The use of LC/MS for the Study of Biotransformation of Xenobiotics in Helminths

Univerzita Karlova v Praze, Farmaceutická fakulta v Hradci Králové Katedra biochemických věd Kandidát Mgr. Ivan Vokřál Školitel Doc. Ing. Barbora Szotáková, Ph.D. Název disertační práce Využití LC/MS pro studium biotransformace xenobiotik u helmintů Parazité každoročně způsobují mnoho ztrát na lidských životech, ale i na hospodářských zvířatech. Jednou z efektivních zbraní, které proti nim můžeme využít, jsou anthelmintika. V řadě případů však anthelmintika přestávají působit a dochází k rozvoji rezistence. Jednou z možných příčin vzniku rezistence je inaktivace léčiva na neaktivní nebo málo aktivní produkty pomocí biotransformace enzymatickým systémem parazita. Cílem této práce bylo přinést nové poznatky o biotransformaci léčiv u helmintů. Důraz byl kladen na vztah biotransformace k rezistenci. Jednou z možných technik, jak tohoto cíle dosáhnout, je studovat přímo produkty biotransformace, tedy metabolity. V dnešní době se tato problematika nejčastěji řeší za pomoci kapalinové chromatografie a hmotnostní spektrometrie a proto i pro splnění našich cílů byla tato technika využita. Byly studovány biotransformace u zástupců tasemnic, motolic a hlístic. Tam, kde to bylo možné, byli studováni zástupci hospodářsky významní jako vlasovka slézová (Haemonchus contortus) nebo motolice kopinatá (Dicrocoelium...Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate Mgr. Ivan Vokřál Supervisor Doc. Ing. Barbora Szotáková, Ph.D. Title of Doctoral Thesis The use of LC/MS for the study of biotransformation of xenobiotics in helminths Annually, parasites cause many losses regarding not only human lives but also the livestock. One of the most effective weapons that can be used against them are anthelmintics. However, in many cases anthelmintics cease to work and resistance development occurs. One of the possible mechanisms of resistance is the biotransformation of the drug to ineffective or less active products by the parasite. The aim of this work was to bring new knowledge about the biotransformation of drugs in helminths. Emphasis was given to the relationship between biotransformation and the resistance of parasites against anthelmintic drugs. A possible technique to achieve this goal is to study directly the products of biotransformation, i.e. metabolites. As this issue is nowadays most frequently solved by liquid chromatography and mass spectrometry, we used this technique to meet our goals. Biotransformation was studied in representatives of tapeworms, flukes and roundworms. Where possible, economically important representatives as Barber's pole...Katedra biochemických vědDepartment of Biochemical SciencesFaculty of Pharmacy in Hradec KrálovéFarmaceutická fakulta v Hradci Králov

UDP-glycosyltransferase family in Haemonchus contortus: Phylogenetic analysis, constitutive expression, sex-differences and resistance-related differences

UDP-glycosyltransferases (UGT), catalysing conjugation of UDP-activated sugar donors to small lipophilic chemicals, are widespread in living organisms from bacteria to fungi, plant, or animals. The progress of genome sequencing has enabled an assessment of the UGT multigene family in Haemonchus contortus (family Trichostrongylidae, Nematoda), a hematophagous gastrointestinal parasite of small ruminants. Here we report 32 putative UGT genes divided into 15 UGT families. Phylogenetic analysis in comparison with UGTs from Caenorhabditis elegans, a free-living model nematode, revealed several single member homologues, a lack of the dramatic gene expansion seen in C. elegans, but also several families (UGT365, UGT366, UGT368) expanded in H. contortus only. The assessment of constitutive UGT mRNA expression in H. contortus adults identified significant differences between females and males. In addition, we compared the expression of selected UGTs in the drug-sensitive ISE strain to two benzimidazole-resistant strains, IRE and WR, with different genetic backgrounds. Constitutive expression of UGT368B2 was significantly higher in both resistant strains than in the sensitive strain. As resistant strains were able to deactivate benzimidazole anthelmintics via glycosylation more effectively then the sensitive strain, UGT368B2 enhanced constitutive expression might contribute to drug resistance in H. contortus

The use of LC/MS for the Study of Biotransformation of Xenobiotics in Helminths

Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate Mgr. Ivan Vokřál Supervisor Doc. Ing. Barbora Szotáková, Ph.D. Title of Doctoral Thesis The use of LC/MS for the study of biotransformation of xenobiotics in helminths Annually, parasites cause many losses regarding not only human lives but also the livestock. One of the most effective weapons that can be used against them are anthelmintics. However, in many cases anthelmintics cease to work and resistance development occurs. One of the possible mechanisms of resistance is the biotransformation of the drug to ineffective or less active products by the parasite. The aim of this work was to bring new knowledge about the biotransformation of drugs in helminths. Emphasis was given to the relationship between biotransformation and the resistance of parasites against anthelmintic drugs. A possible technique to achieve this goal is to study directly the products of biotransformation, i.e. metabolites. As this issue is nowadays most frequently solved by liquid chromatography and mass spectrometry, we used this technique to meet our goals. Biotransformation was studied in representatives of tapeworms, flukes and roundworms. Where possible, economically important representatives as Barber's pole..

The use of LC/MS for the Study of Biotransformation of Xenobiotics in Helminths

Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate Mgr. Ivan Vokřál Supervisor Doc. Ing. Barbora Szotáková, Ph.D. Title of Doctoral Thesis The use of LC/MS for the study of biotransformation of xenobiotics in helminths Annually, parasites cause many losses regarding not only human lives but also the livestock. One of the most effective weapons that can be used against them are anthelmintics. However, in many cases anthelmintics cease to work and resistance development occurs. One of the possible mechanisms of resistance is the biotransformation of the drug to ineffective or less active products by the parasite. The aim of this work was to bring new knowledge about the biotransformation of drugs in helminths. Emphasis was given to the relationship between biotransformation and the resistance of parasites against anthelmintic drugs. A possible technique to achieve this goal is to study directly the products of biotransformation, i.e. metabolites. As this issue is nowadays most frequently solved by liquid chromatography and mass spectrometry, we used this technique to meet our goals. Biotransformation was studied in representatives of tapeworms, flukes and roundworms. Where possible, economically important representatives as Barber's pole..

On pharmacokinetic of single administration of albendazole in mouflon (Ovis musimon)

Name: Ivan Vokřál Title: On pharmacokinetic of single administration of albendazole in mouflon (Ovis musimon) Abstract: This diploma thesis contributes to the knowledge of albendazole pharmacokinetics in mouflon (Ovis gmelini musimon) treated by a single administration of albendazole in a dose of 30,0 mg/kg of body weight. Two separate experiments were realised. The first one was carried out on 12 mouflon ewes divided into 4 groups. Groups of the animals were gradually culled at the time intervals of 4, 6, 8 and 10 hours after the albendazole administration. The samples of the blood (serum), bile from gallbladder and terminal bile ducts were withdrawn. The concentration of albendazole (ABZ) and its major metabolites albendazolsulphoxide (ABZSO) and albendazolsulphone (ABZSO2) were determined in samples of biological materials by HPLC method. To determine the ratio of ABZSO enantiomers, the HPLC method with chiral column was used. The second experiment carried on 2 animals (time interval 6 hours) extended and partially confirmed previous results. The increase in blood concentrations of albendazole and its metabolites until the eighth hour after the administration is evident. In later time intervals decrease in concentrations was documented. The concentrations of ABZSO and ABZSO2 between the bile..

Evaluation of the Potency of Anti-HIV and Anti-HCV Drugs to Inhibit P-Glycoprotein Mediated Efflux of Digoxin in Caco-2 Cell Line and Human Precision-Cut Intestinal Slices

The inhibition of P-glycoprotein (ABCB1) could lead to increased drug plasma concentrations and hence increase drug toxicity. The evaluation of a drug’s ability to inhibit ABCB1 is complicated by the presence of several transport-competent sites within the ABCB1 binding pocket, making it difficult to select appropriate substrates. Here, we investigate the capacity of antiretrovirals and direct-acting antivirals to inhibit the ABCB1-mediated intestinal efflux of [3H]-digoxin and compare it with our previous rhodamine123 study. At concentrations of up to 100 µM, asunaprevir, atazanavir, daclatasvir, darunavir, elbasvir, etravirine, grazoprevir, ledipasvir, lopinavir, rilpivirine, ritonavir, saquinavir, and velpatasvir inhibited [3H]-digoxin transport in Caco-2 cells and/or in precision-cut intestinal slices prepared from the human jejunum (hPCIS). However, abacavir, dolutegravir, maraviroc, sofosbuvir, tenofovir disoproxil fumarate, and zidovudine had no inhibitory effect. We thus found that most of the tested antivirals have a high potential to cause drug–drug interactions on intestinal ABCB1. Comparing the Caco-2 and hPCIS experimental models, we conclude that the Caco-2 transport assay is more sensitive, but the results obtained using hPCIS agree better with reported in vivo observations. More inhibitors were identified when using digoxin as the ABCB1 probe substrate than when using rhodamine123. However, both approaches had limitations, indicating that inhibitory potency should be tested with at least these two ABCB1 probes

Reliable reference gene selection for quantitative real time PCR in Haemonchus contortus

The aim of this work was to identify reliable reference genes for expression studies in adult Haemonchus contortus. Eleven candidate genes were identified and the stability of their expression was assessed in adult males and females of two genetically divergent H. contortus isolates: drug-susceptible (ISE) and multi-drug-resistant (WR). Five genes with the most stable expression patterns were further assessed for suitability as reference genes in anthelmintic-treated H. contortus adults versus non-treated controls. We identified important differences in the expression of a number of candidate genes in anthelmintic-treated samples, confirming the need for careful validation of control genes for such experiments. We propose the use of multiple reference genes for expression studies in this species and found gpd, ama and far most suitable for adult H. contortus

Determination of Antiviral Drugs and Their Metabolites Using Micro-Solid Phase Extraction and UHPLC-MS/MS in Reversed-Phase and Hydrophilic Interaction Chromatography Modes

Two new ultra-high performance liquid chromatography (UHPLC) methods for analyzing 21 selected antivirals and their metabolites were optimized, including sample preparation step, LC separation conditions, and tandem mass spectrometry detection. Micro-solid phase extraction in pipette tips was used to extract antivirals from the biological material of Hanks balanced salt medium of pH 7.4 and 6.5. These media were used in experiments to evaluate the membrane transport of antiviral drugs. Challenging diversity of physicochemical properties was overcome using combined sorbent composed of C18 and ion exchange moiety, which finally allowed to cover the whole range of tested antivirals. For separation, reversed-phase (RP) chromatography and hydrophilic interaction liquid chromatography (HILIC), were optimized using extensive screening of stationary and mobile phase combinations. Optimized RP-UHPLC separation was carried out using BEH Shield RP18 stationary phase and gradient elution with 25 mmol/L formic acid in acetonitrile and in water. HILIC separation was accomplished with a Cortecs HILIC column and gradient elution with 25 mmol/L ammonium formate pH 3 and acetonitrile. Tandem mass spectrometry (MS/MS) conditions were optimized in both chromatographic modes, but obtained results revealed only a little difference in parameters of capillary voltage and cone voltage. While RP-UHPLC-MS/MS exhibited superior separation selectivity, HILIC-UHPLC-MS/MS has shown substantially higher sensitivity of two orders of magnitude for many compounds. Method validation results indicated that HILIC mode was more suitable for multianalyte methods. Despite better separation selectivity achieved in RP-UHPLC-MS/MS, the matrix effects were noticed while using both chromatographic modes leading to signal enhancement in RP and signal suppression in HILIC

Metabolism of albendazole, ricobendazole and flubendazole in Haemonchus contortus adults: Sex differences, resistance-related differences and the identification of new metabolites

Haemonchus contortus (family Trichostrongylidae, Nematoda), a hematophagous gastrointestinal parasite found in small ruminants, has a great ability to develop resistance to anthelmintic drugs. We studied the biotransformation of the three benzimidazole anthelmintics: albendazole (ABZ), ricobendazole (albendazole S-oxide; RCB) and flubendazole (FLU) in females and males of H. contortus in both a susceptible ISE strain and resistant IRE strain. The ex vivo cultivation of living nematodes in culture medium with or without the anthelmintics was used. Ultrasensitive UHPLC/MS/MS analysis revealed 9, 7 and 12 metabolites of ABZ, RCB and FLU, respectively, with most of these metabolites now described in the present study for the first time in H. contortus. The structure of certain metabolites shows the presence of biotransformation reactions not previously reported in nematodes. There were significant qualitative and semi-quantitative differences in the metabolites formed by male and female worms. In most cases, females metabolized drugs more extensively than males. Adults of the IRE strain were able to form many more metabolites of all the drugs than adults of the ISE strain. Some metabolites were even found only in adults of the IRE strain. These findings suggest that increased drug metabolism may play a role in resistance to benzimidazole drugs in H. contortus. Keywords: Drug resistance, Drug metabolism, Anthelmintics, Benzimidazole, Nematod