Dissertations / Theses on the topic 'Human liver microsomes'
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Emery, Maurice George. "Aspects of human CYP 2E1 regulation in health and disease /." Thesis, Connect to this title online; UW restricted, 1999. http://hdl.handle.net/1773/7943.
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McLure, James Alexander, and james mclure@flinders edu au. "Physicochemical determinants of the non-specific binding of drugs to human liver microsomes." Flinders University. Medicine, 2008. http://catalogue.flinders.edu.au./local/adt/public/adt-SFU20081102.165952.
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Accurate determination of the in vitro kinetic parameters Km (Michaelis constant) and Ki (inhibition constant) is critical for the quantitative prediction of in vivo drug clearance and the magnitude of inhibitory drug interactions. A cause of inaccuracy in vitro arises from the assumption that all drug added to an incubation mixture is available for metabolism or inhibition. Many drugs bind non-specifically to the membrane of the in vitro enzyme source.
The aims of this thesis were to: 1) investigate the comparative importance of lipophilicity (as log P), and pKa as determinants of the non-specific binding of drugs to human liver microsomes; 2) develop and validate an ANS fluorescence technique for measuring the non-specific binding of drugs to human liver microsomes; 3) characterise the non-specific binding of a large dataset of physicochemically diverse drugs using the ANS fluorescence procedure; 4) evaluate relationships between selected physicochemical characteristics and the extent of non-specific binding of drugs to human liver microsomes and; 5) computationally model the non-specific binding of drugs to discriminate between high binding (fu(mic) less than 0.5) and low binding (fu(mic) greater than 0.5) drugs.
The comparative binding of the basic drugs atenolol (log P = 0.1; fu(mic) = 1.00), of propranolol (log P = 3.1; fu(mic) = 0.36 - 0.84), and imipramine (log P = 4.8; fu(mic) = 0.42 - 0.82) suggested that lipophilicity is a major determinant of non-specific binding. In contrast, the comparative binding of diazepam (pKa = 3.3; fu(mic) = 0.69 - 0.80), a neutral compound; and the bases propranolol (pKa = 9.5; fu(mic) = 0.36 - 0.84) and lignocaine (pKa = 9.5; fu(mic) = 0.98), indicated that pKa was not a determinant of the extent of non-specific binding. The non-binding of lignocaine, a relatively lipophilic base, was unexpected and confirmed by the non-binding of the structurally related compounds bupivacaine and ropivacaine. These results implicated physicochemical characteristics other than lipophilicity and charge as important for the non-specific binding of drugs to human liver microsomes.
An assay based on 1-anilinonaphthalene-8-sulfonate (ANS) fluorescence was developed using the seven drugs employed in the initial study. Non-specific binding data from equilibrium dialysis and the ANS fluorescence methods were compared and a linear correlation (r2 = 0.92, p less than 0.01) was observed at drug concentrations of 100 and 200 micromolar. The approach was further validated by characterising the microsomal binding of nine compounds (bupropion, chloroquine, chlorpromazine, diflunisal, flufenamic acid, meclofenamic acid, mianserine, triflupromazine, and verapamil) using both binding methods (i.e. equilibrium dialysis and ANS fluorescence). A significant logarithmic relationship (r2 greater than or equal to 0.90) was demonstrated between fu(mic) and the modulus of ANS fluorescence for all drugs and for basic drugs alone at concentrations of 100 and 200 micromolar, while the acidic/neutral drugs showed a significant linear relationship (r2 greater than or equal to 0.84) at these two concentrations (p less than 0.01). The non binding of bupropion provided further evidence that physicochemical properties other than log P and charge were important for non-specific binding of drugs to human liver microsomes.
The ANS fluorescence technique was then used to characterise the non-specific binding of 88 physicochemically diverse compounds. In general, acids and neutrals bound to a low extent (fu(mic) greater than 0.5) whereas bases bound the full fu(mic) range (0.0001 to 1). Statistically significant relationships were observed between the non-specific binding of bases and log P, the number of hydrogen bond donors and hydrogen bond acceptors per molecule, and molecular mass.
Preliminary in silico modeling of the dataset generated by the ANS fluorescence technique, using the program ROCS, provided discrimination of all but one (itraconazole) of the high binding bases. However, there were 14 false positives, resulting in low overall prediction accuracy.
Taken together, the studies conducted in this thesis provide important insights into the physicochemical factors that determine the non-specific binding of drugs to human liver microsomes.
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Schjølberg, Tiril Helgesen. "In Vitro Synthesis of Metabolites of three Anabolic Androgenic Steroids, by Human Liver Microsomes." Thesis, Norges teknisk-naturvitenskapelige universitet, Institutt for bioteknologi, 2013. http://urn.kb.se/resolve?urn=urn:nbn:no:ntnu:diva-22910.
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Anabolic androgenic steroids are substances frequently misused to improve physicalperformance in sports. To reveal substances misused as doping, athlete urinesamples are collected and tested. To identify the steroid and/or its metabolitesin urine, reference compounds must exist for comparison. The time-consumingand ethical concerns about in vivo excretion studies for the examination of thesecompounds, make the use of liver fragment microsomes an attractive alternative.The aim of this thesis was to synthesize and identify metabolites from known andrare anabolic androgenic steroids, by the use of human liver microsomes. Liveris an important organ in steroid metabolism. By incubating AAS with humanliver microsomes and co factors, an in vitro simulation of the liver metabolism wascarried out. A conrmation of metabolites was performed by gas chromatographytandem mass spectrometry in full scan, MRM mode, or both. 6beta-hydroxymethylmetandienon, epimetandienon and 17,17-dimethylmetandienon were successfullysynthesized from metandienon, and the 17beta-hydroxymetandienon was producedfrom the 17,17-dimethyl metabolite. Respectively three and one metabolite(s)were found for the "designer steroids" methylnortestosteron and madol. Metabolitevariations were observed regarding the optimal time of incubation, and enzymaticrequirements of formation.
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Maley, Mary. "The role of individual forms of cytochrome P450 in drug metabolism in human liver microsomes." Thesis, University of Aberdeen, 1996. http://digitool.abdn.ac.uk/R?func=search-advanced-go&find_code1=WSN&request1=AAIU078654.
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Human liver microsomal metabolism of nicardipine was investigated and compared to that of another dihydropyridine, felodipine, and to published results for other compounds belonging to this class of drugs. The metabolism of tamoxifen and two iododerivatives, idoxifene and 4-iodotamoxifen, were also investigated. Nicardipine metabolism by human liver microsomes was dissimilar to that of other dihydropyridines in several respects. For most dihydropyridines studied to date, conversion to the corresponding pyridine is the major metabolic pathway; the results from this study suggested that pyridine formation is not the major pathway of human liver nicardipine metabolism. The oxidation of most dihydropyridines in human liver microsomes is CYP3A-dependent. In this study, the results from correlation studies and inhibition experiments implicated only CYP3A in nicardipine metabolism, however, not to the same extent as for other dihydropyridines. N-demethylation is the major metabolic route for tamoxifen in human liver and is dependent on the activity of CYP3A. The results from this study suggested that CYP3A is not largely involved in the metabolism of idoxifene and 4-iodotamoxifen in human liver microsomes. Incubations of idoxifene with human liver microsomes resulted in the formation of two metabolites, neither of which could be identified. Correlation and inhibition studies indicated that CYP1A, 2C, 2D and 3A were not involved in idoxifene metabolism in human liver microsomes, although, there was some evidence to support CYP2A involvement. Incubation of 4-iodotamoxifen with microsomes resulted in the formation of up to four metabolites, two of which could be identified. The formation of N-desmethyl 4-iodotamoxifen, the second largest metabolite, appeared to be dependent on CYP3A in human liver microsomes. Correlation studies did not implicate any P450 in the other pathways of 4-iodotamoxifen metabolism in human liver microsomes.
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Abu-Omar, Ghada M. "Drug interactions and metabolism of cyclosporin A and steroids by human liver microsomes in vitro." Thesis, University of Aberdeen, 1992. http://digitool.abdn.ac.uk/R?func=search-advanced-go&find_code1=WSN&request1=AAIU545502.
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1) The purpose of this work is to study the enzymology of cyclosporin A (CsA) metabolism by human liver in vitro , in particular investigating the basis for interindividual variations and drug interactions involving CsA metabolism, and to apply this knowledge to the development of a non-invasive test for predicting an individual's ability to metabolise CsA in vivo . The ultimate aim is to help to improve CsA therapy by understanding the factors which determine its metabolism in patients. 2) Human liver microsomes metabolised cyclosporin A (Km 25M) to eight identifiable metabolites, which were detected following incubation with [3H]-CsA in the presence of NADPH and aerobic O2. An interindividual variation in the generation of CsA metabolites was also observed. In addition, the formation rates of primary and secondary CsA metabolites were induced to different extents by 2 and 4 fold respectively, in liver microsomes prepared from patients treated with anticonvulsant treatment. Metabolite M1 formation was also induced by anticonvulsant treatment. 3) A significant correlation was observed between the total rate of CsA metabolism and cytochrome P-450IIIA concentration as measured by Western blot analysis, in 9 human liver microsomal preparations; 6 were obtained from untreated individuals and 3 from anticonvulsant-treated patients. This correlation was also present when the primary and the secondary metabolite profiles were considered separately. The kinetics of CsA metabolism by human liver microsomes demonstrated different Km and Vmax values for the formation rates of primary and secondary CsA metabolites. This suggests the possible involvement of two enzymes within the P-450IIIA family, responsible for the formation of primary and secondary metabolites, respectively. 4) Anti P-450IIIA antibodies inhibited the total rate of CsA metabolism in microsomal preparations from anticonvulsant-treated patients but to a lesser extent than those from untreated individuals. This suggests an involvement of other form(s) of P-450in CsA metabolism, responsible for between 20-30&'37 of CsA metabolic activity in human liver microsomes. However, these are unlikely to be P-450IIB1, P-450IA1 or P-450IIC, as none of the antibodies raised against these P450s significantly inhibited the metabolism of CsA. 5) From induction, inhibition and correlation studies, cytochrome P-450IIIA has been confirmed as being responsible for the major part of CsA metabolism in human and rat liver microsomes.
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Dowsley, Taylor Forbes. "CYP2E1-dependent bioactivation of 1,1-dichloroethylene to reactive intermediates in murine and human lung and liver microsomes." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp02/NQ38304.pdf.
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Strömqvist, Malin. "Development of quantitative methods for the determination of vemurafenib and its metabolites in human plasma." Thesis, Linköpings universitet, Kemi, 2014. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-110076.
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Vemurafenib is a potent serine/threonine kinase inhibitor and is registered as Zelboraf® for the treatment of metastatic melanomas harboring BRAFV600E mutations. There is a large individual variation in drug response and the side effects observed among patients treated with Zelboraf® has proven to be severe. LC-MS/MS methods were developed to measure vemurafenib and its metabolites in human plasma for prediction of treatment outcome and side effects in order to individualize treatment with Zelboraf®. A novel, rapid quantification method was developed for vemurafenib using a stable isotope labeled internal standard. The method was validated according to international guidelines with regard to calibration range, accuracy, precision, carry-over, dilution integrity, selectivity, matrix effects, recovery and stability. All parameters met the set acceptance criteria. The first method suitable for quantifying vemurafenib metabolites in human plasma is presented. Lacking commercially available reference substances, human liver microsomes were used to produce the metabolites. In patient samples at steady-state five previously in vitro identified metabolites were quantified for the first time.
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Shepard, Dale Randall. "The Metabolism of Phenytoin by Human Liver Microsomes and Cytochrome P450s Expressed in Saccharomyces Cerevisiae and COS-1 Cells /." The Ohio State University, 1995. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487931512618872.
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Uwimana, Eric. "Probing the PCB metabolome: metabolism of chiral and non-chiral polychlorinated biphenyls to chiral hydroxylated metabolites in humans and rats." Diss., University of Iowa, 2018. https://ir.uiowa.edu/etd/6657.
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Polychlorinated biphenyls (PCBs) continue to pose a health concern because of their predominance in the diet and air as well as in environmental samples and humans. PCB congeners with 3 or 4 chlorine substituents in ortho position have been associated with neurodevelopmental disorders. Hydroxylated metabolites (OH-PCBs) of these PCBs are also potentially toxic to the developing brain. Metabolism studies have mainly focused on animal models. However, preliminary data from this dissertation work have revealed PCB metabolism differences between laboratory animal models and humans in terms of metabolite profiles, chiral signatures. More concerning, biotransformation of chiral PCBs is poorly investigated in humans. The objective of this dissertation research was to study the biotransformation of chiral and prochiral PCBs to chiral hydroxylated metabolites in humans and rats and to identify individual human P450 enzymes involved in the metabolism of these PCBs. I chose chiral PCB congeners 2,2',3,4',6-pentachlorobiphenyl (PCB 91); 2,2',3,5',6-pentachlorobiphenyl (PCB 95), 2,2',3,3',4,6'-hexachlorobiphenyl (PCB 132) and 2,2',3,3',6,6'-hexachlorobiphenyl (PCB 136) for this investigation because they are environmentally relevant and their metabolism has been studied in rodents and other laboratory animal species (Kania-Korwel et al., 2016a). Prochiral PCB congeners 2,2′,4,6′-tetrachlorobiphenyl (PCB 51) and 2,2′,4,5,6′-pentachlorobiphenyl (PCB 102) were selected because their considerable presence in technical PCB mixtures.
To test the hypothesis that P450 enzyme and species differences mediate the congener-specific enantioselective metabolism of chiral PCBs to hydroxylated metabolites, I sought to establish structure-metabolism relationships by studying the enantioselective metabolism of structurally diverse chiral PCBs by human liver microsomes (HLMs). Racemic PCB 91, PCB 95 and PCB 132 were incubated in vitro with pooled or individual donor HLMs at 37 °C, and levels and chiral signatures of the parent PCB and its hydroxylated metabolites were determined by high-resolution gas chromatography equipped with time-of-flight mass spectrometry (GC/TOF-MS) or electron capture detection (GC-ECD). Hydroxylated metabolites formed were identified and metabolic schemes for these PCBs proposed. I found inter-individual differences in the formation of OH-PCBs by individual donor HLMs. Comparison of the metabolite profiles of PCB 91, PCB 95, PCB 132 and PCB 136 (PCB 136 metabolism by HLMs was investigated by other researchers) revealed congener-specific differences in the oxidation of PCBs by human cytochrome P450 enzymes. PCB 91 and PCB 132 were mainly hydroxylated in meta position, with the 1,2-shift metabolites being the major metabolites formed from both PCB congeners by HLMs. In contrast, PCB 95 and PCB 136 were primarily hydroxylated in the para position. Moreover, we determined human P450 isoforms involved in the metabolism of neurotoxic PCBs using in silico and in vitro approaches. In silico predictions suggested that chiral PCBs are metabolized by CYP1A2, CYP2A6, CYP2B6, CYP2E1, and CYP3A4. Experimentally we found that CYP2A6, CYP2B6 and to a minor extent CYP2E1 were the enzymes involved in the metabolism of these chiral PCBS.
We also investigated nonchiral sources of chiral OH-PCBs by studying the P450- and species-dependent biotransformation of prochiral PCB 51 and PCB 102 to chiral OH-PCB metabolites. Prochiral PCB 51 and PCB 102 were incubated with liver microsomes prepared from male Sprague-Dawley rats pretreated with various inducers of P450 enzymes including phenobarbital (PB), dexamethasone (DEX), isoniazid (INH), β-naphthoflavone (BNF), clofibric acid (CFA) or corn oil (CO); and untreated male cynomolgus monkeys, Hartley albino guinea pigs, New Zealand rabbits, golden Syrian hamsters; and untreated female Beagle dogs. PCB 51 and PCB 102 were metabolized to 2,2',4,6'-tetrachlorobiphenyl-3'-ol (OH-PCB 51) and 2,2',4,5,6'-pentachlorobiphenyl-3'-ol (OH-PCB 102), respectively. The formation of both metabolites was P450 isoforms- and species-dependent. Moreover, OH-PCB 51 and OH-PCB 102 were chiral and were formed enantioselectively in all microsomes investigated.
Taken together, my findings demonstrate (1) considerable inter-individual variability in the congener-specific metabolism of PCBs to OH-PCBs; (2) the enantioselective formation of OH-PCBs by human CYP2A6, CYP2B6, and CYP2E1; and (3) that chiral PCB metabolites are formed enantioselectively from prochiral PCB congeners. Interestingly, the metabolism of PCBs by CYP2A6 appears to involve arene oxide intermediates, as suggested by the formation of 1,2-shift products as major metabolites of PCB 91 and PCB 132. In contrast, 1,2-shift products are minor PCB metabolites formed in rodents. Therefore extrapolation of hepatic metabolism across species may not be consistent and these differences should be considered in future toxicity and risk assessment studies.
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Larabi, Islam Amine. "Nouveaux produits de synthèse : analyse, consommation et métabolisme ; Applications cliniques et médicolégales Rapid and simultaneous screening of new psychoactive substances and conventional drugs of abuse. A comparative study of Biochip Array Technology versus LC-MS/MS in whole blood and urine Development of a sensitive untargeted liquid chromatography– high resolution mass spectrometry screening devoted to hair analysis through a shared MS2 spectra database: A step toward early detection of new psychoactive substances Validation of an UPLC-MS/MS method for the determination of sixteen synthetic cannabinoids in human hair. Application to document chronic use of JWH-122 following a non-fatal overdose Development and validation of liquid chromatography-tandem mass spectrometry targeted screening of 16 fentanyl analogs and U-47700 in hair: Application to 137 authentic samples Prevalence and Surveillance of Synthetic Cathinones Use by Hair Analysis: An Update Review Prevalence of New Psychoactive Substances(NPS) and conventional drugs of abuse (DOA) in high risk populations from Paris(France) and its suburbs A cross sectional study by hair testing(2012–2017) Evaluation of drug abuse by hair analysis and self-reported use among MSM under PrEP: Results from a sub-study of the ANRS-IPERGAY trial. Hair testing for 3‑fluorofentanyl, furanylfentanyl, methoxyacetylfentanyl, carfentanil, acetylfentanyl and fentanyl by LC–MS/MS after unintentional overdose Drug‐facilitated sexual assault (DFSA) involving 4‐methylethcathinone (4‐MEC),3,4‐Methylenedioxypyrovalerone (MDPV), and doxylamine highlighted by hair analysis Metabolic Profiling of Deschloro-N-ethyl-ketamine (O-PCE) and identification of new target metabolites in urine and hair using human liver microsomes and high-resolution accurate mass spectrometry." Thesis, université Paris-Saclay, 2020. http://www.theses.fr/2020UPASL029.
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L’objectif de ce travail a été de développer deux approches analytiques dédiées à l’analyse toxicologique des nouveaux produits de synthèse (NPS) dans différentes matrices biologiques (sang, urine et cheveux). La première est basée sur le criblage non ciblé par chimiluminescence sur biopuces et chromatographie liquide couplée à la spectrométrie de masse haute résolution (LC-HRMS) et la deuxième correspond à un criblage ciblé par spectrométrie de masse en tandem (LC-MS/MS). Ces deux approches ont ensuite été appliquées dans des études observationnelles pour évaluer la consommation de NPS dans des populations à risques de surdosage, de pharmacodépendance ou de soumission chimique dans un contexte clinique ou médico-judiciaire.La dernière partie a été consacrée au développement d’un nouvel outil analytique de traitement des données issues de la LC-HRMS qui a permis d’étudier le métabolisme de 9 NPS in vitro sur des cultures de microsomes du foie humain (HLM) et in vivo sur des échantillons biologiques d’usagers de ces drogues. Cette dernière approche a permis la création d’une bibliothèque de spectres de haute résolution composée de 228 métabolites dont certains ont été proposés comme marqueurs pertinents d’exposition aux NPS dont ils sont issus.Ce travail a été concrétisé par la rédaction de 10 publications scientifiques et a permis d’initier plusieurs collaborations pluridisciplinairesThe aim of the present work was to develop two analytical approaches dedicated to the analysis of new psychoactive substances in different biological matrices (blood, urine and hair). The first approach is based on untargeted screening by both biochip array technology chemiluminescence assay and liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS) and the second corresponds to a targeted screening by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). These two approaches were then applied in observational studies to assess the consumption of NPS in high risk populations (overdose, drug abuse, drug facilitated crimes) in clinical and forensic settings. The last part of the work was devoted to the development of a new analytical tool for LC-HRMS data processing which made it possible to study the metabolism of 9 NPS In vitro on human liver microsomes (HLM) and In vivo in biological samples from drug users. This approach has enabled the creation of HRMS spectral library containing 228 metabolites, some of which have been proposed as relevant markers of NPS exposure.This work has resulted on 10 scientific publications and allowed to initiate many multidisciplinary collaborations
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Kroetz, Deanna L. "Inhibition of human liver microsomal epoxide hydrolase /." Thesis, Connect to this title online; UW restricted, 1990. http://hdl.handle.net/1773/7958.
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Vollmar, Christian Verfasser], and Hans H. [Akademischer Betreuer] [Maurer. "New cathinone-derived designer drugs 3-bromomethcathinone and 3-fluoromethcathinone : studies on their metabolism in rat urine and human liver microsomes using GC-MS and LC-high-resolution MS and their detectability in urine / Christian Vollmar. Betreuer: Hans H. Maurer." Saarbrücken : Saarländische Universitäts- und Landesbibliothek, 2014. http://d-nb.info/1053725442/34.
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Habenschus, Maísa Daniela. "Estudos de inibição das enzimas do citocromo P450 pelo produto natural (-)-grandisina utilizando microssomas hepáticos de humanos." Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/59/59138/tde-06072016-095943/.
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A (-)- grandisina (GRA) é um produto natural da classe das lignanas e é encontrada em muitas espécies de plantas das regiões Norte e Nordeste do Brasil. Por apresentar diversas propriedades biológicas, como atividade tripanocida, anti-inflamatória, antinociceptiva, e principalmente atividade antileucêmica e antitumoral contra tumores de Ehrlich, a GRA pode ser considerada um potencial candidato a fármaco. Porém, para que a GRA se torne um fármaco são necessárias diversas etapas de estudos, incluindo estudos pré-clínicos de interações medicamentosas (DDI). As DDI ocorrem principalmente devido a inibições diretas e tempo-dependentes das enzimas do citocromo P450 (CYP450), uma superfamília de enzimas responsável por metabolizar cerca de 75% dos fármacos em uso. Os estudos pré-clínicos de DDI envolvem o conhecimento do potencial inibitório do candidato a fármaco sobre essas enzimas e esses estudos podem ser realizados empregando diversos modelos in vitro, como, por exemplo, microssomas hepáticos de humanos (HLM). Assim, nesse estudo foi avaliado o efeito inibitório da GRA sobre a atividade das principais isoformas do CYP450 e também foram determinadas as isoformas que contribuem para a formação dos metabólitos da GRA. Os resultados demonstraram que múltiplas isoformas participam da formação dos metabólitos da GRA, com destaque para a CYP2C9, que participa da formação de todos os metabólitos. Em relação aos estudos de inibição, foi possível concluir que a GRA é um inibidor fraco da CYP1A2 e CYP2D6, com valores de IC50 maiores do que 200 µM e 100 µM, respectivamente, e um inibidor moderado e competitivo da CYP2C9, com IC50 igual a 40,85 µM e Ki igual a 50,60 µM. Para a CYP3A4 o potencial inibitório da GRA foi avaliado utilizando dois substratos distintos. A GRA demonstrou ser tanto um inibidor dose-dependente moderado e competitivo dessa isoforma, quanto um inibidor tempo-dependente baseado em mecanismo com potencial de inativação equiparável ao do irinotecano, inibidor baseado em mecanismo clinicamente significativo. Utilizando a nifedipina como substrato os valores de IC50 e Ki foram 78,09 µM e 48,71 µM, respectivamente. Já os valores dos parâmetros cinéticos de inativação foram KI= 6,40 µM, kinact= 0,037 min-1 e Clinact= 5,78 mL min-1 µmol-1. Para os ensaios empregando o midazolam os valores de IC50 e Ki foram 48,87 µM e 31,25 µM, respectivamente, e os valores dos parâmetros cinéticos de inativação foram KI= 31,53 µM, kinact= 0,049 min-1 e Clinact= 1,55 mL min-1 µmol-1. Com relação a CYP2E1, por sua vez, foi possível observar que a GRA tem capacidade de aumentar a atividade dessa isoforma significativamente a partir da concentração de 4 µM. Portanto, conclui-se que não há risco da GRA apresentar interações medicamentosas com fármacos metabolizados pela CYP1A2 e CYP2D6, enquanto que para a CYP2C9, apesar da GRA ser um inibidor moderado dessa isoforma, o risco é baixo. Já para medicamentos metabolizados pela CYP2E1 e CYP3A4 o risco de DDI existe e isso deve ser cuidadosamente monitorado in vivo, principalmente porque a CYP3A4 é a isoforma responsável por catalisar o metabolismo da maioria dos fármacos.(-)-grandisin (GRA) is a lignanic natural product found in many species of plants from North and Northeast of Brazil. This compound has several biological properties, such as trypanocide, anti-inflammatory, antinociceptive, antileukemia activity and antitumor activity against Ehrlich tumor. Because of these biological properties, GRA is considered a potential drug candidate, however, before becoming a new drug, GRA has to undergo various tests, including preclinical drug-drug interactions (DDI) studies. Most of the times, DDI occur because of direct and time-dependent inhibitions of cytochrome P450 (CYP450) enzymes, an enzyme superfamily responsible for metabolizing the vast majority of drugs administered. Preclinical drug-drug interactions studies involve the evaluation of the potential of a drug candidate to inhibit this superfamily of enzymes and these studies can be conducted using in vitro models, such as human liver microsomes (HLM). Therefore, in this project, the inhibitory effect of GRA on the activity of some CYP450 isoforms was evaluated and the isoforms that catalyze the formation of GRA\'s metabolites were also determined. Results showed that multiple CYP450 isoforms participate in the GRA\'s metabolites formation, highlighting CYP2C9, which catalyzes the formation of all metabolites. The inhibition studies showed that GRA is a weak inhibitor of CYP1A2 and CYP2D6, with IC50 values greater than 200 µM and 100 µM, respectively, and a moderate and competitive inhibitor of CYP2C9, with IC50 value equal to 40.85 µM and Ki value equal to 50.60 µM. The capability of GRA to inhibit CYP3A4 was evaluated using two different substrates. GRA showed to be a moderate and competitive dose- dependent inhibitor of this isoform and also a mechanism-based time-dependent inhibitor with potential of inactivation comparable to irinotecan, a clinically significant mechanism-based inhibitor. IC50 and Ki values obtained using nifedipine as substrate were 78.09 µM and 48.71 µM, respectively, and inactivation kinetics parameters were KI= 6.40 µM, kinact= 0,037 min-1 e Clinact= 5.78 mL min-1 µmol-1. On the other hand, IC50 and Ki values using midazolam as substrate were 48.87 µM and 31.25 µM, respectively, and the values of inactivation kinetics parameters were KI= 31.53 µM, kinact= 0,049 min-1 and Clinact= 1.55 mL min-1 µmol-1. With respect to CYP2E1, it was observed that GRA increases its activity significantly from a concentration of 4 µM. Therefore, it is possible to conclude that there is no risk of DDI between GRA and drugs metabolized by CYP1A2 and CYP2D6, while for CYP2C9, although GRA is a moderate inhibitor of this isoform, the risk is low. Finally, for drugs metabolized by CYP3A4 and CYP2E1 there is risk of DDI and this should be carefully monitored in humans, mainly because CYP3A4 is an isoform responsible for catalyzing the metabolism of most drugs in use.
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Ameline, Alice. "Aspects analytiques, cliniques et médico-judiciaires des nouvelles substances psychoactives." Thesis, Strasbourg, 2019. http://www.theses.fr/2019STRAJ018/document.
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En raison de la diffusion incontrôlée sur le e-commerce, la sécurité et l’alternative légale aux stupéfiants habituels, les nouvelles substances psychoactives (NPS), d’apparition récente (2008), sont au cœur des phénomènes récents d’addiction et de décès mal expliqués. Au-delà des différents défis dans nos sociétés (prévention, législation), la capacité d’identifier les NPS dans des échantillons biologiques pour caractériser leur utilisation, présente de nombreux challenges analytiques. L’objectif principal de cette thèse a été de collecter des échantillons biologiques (sang, urine, cheveux) provenant de cas d’exposition à des NPS et d’y caractériser les substances présentes à l’aide de méthodes analytiques originales, dans le but d’enrichir les librairies de spectres de masse et d’améliorer, en conséquence, la détection de la consommation de NPS. En particulier, il s’agissait d’augmenter la fenêtre de détection de la prise de NPS en se focalisant sur les métabolites qui sont, le plus souvent, les produits majeurs d’élimination. Le développement analytique, par chromatographie liquide ultra haute performance couplée à la spectrométrie de masse en tandem (UHPLC-MS/MS), a demandé plusieurs mois d’optimisation afin d’obtenir une méthode robuste, exhaustive et sensible. Actuellement, la librairie de spectres MS comporte 114 NPS et est mise à jour régulièrement. A la suite de ce développement, ma thèse a porté sur l’étude de cas d’intoxication vus au service des urgences du CHU de Strasbourg, mais aussi en médecine légale, avec des situations de décès et d’identification de produits inconnus provenant de saisies (poudres et cristaux). Il a également été nécessaire de développer des outils analytiques complémentaires, tels que la caractérisation de métabolite(s) par étude sur microsomes hépatiques humains (HLMs), et l’utilisation de la spectroscopie par résonance magnétique nucléaire (RMN) afin d’identifier avec certitude certains composés et de déterminer leur degré de pureté. Les outils analytiques développés et la stratégie mise en place ont permis la rédaction de 18 publications, ainsi que l’agencement de nombreuses collaborationsDue to the uncontrolled spread on the Internet and their legal alternative to usual drugs, the new psychoactive substances (NPS), recently appeared (2008), are at the center of recent phenomena of addiction and badly explained deaths. Beyond different challenges in our societies (prevention, legislation), the ability to identify NPS in biological samples, in order to characterize their use, presents many analytical challenges. The main objective of this thesis was to collect biological samples (blood, urine, hair) from cases of exposure to NPS and to characterize the substances present using original analytical methods, in order to enlarge the libraries of mass spectra and improve, as a result, the detection of NPS consumption. In particular, it was intended to increase the detection sensitivity of NPS intake by focusing on the metabolites that are often the major products of elimination. This analytical development, by ultra-high liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS), required several months of optimization in order to obtain a robust, exhaustive and sensitive method. At present, the mass spectra database has 114 NPS and is regularly updated. Thereafter, ma thesis focused on the study of cases of intoxication observed in the emergency department of Strasbourg, but also in legal medicine with situations of deaths and identification of unknown products collected from seizures (powders and crystals). It has also been necessary to implement complementary analytical tools, such as the characterization of metabolites by human liver microsomes (HLMs), and the use of nuclear magnetic resonance (NMR) spectroscopy to accurately identify the compounds and establish their purity degrees. The analytical tools developed, and the strategy adopted, allowed the writing of 18 publications, as well as the setting up of numerous collaborations
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Al, Ali Ahmad. "Le dosage des cytochromes P450 (CYPs) humains par spectrométrie de masse : applications en toxicologie." Thesis, Paris 5, 2014. http://www.theses.fr/2014PA05P603/document.
Full textAbstract:
Les cytochromes P450 (CYPs) jouent un rôle essentiel dans le métabolisme oxydatif de nombreux composés endogènes et exogènes. L’expression de CYPs est extrêmement variable en fonction de facteurs physiopathologiques, génétiques et environnementaux. Le métabolisme des xénobiotiques par les CYPs dépend en partie de la nature, de la quantité et de l’activité d’isoformes des CYPs impliqués. L'analyse quantitative de l'expression de CYP dans les organes du métabolisme, tels que le foie, sont d'une importance particulière étant donné que la biotransformation réalisée par les CYPs est souvent un facteur critique qui affecte l'efficacité, la disponibilité et la toxicité des médicaments chez l'homme. La technique actuelle de dosage la plus courante est l’immunoquantification par Western Blot. Cette technique est limitée par la disponibilité et la spécificité de l'anticorps. Les techniques de protéomique par spectrométrie de masse, permettant d’analyser de très faibles quantités de protéines en mélange, sont les méthodes de choix pour l’identification et la quantification des CYPs dans différents organes. Nous avons développé et validé une méthode pour doser 6 CYPs (1A2, 2C9, 2D6, 2J2, 3A4 et 3A5) par spectrométrie de masse en couplage chromatographique. Cette méthode, simple, rapide de sensibilité satisfaisante et peu coûteuse, a été validée dans différents types de matrices biologiques (lignées cellulaires hépatiques et neuronales, baculosomes). Ensuite, elle a été appliquée à grande échelle pour l’analyse de 50 foies humains (microsomes et mitochondries) afin d’étudier la relation phénotype/génotype pour les CYPs. Cette méthode pourra être appliquée à d’autres CYPS, est un outil utile qui permettra d’améliorer la compréhension et la prédiction pharmacocinétique et toxique de médicaments et d’autres produits chimiquesCytochromes P450 (CYPs) play a key role in the oxidative metabolism of many endogenous and exogenous compounds. The expression of CYPs is extremely variable depending on patho-physiological, genetic and environmental factors. The metabolism of xenobiotics by CYPs depends on the nature the quantity and the activity of CYP isoforms involved. Quantitative analysis of CYP expression in organs such as liver, are of particular importance since the biotransformation performed by CYPs is often a critical factor that affects the efficiency, availability and drug toxicity in humans. The most common technique is the immune-quantitation (Western Blot). This technique is limited by the availability and specificity of the antibody. Mass spectrometry-based proteomics, able to analyze very small amounts of protein in a mixture, are the methods of choice for identification and quantification of CYPs in different organs. We developed and validated a method for dosing 6 CYPs (1A2, 2C9, 2D6, 2J2, 3A4 and 3A5) by liquid chromatography coupled with mass spectrometry. This simple, rapid, low-cost method has an adequate sensitivity, and has been validated in different types of biological matrices (liver and neuronal cell lines, baculosomes). It has been applied at large-scale to analyze these 6 CYPs in 50 human livers samples (microsomes and mitochondria) to study the phenotype/genotype relationship. This method, which could easily be applied to other CYPs, provides an important tool to improve the understanding and prediction of pharmacokinetics and toxicity profile of drugs and other chemicals
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Tsai, I.-Lin, and 蔡易霖. "Identify the metabolites of methylone and ethylone using human liver microsomes and human urine." Thesis, 2019. http://ndltd.ncl.edu.tw/handle/8b6k2p.
Full textAbstract:
碩士國立臺灣大學
法醫學研究所
107
Synthetic cathinone is the most frequently abused new psychoactive substances (NPS) in Taiwan. Methylone and ethylone, which were categorized as scheduled III drug in 2012 and 2016 in Taiwan, are two derivatives of synthetic cathinone. It is important to investigate the metabolism of methylone and ethylone in order to find specific markers in human specimen. However, the metabolic profile of these two compounds are still limited especially for phase II metabolites. In previous study, methylone and ethylone each formed 7 metabolites through 5 metabolic pathways. In this study, we in vitro synthesized phase I and phase II metabolites by human liver microsomes and cytosols, and observed metabolites by fragment pattern and accurate mass using liquid chromatography-mass spectrometry (LC-MS) and liquid chromatography-high resolution mass spectrometry (LC-HRMS). Total eight phase I metabolites and three phase II metabolites were detected. Three in vitro phase II metabolites, sulfate conjugate of dihydroxymethcathinone (DHMC-S) from methylone, sulfate conjugate of dihydroxyethcathinone (DHEC-S) from ethylone and glucuronide conjugate of dihydroxymethcathinone (DHEC-G) from ethylone have never reported before. The method was established to identify the metabolites in human specimens. Urine samples from methylone and ethylone abusers were analyzed to evaluate the composition of metabolites. Dihydromethylone and glucuronide conjugate of 3-hydroxy-4-methoxymeth-cathinone (3-OH-4-MeO-MC-G) or its isomer 4-OH-3-MeO-MC-G were major metabolites of methylone, and Dihydroethylone and DHEC-S were major metabolites of ethylone in abusers urine samples. But more urine sample should be analyzed to make the metabolic trend more obvious.
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McLure, James Alexander. "Physicochemical determinants of the non-specific binding of drugs to human liver microsomes." 2008. http://catalogue.flinders.edu.au/local/adt/public/adt-SFU20081102.165952/index.html.
Full text
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Madeira, Maria. "The effect of cimetidine on dextromethorphan O-demethylase activity of human liver microsomes and recombinant CYP2D6." Thesis, 2003. http://hdl.handle.net/2429/14177.
Full textAbstract:
Cimetidine is a liistamine H₂-receptor antagonist used in the treatment of peptic ulcer disorders. There have been numerous clinical observations of cimetidine drug-drug interactions, in some cases involving known substrates of cytochrome P450 (P450) 2D6. One explanation for these observations is impaired hepatic metabolism via inhibition o f P450s, the primary mediators of hepatic phase I metabolism of drugs. Cimetidine has been demonstrated to inhibit various P450 enzymes in vitro, including CYP2D6, but the inhibition constants are generally higher than the plasma concentrations measured following in vivo administration. In rats, the in vitro selectivity and potency of cimetidine can be increased by preincubating the inhibitor and NAPDH with hepatic microsomes, and the formation of a metabolite-intermediate complex has been implicated. The effect of preincubation on inhibition of CYP2D6 by cimetidine has not been investigated. The objective of this project was to further characterize the effect of cimetidine on CYP2D6 in vitro using human liver microsomes and recombinant CYP2D6. Dextromethorphan O-demethylase activity was used as a probe of CYP2D6 activity. Metabolite formation was quantified using a high performance liquid chromatography assay with fluorescence detection. The V[sub max] and K[sub m] of this reaction were 0.82+0.06 nmol/min/nmol P450 and 4.1 ±0.1 μM, respectively, in microsomes; and 15.9±0.8 nmol/min/nmol P450 and 1.4±0.6 μM, respectively, with recombinant CYP2D6. With human liver microsomes, cimetidine was a competitive inhibitor of CYP2D6 with a K, of 38.0±5.3 μM; the observed inhibition was not effected by preincubation. With the recombinant enzyme, cimetidine acted as a mixed inhibitor with a K[sub i] of 102.5±16.8 μM. Preincubation with the inhibitor and NADPH led to a further decrease in activity of 11% to 18% in a manner that was consistent with mechanism-based inactivation. The effect of preincubation was attenuated by including quinidine in the preincubation reaction. The k[sub inact] and K[sub I] were estimated to be 0.03 min⁻¹ and 19.2 μM, respectively, while the t [sub ½] of inactivation was 25 minutes. In the present study, cimetidine acted as an inhibitor of dextromethorphan O-demethylation in both in vitro systems. With recombinant CYP2D6, loss of activity was enhanced with preincubation. In both cases, the effect of cimetidine as an inhibitor of CYP2D6 was modest and does not fully explain the clinical findings.
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Riley, Anna Ruth. "Propoxyphene, Norpropoxyphene, and Proadifen (SKF-525A) Are Mechanism Based Inhibitors of CYP3A4, CYP3A5, and CYP3A in Human Liver Microsomes." Thesis, 2008. http://hdl.handle.net/1805/1855.
Full textAbstract:
Indiana University-Purdue University Indianapolis (IUPUI)The purpose of this study is to determine if propoxyphene and norpropoxyphene are mechanism-based (irreversible) inhibitors of CYP3A, and to determine if propoxyphene and norpropoxyphene are reversible inhibitors of CYP3A. Mechanismbased inhibition is a type of irreversible inhibition that results from an inhibitor or its metabolite binding to an enzyme during drug metabolism, which renders the enzyme nonfunctional. Propoxyphene is an analgesic that is frequently prescribed in the United States and Europe. It is metabolized by CYP3A enzymes, and is an irreversible inhibitor of CYP3A4. The major metabolite of propoxyphene is norpropoxyphene, which has not been extensively studied for enzyme inhibition. Proadifen (SKF-525a) is not a marketed drug, but it is a known CYP inhibitor that is structurally similar to propoxyphene and norpropoxyphene. Propoxyphene, norpropoxyphene, and proadifen were characterized in these studies with CYP3A4(+b5), CYP3A5(+b5) and pooled human liver microsomes. Time-dependent and concentration-dependent loss of activity of CYP3A was measured by formation of testosterone product. Propoxyphene and norpropoxyphene exhibited the greatest inhibition with CYP3A in human liver microsomes, followed by CYP3A4(+b5), and CYP3A5(+b5). Both compounds formed metabolic-inhibitor complexes with vi CYP3A4(+b5) and CYP3A5(+b5), but not with human liver microsomes. Proadifen was a more potent inhibitor of CYP3A4(+b5) than of human liver microsomes and CYP3A5(+b5). The KI values of propoxyphene and CYP3A4(+b5) and human liver microsomes fall within the range of reported therapeutic blood levels of propoxyphene, with reversible inhibition constants (Ki values) above therapeutic blood concentrations for propoxyphene and norpropoxyphene. The KI values of norpropoxyphene and CYP3A4(+b5) and human liver microsomes are higher than most reported blood levels, except for blood levels after repeated dosing of propoxyphene at high concentrations. The predicted change in the area under the plasma concentration versus time curve of an orally administered CYP3A substrate with propoxyphene (AUC'po/AUCpo) was calculated for common CYP3A substrates. The AUC'po/AUCpo ratios are four to twenty-five times higher with co-administration of propoxyphene based on in vitro kinetic parameters. Propoxyphene and norpropoxyphene may cause adverse events when chronically administered at high doses and/or when co-administered with other CYP3A substrates.
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Hsun-Yi, Tseng, and 曾薰儀. "Study on Metabolism of Territrem A by Human Liver Microsomes and Human Cytochrome P450 3A4 expressed in V79 Chinese Hamster cells." Thesis, 2000. http://ndltd.ncl.edu.tw/handle/81149866620663181463.
Full textAbstract:
碩士國立臺灣大學
毒理學研究所
88
Abstract Territrem A (TRA) is a tremogenic mycotoxin isolated from the chloroform extracts of the sub-merged rice culture of Aspergillus terreus 23-1. The previous studies indicated that three metabolites, designated as MA1, MAX and MA2, were obtained when TRA was the substrate in liver microsomes of adult male Wistar rats which was pretreated with phenobarbital or dexamethasone. However, only MA1 was formed from TRA in liver microsomes of female rats. From chemical and immuno inhibition studies, we had suggested that CYP3A1/2 mainly involved in TRA metabolism. In the present study, I investigate the territrem A metabolism in human liver microsomes and in V79 Chinese Hamster cells, designed as V79MZh3A4, in which human cytochrome P450 3A4 were expressed. The aims of this study were to elucidate the questions as following: (1) TRA metabolism in different species. (2) TRA metabolism in sex difference of human. (3) Role of CYP3A4 in TRA metabolism. From the studies, I have obtained the following results. (1) Both human livers and V79MZh3A4 cells could express the activities of testosterone 6hydroxylase. Otherwise, the testosterone 6hydroxylase was not being determined in V79MZ cells. (2) Inmmunobloting assay showed that CYP3A4 protein was detected in human liver microsomes and V79MZh3A4 cells, but not in V79MZ cells. (3) When TRA was used as the substrate, MA1 and trace amount MAX were formed in male and female human liver microsomes. (4) Furthermore the results obtained was using live V79MZh3A4 cells showed only MA1 was found. When MA1 was used as the substrate in the same experimental condition, no metabolites were found. (5) In enzyme kinetics study, I found that V79MZh3A4 cells could metabolize TRA judged from the value of Vmax / Km of MA1 calculatedly activity. (6) The specific cytochrome P450 inhibitor of CYP3A4, such as ketoconazole, inhibited the activities of testosterone 6 - hydroxylase as well as MA1 in human liver microsomes was increased of dose of inhibition. Therefore, it is suggested that CYP3A4 has a major role in metabolic pathway from TRA to MA 1.
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Liu, X., L. Hu, G. Ge, B. Yang, J. Ning, S. Sun, L. Yang, Klaus Pors, and J. Gu. "Quantitative analysis of cytochrome P450 isoforms in human liver microsomes by the combination of proteomics and chemical probe-based assay." 2014. http://hdl.handle.net/10454/10502.
Full textAbstract:
NoCytochrome P450 (CYP) is one of the most important drug-metabolizing enzyme families, which participates in the biotransformation of many endogenous and exogenous compounds. Quantitative analysis of CYP expression levels is important when studying the efficacy of new drug molecules and assessing drug-drug interactions in drug development. At present, chemical probe-based assay is the most widely used approach for the evaluation of CYP activity although there are cross-reactions between the isoforms with high sequence homologies. Therefore, quantification of each isozyme is highly desired in regard to meeting the ever-increasing requirements for carrying out pharmacokinetics and personalized medicine in the academic, pharmaceutical, and clinical setting. Herein, an absolute quantification method was employed for the analysis of the seven isoforms CYP1A2, 2B6, 3A4, 3A5, 2C9, 2C19, and 2E1 using a proteome-derived approach in combination with stable isotope dilution assay. The average absolute amount measured from twelve human liver microsomes samples were 39.3, 4.3, 54.0, 4.6, 10.3, 3.0, and 9.3 (pmol/mg protein) for 1A2, 2B6, 3A4, 3A5, 2C9, 2C19, and 2E1, respectively. Importantly, the expression level of CYP3A4 showed high correlation (r = 0.943, p < 0.0001) with the functional activity, which was measured using bufalin-a highly selective chemical probe we have developed. The combination of MRM identification and analysis of the functional activity, as in the case of CYP3A4, provides a protocol which can be extended to other functional enzyme studies with wide application in pharmaceutical research.
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Numa, Andres Masato. "The effect of Ginkgo Biloba extract on valproic acid metabolism by human liver microsomes from donors with the CYP2C9*1/*1 genotype." Thesis, 2005. http://hdl.handle.net/2429/17242.
Full textAbstract:
Ginkgo biloba extract (GBE) is a popular herbal preparation used primarily in the
treatment of dementia, peripheral vascular diseases, and neurosensory problems. In this study,
the effect of GBE on the oxidative metabolism of the anti-epileptic valproic acid (VPA) was
investigated. Human liver microsomes (HLM) from donors with the CYP2C9*1/*1 genotype
were incubated with VPA and GBE, and the formation of 4-ene-VPA, 4-OH-VPA, 5-OH-VPA,
and 3-OH-VPA were monitored by GC/MS in NICI mode. GBE inhibited the formation of all
four metabolites in a dose-dependent manner. GBE from three different sources showed similar
inhibition of metabolite formation.
Pre-incubation of H LM with a monoclonal antibody against CYP2C9 significantly
reduced the formation of 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA, demonstrating that CYP2C9
is the major isoform responsible for their formation. Pre-incubation of H L M with monoclonal
antibodies against CYP2B6 and CYP2A6 reduced their formation by a smaller amount,
suggesting that they are minor isoforms involved in their formation. Pre-incubation of H LM with
monoclonal antibodies against CYP2B6 and CYP2A6 followed by incubation with VPA and
GBE dramatically reduced their formation. These results show that the inhibition of 4-ene-VPA,
4-OH-VPA, and 5-OH-VPA formation in H LM by GBE is due mostly to inhibition of CYP2C9.
These results were confirmed by incubations with recombinant enzymes. GBE inhibited
4-ene-VPA, 4-OH-VPA, and 5-OH-VPA formation by recombinant CYP2C9; 4-ene-VPA, 4-
OH-VPA, and 5-OH-VPA formation by recombinant CYP2B6; and 3-OH-VPA formation by
recombinant CYP2A6.
To elucidate which constituents of GBE are responsible for the inhibition seen with the
whole extract, individual terpene trilactones (bilobalide and ginkgolides A, B, C, and J) and flavonol glycosides (isorhamnetin-3-O-nitinoside, kaempferol-3-O-rutinoside, and quercetin-3-
O-rutinoside) were incubated with VP A. However, at the concentrations present in GBE, these
constituents failed to inhibit VPA metabolism. The aglycones of isorhamnetin, kaempferol, and
quercetin inhibited VPA metabolism. Although the aglycones were not detected in GBE, they
may be of importance in vivo, as flavonol glycosides are hydrolyzed to their respective aglycones
in the small intestine.
In conclusion, GBE inhibited the CYP2C9-, CYP2B6-, and CYP2A6-mediated
metabolism of VPA. However, the effect could not be explained by the individual terpene
trilactones or flavonol glycosides tested.Pharmaceutical Sciences, Faculty of
Graduate
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Li, Yibai. "Pharmacogenetics of ketamine metabolism and immunopharmacology of ketamine." Thesis, 2014. http://hdl.handle.net/2440/106345.
Full textAbstract:
Ketamine is an anaesthetic agent that is being increasingly used at sub-anaesthetic doses as an analgesic or co-analgesic in the management of postoperative pain and chronic pain. In most countries, ketamine is administered as a racemic compound consisting of two enantiomers: (S)- and (R)-ketamine at a ratio of 1:1. Ketamine analgesia is frequently restricted by the low efficacy and large interindividual variability in drug response, which may be associated with the differences in the plasma pharmacokinetics. Previous in vitro studies suggested that ketamine is primarily cleared to its active metabolite, norketamine, by two hepatic CYP enzymes: CYP2B6 and CYP3A4, whose expression and catalytic activities show vary large variability in humans due to genetic and environmental influences. Therefore, it is logical that the variability in these enzymes contributes to the variability in ketamine pharmacokinetics. Additional variability in analgesic response may arise from the heterogeneous nature of pain, as ketamine is expected to be more effective against hyperalgesia and allodynia (neuropathic pain). Although these anti-hyperalgesic and anti-allodynic mechanisms have been primarily associated with the non-competitive antagonism of neuronal NMDA receptors, it has been speculated that the attenuation of proinflammatory response may also contribute to ketamine analgesia, since there is evidence to suggest important roles of proinflammatory cytokines in the pathogenesis of neuropathic pain. Thus, the major aims of this thesis were to examine the influence of variability in enzyme activity, especially that due to CYP2B6 genetic polymorphisms, on ketamine pharmacokinetics in vitro and ex vivo in chronic pain patients. The secondary aims of this thesis were to examine the effects of ketamine and norketamine enantiomers on proinflammatory cytokine production in vitro, using interleukin-6 (IL-6) as a marker of cytokine production; and to explore the mechanistic characterisation of the drug actions using both in silico docking simulations and in vitro experiments. The in vitro experiments showed that, at clinically relevant concentrations, CYP2B6 but not CYP3A4 is the major isoform responsible for ketamine metabolism to norketamine in human liver microsomes (HLM). Moreover, the presence of the CYP2B6*6 allele, the most common allelic variant of the CYP2B6 gene, reduced the intrinsic clearance of both ketamine enantiomers in HLMs and cDNA-expressed proteins by at least 62%. This substantial CYP2B6*6 allele-induced decrease in ketamine intrinsic clearance in vitro was also observed ex vivo in chronic pain patients who received 24 h continuous subcutaneous infusions of 100 mg to 500 mg ketamine. The impact of the CYP2B6*6 allele, by itself and in combined with the age of the patient, explained approximately 40% and 60% of interindividual variation in plasma ketamine concentrations at steady-state, respectively, whereas sex, disease and other medications had no significant influences. The decrease of ketamine clearance may be associated with the adverse effects of ketamine, as patients who experienced adverse effects showed approximately 15% lower steady-state plasma clearance of ketamine than those who did not. However, no evidence linking the plasma pharmacokinetics of ketamine and norketamine and the analgesic efficacy was found. One possible explanation for this lack of concentration-response relationship is the overwhelming effect of heterogeneous nature of pain on analgesic response, since ketamine analgesic efficacy was higher in patients suffering from neuropathic pain than other pain types. This finding may reflect the fact that the analgesic activity of ketamine is more likely due to the attenuation of pain hypersensitivity rather than the direct suppression of nociceptive transmission. The in vitro experiments on the inhibition of IL-6 by ketamine and norketamine enantiomers showed that pre-incubation with these drugs, at biologically relevant concentrations (1 to 100 μM), stereoselectively attenuated stimulated IL-6 production in recombinant cells in a concentration- and time-dependent manner. (R)-ketamine inhibited stimulated IL-6 production by approximately 60% at all exposure duration, an inhibitory effects that were up to 2-fold greater than (S)-ketamine after short term exposure (less than 2 h). However (S)-ketamine was as potent as (R)-ketamine after long-term exposure (4 to 8 h), as its inhibitory effects were significantly enhanced with exposure duration. In addition, (S)-norketamine also attenuated IL-6 response in a time-dependent manner with approximately half the potency of (S)- ketamine. Further in vitro experiments and in silico docking simulation suggested that this time-dependent effects of (S)-ketamine and (S)-norketamine may indicates a mechanisticallybased difference between acute and chronic effects of (S)-enantiomers on IL-6 production. This findings extend the current knowledge of the innate immune pharmacology of ketamine that may lead to a new direction for future research into ketamine analgesia. In summary, this thesis demonstrates a substantial impact of the CYP2B6*6 allelic variant on the clearance of ketamine, which may contribute to the interindividual variability in drug concentration. However, other factors such as the heterogeneity in the nature of pain and the inflammatory state should be taken into consideration to provide a more accurate prediction on ketamine analgesic response.Thesis (Ph.D.) (Research by Publication) -- University of Adelaide, School of Medical Sciences, 2014.
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Seibert, C., B. R. Davidson, B. J. Fuller, Laurence H. Patterson, W. J. Griffiths, and Y. Wang. "Multiple-approaches to the identification and quantification of cytochromes P450 in human liver tissue by mass spectrometry." 2009. http://hdl.handle.net/10454/6179.
Full textAbstract:
Here we report the identification and approximate quantification of cytochrome P450 (CYP) proteins in human liver microsomes as determined by nano-LC-MS/MS with application of the exponentially modified protein abundance index (emPAI) algorithm during database searching. Protocols based on 1D-gel protein separation and 2D-LC peptide separation gave comparable results. In total, 18 CYP isoforms were unambiguously identified based on unique peptide matches. Further, we have determined the absolute quantity of two CYP enzymes (2E1 and 1A2) in human liver microsomes using stable-isotope dilution mass spectrometry, where microsomal proteins were separated by 1D-gel electrophoresis, digested with trypsin in the presence of either a CYP2E1- or 1A2-specific stable-isotope labeled tryptic peptide and analyzed by LC-MS/MS. Using multiple reaction monitoring (MRM) for the isotope-labeled tryptic peptides and their natural unlabeled analogues quantification could be performed over the range of 0.1-1.5 pmol on column. Liver microsomes from four individuals were analyzed for CYP2E1 giving values of 88-200 pmol/mg microsomal protein. The CYP1A2 content of microsomes from a further three individuals ranged from 165 to 263 pmol/mg microsomal protein. Although, in this proof-of-concept study for CYP quantification, the two CYP isoforms were quantified from different samples, there are no practical reasons to prevent multiplexing the method to allow the quantification of multiple CYP isoforms in a single sample.
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Sutherland, Mark H., Jason H. Gill, Paul M. Loadman, Jonathan P. Laye, Helen M. Sheldrake, Nicola A. Illingworth, Mohammed N. Alandas, et al. "Antitumor activity of a duocarmycin analogue rationalized to be metabolically activated by cytochrome P450 1A1 in human transitional cell carcinoma of the bladder." 2012. http://hdl.handle.net/10454/6210.
Full textAbstract:
NoWe identify cytochrome P450 1A1 (CYP1A1) as a target for tumor-selective drug development in bladder cancer and describe the characterization of ICT2700, designed to be metabolized from a prodrug to a potent cytotoxin selectively by CYP1A1. Elevated CYP1A1 expression was shown in human bladder cancer relative to normal human tissues. RT112 bladder cancer cells, endogenously expressing CYP1A1, were selectively chemosensitive to ICT2700, whereas EJ138 bladder cells that do not express CYP1A1 were significantly less responsive. Introduction of CYP1A1 into EJ138 cells resulted in 75-fold increased chemosensitivity to ICT2700 relative to wild-type EJ138. Negligible chemosensitivity was observed in ICT2700 in EJ138 cells expressing CYP1A2 or with exposure of EJ138 cells to CYP1B1- or CYP3A4-generated metabolites of ICT2700. Chemosensitivity to ICT2700 was also negated in EJ138-CYP1A1 cells by the CYP1 inhibitor alpha-naphthoflavone. Furthermore, ICT2700 did not induce expression of the AhR-regulated CYP1 family, indicating that constitutive CYP1A1 expression is sufficient for activation of ICT2700. Consistent with the selective activity by CYP1A1 was a time and concentration-dependent increase in gamma-H2AX protein expression, indicative of DNA damage, associated with the activation of ICT2700 in RT112 but not EJ138 cells. In mice-bearing CYP1A1-positive and negative isogenic tumors, ICT2700 administration resulted in an antitumor response only in the CYP1A1-expressing tumor model. This antitumor response was associated with detection of the CYP1A1-activated metabolite in tumors but not in the liver. Our findings support the further development of ICT2700 as a tumor-selective treatment for human bladder cancers.