[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] 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. · 2.51 Impact Factor
[Show abstract][Hide abstract] 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. · 5.54 Impact Factor
[Show abstract][Hide abstract] 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. · 2.49 Impact Factor
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] ABSTRACT: Mechanical properties of biological structures affect functional ability of organism. Current knowledge is prevailingly concentrated on static characteristics. The present work analyzed dynamic mechanical responses of various biological materials. Following biological structures were measured: samples of aorta walls of human origin and from model organisms, human body surface, and samples of bones of various types and origin. Linear approximation leads in case of aortas and bones to simple Voight's model. Modules of elasticity (in tensile loading) of aortas were from 10(2) kPa to 10(3) kPa. Module of elasticity of bones were from 10(6) Pa to 10(10) Pa. Viscous coefficients of aortas were from 102 Pa.s to 10(3) Pa.s. Viscous coefficients of bones were from 10(0) Pa.s to 10(2) Pa.s. Nonlinearities: We found that following types of nonlinearities are significant: strain-stress relationship, time-dependent changes in elastic as well as viscose bodies. Strain and stress is well approximated by quadratic function sigma = a epsilon2 + b epsilon + c with parameters a = 1833, b = 135, c = 20.0 (porcine aorta). Time-dependence in elastic coefficient: At the beginning of responses the elastic coefficient was of 42% lower then at 0.02 s of duration of the response (porcine aortas). Analogical results follow also from experiments on other structures (skin, bones).
Acta medica (Hradec Králové) / Universitas Carolina, Facultas Medica Hradec Králové 02/2004; 47(4):297-300.
[Show abstract][Hide abstract] 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. · 5.22 Impact Factor
[Show abstract][Hide abstract] 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.