hradec Králové
      Königgrätz, Královéhradecký, Czech Republic