lsoflurane interacts with cytochrome P-450 in rat and human hepatic microsomes and the Δ6- and Δ5-desaturases in rat hepatic microsomes. The interaction of isoflurane with cytochrome P-450 results in its metabolism to fluoride ion and organofluorine metabolites. The cytochrome P-450 isozymes catalysing the defluorination of isoflurane were assessed in hepatic microsomes from phenobarbital-, β-naphthoflavone- and pregnenolone-16α-carbonitrilepretreated and untreated rats. One or more of the cytochrome P-450 isozymes induced by phenobarbital and pregnenolone-16α-carbonitrile appear to defluorinate isoflurane, but those induced by β-naphthoflavone do not. From a comparison of the extent of defluorination of isoflurane in hepatic microsomes from phenobarbital- and pregnenolone-16α-carbonitrile-pretreated rats, and their Kₘ and Vₘₐₓ values, it appears that isoflurane is defluorinated by one or more isozymes induced by both phenobarbital and pregnenolone-16α-carbonitrile. The major isozyme is probably cytochrome P-450PCN1. The metabolites of isoflurane were identified in human and phenobarbital-induced rat hepatic microsomes. In microsomes from phenobarbital-pretreated rats, isoflurane is metabolised to fluoride ion and trifluoroacetaldehyde; trifluoroacetic acid is not produced in measureable amounts. The trifluoroacetaldehyde produced binds to microsomal constituents. In human hepatic microsomes, the organofluorine metabolite is identified as trifluoroacetic acid. It is proposed that isoflurane is metabolised by different pathways in human and phenobarbital-induced rat hepatic microsomes. The interaction of isoflurane with the cyanide-sensitive factors was assessed by several criteria. Firstly, using the reoxidation of cytochrome b₅ as an index of fatty acid desaturase activity, isoflurane appears to interact with the Δ6- and/or Δ5-desaturases, but not the Δ9-desaturase. Secondly, these results were confirmed and clarified by the use of direct assays to measure the fatty acid desaturase activity. Using the direct assay, we confirmed that isoflurane did not inhibit the Δ9-desaturase and inhibited Δ6-desaturation of linoleic acid, but not the Δ6-desaturation of α-linolenic acid. The inhibition of the Δ6-desaturation of linoleic acid occurred at low millimolar concentrations of isoflurane. lsoflurane inhibits the Δ5-desaturation of eicosa-8, 11, 14-trienoic acid to a small extent which is only apparent at much higher concentrations of isoflurane than that which inhibits the Δ6-desaturase. Further studies focussed on measurement of the activity of Δ6-desaturase in order to attempt to study the kinetics of the inhibition of the Δ6-desaturase by isoflurane: Δ6-desaturase activity was assessed using hepatic microsomes as the source of the enzyme and linoleic acid as substrate precursor. In the course of these studies, we identified a number of factors that affected the apparent activity of the Δ6-desaturase in hepatic microsomes. These included significant levels of endogenous substrate and competing reactions in the hepatic microsomes. Endogenous substrate levels were quantified and corrected for. We then resorted to computer modelling to extract the kinetics of the Δ6-desaturase free of contributions from acyl-CoA synthetase and lysophospholipid acyltransferase, as well as enzyme decay. The kinetics of isoflurane inhibition of the Δ6-desaturase were then superimposed and studied by computer modelling.

Introduction Cytochromes P450 (CYPs) are the major family of enzymes responsible for detoxification and metabolism of a wide range of both endogenous and xenobiotics chemicals in living organisms. The use of CYPs to activate prodrugs to cytotoxins selectively in tumours has been explored including AQ4N, Phortress and Aminoflavone. CYP1A1, CYP1B1, CYP2W1, and CYP4F11 have been identified as expressed in tumour tissue and surrounding stroma at high frequency compared to most normal tissues. Aim is to investigate the differential metabolism of novel chloromethylindoline by high frequency expressed CYPs in tumours. This differential may be exploited to elicit a selective chemotherapeutic effect by metabolising inert small molecules to potent cytotoxins within the tumour environment. Materials and Methods Sensitive and specific LC/MS/MS techniques have been developed to investigate the metabolism of chloromethylindolines. Recombinant enzymes and transfected cell lines were used to investigate the metabolic profiles with a focus on production of the cytotoxic derivatives of chloromethylindolines. Results Detailed metabolic studies show that (1-(Chloromethyl)-1,2-dihydropyrrolo [3,2-e]indol-3(6H)-yl)(5-methoxy-1H-indol-2-yl) methanone (ICT2700) and other chloromethylindolines are converted by CYP1A1 mediated hydroxylation at the C-5 position leading to highly potent metabolites. In vitro cytotoxicity studies showed differentials of up to 1000-fold was achieved between CYP1A1 activated compared to the non-metabolised parent molecules. The reactivity of metabolites of ICT2700 was also explored using glutathione as a nucleophile. The metabolites were identified by a combination of LC/MS and LC MS/MS techniques. Investigations using mouse and human liver microsomes show that a large number of metabolites are created though none were shown to be associated with a potential anticancer effect. Studies focused on CYP2W1 show that this isoform metabolised ICT2706 to a cytotoxic species and a pharmacokinetic study showed a good distribution of ICT2706 into mouse tissues including tumour. However metabolism of ICT2726 by CYP2W1 resulted only in a non-toxic metabolite profile and may have potential as a biomarker for functional CYP2W1 in tissues. Preliminary studies show that palmitic acid hydroxylation is a useful marker of functional CYP4F11. Summary and conclusion The in vitro results show that the chloromethylindolines are a novel class of agent with potential as prodrugs that following specific hydroxylation by CYP1A1 and CYP2W1 are converted to ultra-potent cytotoxins. Other metabolites are also evident which are not cytotoxic. Studies in vivo show that selected chloromethylindolines possess a good pharmacokinetic profile and show potential as prodrug anticancer agents that require activation by CYP1A1 or CYP2W1. The methods, results, progress and suggestions for future work are presented in this thesis.

Introduction: Cytochromes P450 (CYPs) are the major family of enzymes responsible for detoxification and metabolism of a wide range of both endogenous and xenobiotics chemicals in living organisms. The use of CYPs to activate prodrugs to cytotoxins selectively in tumours has been explored including AQ4N, Phortress and Aminoflavone. CYP1A1, CYP1B1, CYP2W1, and CYP4F11 have been identified as expressed in tumour tissue and surrounding stroma at high frequency compared to most normal tissues. Aim is to investigate the differential metabolism of novel chloromethylindoline by high frequency expressed CYPs in tumours. This differential may be exploited to elicit a selective chemotherapeutic effect by metabolising inert small molecules to potent cytotoxins within the tumour environment. Materials and Methods: Sensitive and specific LC/MS/MS techniques have been developed to investigate the metabolism of chloromethylindolines. Recombinant enzymes and transfected cell lines were used to investigate the metabolic profiles with a focus on production of the cytotoxic derivatives of chloromethylindolines. Results: Detailed metabolic studies show that (1-(Chloromethyl)-1,2-dihydropyrrolo [3,2-e]indol-3(6H)-yl)(5-methoxy-1H-indol-2-yl) methanone (ICT2700) and other chloromethylindolines are converted by CYP1A1 mediated hydroxylation at the C-5 position leading to highly potent metabolites. In vitro cytotoxicity studies showed differentials of up to 1000-fold was achieved between CYP1A1 activated compared to the non-metabolised parent molecules. The reactivity of metabolites of ICT2700 was also explored using glutathione as a nucleophile. The metabolites were identified by a combination of LC/MS and LC MS/MS techniques. Investigations using mouse and human liver microsomes show that a large number of metabolites are created though none were shown to be associated with a potential anticancer effect. Studies focused on CYP2W1 show that this isoform metabolised ICT2706 to a cytotoxic species and a pharmacokinetic study showed a good distribution of ICT2706 into mouse tissues including tumour. However metabolism of ICT2726 by CYP2W1 resulted only in a non-toxic metabolite profile and may have potential as a biomarker for functional CYP2W1 in tissues. Preliminary studies show that palmitic acid hydroxylation is a useful marker of functional CYP4F11. Summary and conclusion: The in vitro results show that the chloromethylindolines are a novel class of agent with potential as prodrugs that following specific hydroxylation by CYP1A1 and CYP2W1 are converted to ultra-potent cytotoxins. Other metabolites are also evident which are not cytotoxic. Studies in vivo show that selected chloromethylindolines possess a good pharmacokinetic profile and show potential as prodrug anticancer agents that require activation by CYP1A1 or CYP2W1. The methods, results, progress and suggestions for future work are presented in this thesis.

Os diterpenos labdanos ácidos ent-poliáltico (AP) e ent-diidroagático (ADA) são metabólitos secundários amplamente distribuídos em diversas espécies vegetais, sendo particularmente comuns nos exsudatos extraídos dos troncos das árvores do gênero Copaifera (Leguminosae- Caesalpinioideae). Estas oleorresinas apresentam diversas atividades biológicas e são utilizadas na medicina popular desde a época dos índios pré-colombianos. Apesar de sua ampla utilização, não há estudos acerca de seu metabolismo. Estudos de biotransformação com constituintes bioativos como o AP e ADA podem contribuir para o entendimento de reações metabólicas in vivo das oleorresinas. Além de indispensáveis na fase pré-clinica de desenvolvimento e otimização de fármacos, os estudos de biotransformação in vitro também podem propiciar a obtenção de novas estruturas químicas farmacologicamente ativas. Assim, os diterpenos AP e ADA foram submetidos a estudos de biotransformação com diferentes tipos de biocatalisadores: fungos filamentosos, micro-organismos do trato gastrointestinal e microssomas hepáticos humanos. Foram obtidos quinze metabólitos de biotransformação com os fungos Cunninghamella elegans, Cunninghamella echinulata e Aspergillus brasiliensis, sendo treze estruturas ainda não descritas na literatura. A reação enzimática mais comum promovida pelos fungos sobre os diterpenoides foi a hidroxilação, porém isomerização da ligação dupla e acetilação também foram identificadas. Apesar de terem sido detectadas diminuições acentuadas nas concentrações dos diterpenos nas culturas com os microorganismos do trato gastrointestinal, os rendimentos dos processos de biotransformação na escala de produção desenvolvida não propiciaram metabólitos em concentrações satisfatórias para isolamento. Apesar dos baixos rendimentos, foi possível propor através de espectrometria de massas a estrutura de quatro derivados oxidados e/ou metilados do ADA após incubação com Saccharomyces boulardii, Lactobacillus fermentum, Escherichia coli, cultura mista com probióticos do gênero Lactobacillus e cultura mista com os probióticos do gênero Bifidobacterium. O ácido ent-agático foi identificado entre os metabólitos propostos, indicando a necessidade de mais estudos acerca do metabolismo dos constituintes das oleorresinas de Copaifera, uma vez que o ácido agático já foi reportado na literatura como abortivo em mamíferos. Para os metabólitos obtidos com microssomas hepáticos humanos foram propostas quatro estruturas oxidadas no metabolismo de fase I e quatro estruturas conjugadas com ácido glucurônico no metabolismo de fase II e fase I e II combinados. Os precursores e os derivados majoritários de biotransformação foram submetidos a ensaios de citotoxidade com células tumorais das linhagens Caco-2, HeLa e MCF-7, além da linhagem normal MCF-10A. As mudanças químicas promovidas pelos fungos na estrutura do ADA não acarretaram mudanças significativas em sua atividade biológica, enquanto a maioria dos derivados do AP apresentou menor efeito citotóxico. Apenas o metabólito P06(AP) apresentou atividade cerca de quatro vezes maior (IC50 = 62,6 ?M) que o precursor, sugerindo que a migração da dupla e a introdução de grupo acetato na estrutura do AP pode ser uma estratégia para obtenção de derivados mais ativos frente a linhagem HeLa. De modo geral, os resultados obtidos neste estudo reforçam a importância da biotransformação como estratégia para obtenção de novas estruturas químicas e contribuem para o entendimento do metabolismo de constituintes bioativos das oleorresinas medicinais de Copaifera / The labdane diterpenes ent-polyalthic (AP) and ent-dihydroagathic (ADA) acids are secondary metabolites widely spread in several plants. Both diterpenes are particularly common in the oleoresins extracted from the tree trunks of Copaifera sp. (Leguminosae- Caesalpinioideae). These oleoresins have several biological activities and have been used in folk medicine since the pre-Columbian Indians times. Despite their wide use, research on the metabolism of the oleoresins is lacking. Biotransformation studies with their bioactive constituents such as AP and ADA may contribute to the understanding of in vivo metabolic reactions. In vitro biotransformation studies are mandatory in the preclinical stage of drug development and optimization and can also provide new pharmacologically active derivatives. Given that, the diterpenes AP and ADA were submitted to biotransformation studies with different types of biocatalysts: filamentous fungi, microorganisms from the gastrointestinal tract and human liver microsomes. The biotransformation with the fungi Cunninghamella elegans, Cunninghamella echinulata and Aspergillus brasiliensis afforded two known structures and thirteen new metabolites. The most common enzymatic reaction by the fungi was hydroxylation, but isomerization of the double bond and acetylation were also detected. Although the microorganisms from the gut microbiota were able to reduce the concentration of the diterpenes in the cultures, the biotransformation yields of the processes were not enough for metabolite isolation. Despite the low yields, four oxygenated and/or methylated metabolites were proposed by mass spectrometry, after incubation of ADA with Saccharomyces boulardii, Lactobacillus fermentum, Escherichia coli, mixed culture with Lactobacillus sp. probiotics and mixed culture with Bifidobacterium sp. probiotics. The diterpene ent-agathic acid was identified among the metabolites, indicating the need for further studies on the metabolism of Copaifera oleoresin constituents, since agathic acid has already been reported as abortive in mammals. Four oxygenated structures and four metabolites conjugated with glucuronic acid were proposed for the phase I and phase II metabolism with human liver microsomes, respectively. The antiproliferative effects of the diterpenes and their major biotransformation derivatives were evaluated against the cancer cell lines Caco-2, HeLa and MCF-7 and the normal cell line MCF-10A. The chemical changes promoted by the fungi in the structure of ADA resulted in no significant changes in its biological activity. Most AP\'s derivatives displayed lower cytotoxic effects, except for the metabolite P06(AP), which showed to be four times more active (IC50 = 62.6 ?M) than its precursor. The migration of the double bond and the introduction of the acetate group in AP\'s skeleton were associated with the greater biological activity. The results obtained herein reinforce the use of biotransformation as a strategy to obtain new chemical structures and contribute to the understanding of the metabolic pathways of bioactive constituents of Copaifera medicinal oleoresins.

FORTES, S.S. Estudo de metabolismo in vitro e inibição enzimática do produto natural Licarina A empregando microssomas hepático de humanos. 2017. Tese (Doutorado) - Faculdade de Filosofia Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, 2017. Muitos fármacos comercializados tiveram sua origem em produtos naturais e seus derivados. Devido ao grande potencial farmacológico destas novas moléculas pesquisadas, uma etapa importante e inicial no desenvolvimento de um novo fármaco é a avaliação do seu comportamento frente as enzimas do citocromo P450 (CYP 450), incluindo os estudos de interações medicamentosas. Neste contexto, um substrato que merece destaque é a Licarina A (Lic A). Este composto é uma neolignana encontrada em algumas espécies de plantas e vêm demonstrando várias propriedades biológicas promissoras, dentre elas destaca-se a atividade anti-leishimania. No entanto, para que esta substância com comprovada atividade se torne um fármaco é necessário realizar, na fase pré-clínica, estudos sobre seu perfil metabólico frente às enzimas do CYP450 e estudos de interação medicamentosa. Portanto, esta Tese teve como objetivo determinar os parâmetros enzimáticos utilizando microssomas hepáticos de humanos através do estudo de metabolismo in vitro com esta molécula e realizar estudos de interação medicamentosa através dos estudos de inibição enzimática e pesquisar a isoforma do CYP450 que metaboliza predominantemente este produto natural através do emprego de enzimas recombinantes de humanos. Primeiramente, foi desenvolvido um método analítico para a determinação do produto natural Licarina A em meio microssomal. As análises foram realizadas por cromatografia liquida de alta eficiência empregando a coluna Ascentis C18 e fase móvel composta por metanol: solução aquosa de ácido fórmico 0,1% (75:25, v/v); a vazão empregada foi de 1,0 mL min-1. O método foi validado na faixa de concentração de 0,383 a 76,65 ?mol L-1, com coeficiente de correlação linear de 0,99 e limite de quantificação de 0,383 ?mol L-1. A precisão e exatidão apresentaram resultados dentro do recomendável pela ANVISA. Após validação do método, estabeleceram-se as condições lineares para a depleção da Lic A no meio microssomal e posteriormente, a cinética foi determinada em condições de velocidade inicial utilizando para tanto 0,20 mg mL-1 de concentração de proteínas microssomais e 20 minutos de tempo de incubação. O comportamento observado na cinética enzimática para a depleção da Lic A foi um comportamento atípico, caracterizada pelo modelo cinético de Hill. Os valores de Vmax, S50 e coeficiente de Hill foram, 1,651 ?mol mg-1 min-1, 3,87 ?mol L-1 e 2,0 respectivamente. A partir dos parâmetros cinéticos o valor de clearance intrínseco (CLint) para a Lic A foi de 0,22 mL min-1 mg-1. Posteriormente, a correlação in vitro in vivo foi realizada e foi observado um clareance hepático (CLhep) de 20 mL min-1 kg-1 e taxa de extração hepática (E) de 1. As isoformas do CYP450 envolvidas no metabolismo da Lic A foram CYP 1A2 e 2B6. Os estudos de inibição mostraram que a Lic A é um inibidor fraco frente as isoformas do CYP450 estudadas, com valores de IC50 maiores do que 80 ?mol L-1. Embora já tenha sido estudada diferentes vias metabólicas da licarina A com vários metabólitos identificados, esta foi a primeira vez que foi observado a formação de um metabólito in vitro com o uso de microssomas hepático humano. Com o auxílio da espectrometria de massa foi possível a identificação do metabólito de m/z 343 [M+H]+, possivelmente um composto epoxidado, da licarina A. / FORTES, S.S. In vitro metabolism and enzymatic inhibition study of the natural product Licarin A employing human liver microsomes. 2017. Thesis (Doctoral) - Faculdade de Filosofia Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, 2017. Many marketed drugs had their origin in natural products and their derivatives. Due to the biological potential of these new molecules, an important initial step in the development of a new drug is the evaluation of its behavior in front of cytochrome P450 enzymes (CYP 450), including studies of drug interactions. In this context, a substrate that deserves attention is Licarin A (Lic A). This compound is a neolignan found in some species of plants and several promising biological properties have been describing for this natural product, among them anti-leishimania activity. However, for this substance to become a drug, it is necessary to perform, in the preclinical phase, studies regarding its metabolic profile and drug interactions. Therefore, this thesis aimed to determine the enzymatic parameters by using human liver microsomes through in vitro metabolism study with this molecule and to conduct drug interaction studies through the enzyme inhibition studies and finally, to investigate the CYP450 isoforms that metabolize predominantly this natural product through the use of recombinant human enzymes. Firstly, an analytical method was developed for the quantification of the natural product Licarin A in microsomal medium. The analyzes were performed by high performance liquid chromatography employing an Ascentis C18 column and mobile phase composed of methanol: 0.1% formic acid aqueous solution (75:25, v / v); the flow rate used was 1.0 mL min-1. The method was validated in the concentration range of 0.333 to 76.65 ?mol L-1, with a linear correlation coefficient of 0.99 and a quantification limit of 0.333 ?mol L-1. Accuracy and precision showed results in agreement with ANVISA guidelines. After method validation, the linear conditions for depletion of Lic A in the microsomal medium were established. Subsequently, the kinetics were determined under initial velocity conditions using 0.20 mg mL-1 of microsomal protein concentration and 20 minutes of incubation time. The behavior observed in the enzymatic kinetics for the depletion of Lic A was an atypical behavior, characterized by the Hill kinetic model. The values of Vmax, S50 and Hill coefficient were 1.651 ?mol mg-1 min-1, 3.87 ?mol L-1 and 2.0, respectively. From the kinetic parameters, the intrinsic clearance (CLint) for Lic A was 0.22 mL min-1 mg-1. Subsequently, in vitro in vivo correlation was performed and a hepatic clareance (CLhep) of 20 mL min-1 kg-1 and a hepatic extraction rate (E) of 1 was observed. The CYP450 isoforms involved in the metabolism of Lic A were CYP 1A2 and 2B6. Inhibition studies have shown that Lic A is a weak CYP450 inhibitor, with IC50 values greater than 80 ?mol L-1. Although different metabolic pathways of licanin A have been studied and several metabolites were identified, this is the first report about the formation of an in vitro metabolite after metabolism by human liver microsomes. With the aid of mass spectrometry it was possible to identify the metabolite of m/z 343 [M+H]+, possibly an epoxidized compound, of licanin A.

As principais propriedades farmacológicas da Casearia sylvestris, uma espécie de árvore cujas folhas são utilizadas na medicina popular, já foram descritas na literatura. Recentemente foi demonstrada a potente atividade citotóxica in vitro da casearina X (CAS X), o diterpeno clerodânico majoritário isolado das folhas de C. sylvestris, contra linhagens de células tumorais humanas. Apesar dos resultados promissores, sua potente atividade citotóxica in vitro não pode ser extrapolada para uma potente atividade in vivo, a menos que possua boa biodisponibilidade e duração desejável do seu efeito. Tendo em vista que o avanço nas pesquisas de produtos naturais requer a avaliação pré-clínica de propriedades farmacocinéticas, no presente trabalho foi realizada a caracterização in vitro do metabolismo e da absorção intestinal da CAS X, com o objetivo de prever sua biodisponibilidade in vivo. Para os estudos de metabolismo in vitro, foi utilizado o modelo microssomal hepático de ratos e de humanos. Foi desenvolvido um método analítico para a quantificação da CAS X em microssomas, empregando a precipitação de proteínas com acetonitrila no preparo das amostras e a cromatografia líquida de alta eficiência para as análises. O método foi validado de acordo com os guias oficiais da Agência Nacional de Vigilância Sanitária e da European Medicine Agency (EMA). A CAS X demonstrou ser substrato para as reações de hidrólise mediada pelas carboxilesterases (CES) e apresentou um perfil cinético de Michaelis-Menten. Foram estimados os parâmetros de Vmax e KM, demonstrando que o clearance intrínseco em microssomas hepático de humanos foi 1,7 vezes maior que o de ratos. O clearance hepático foi estimado por extrapolação in vitro-in vivo, resultando em mais de 90% do fluxo sanguíneo hepático em ambas as espécies. Um estudo qualitativo para a pesquisa de metabólitos foi feito utilizando espectrometria de massas, pelo qual foi possível sugerir a formação da casearina X dialdeído como produto de metabolismo. Nos estudos de absorção intestinal in vitro foi utilizado o modelo de monocamadas de células Caco-2. Um método analítico por cromatografia líquida acoplada a espectrometria de massas foi desenvolvido e validado de acordo com o EMA, para as etapas de quantificação da CAS X no sistema de células. Os parâmetros cinéticos de permeabilidade aparente absortiva e secretória da CAS X foram estimados em um sistema celular, no qual a atividade hidrolítica da CES foi inibida. Assim, a CAS X foi capaz de permear a monocamada de células Caco-2, provavelmente por transporte ativo, sem a ocorrência de efluxo, mas com significativa retenção do composto dentro das células. Em conjunto, os ensaios in vitro realizados demonstraram a susceptibilidade da CAS X ao metabolismo de primeira passagem, como substrato para as CES específicas expressas no fígado e intestino. / Casearia sylvstris leaves are commonly used by folk medicine and its main pharmacological properties were already described in the literature. Casearin X (CAS X) is the major clerodane diterpene isolated from the leaves of C. sylvestris. Recently, the in vitro cytotoxic activity of the CAS X was demonstrated against human tumor cells lineages. Despite promising results, the CAS X in vitro cytotoxic activity cannot be extrapolated to an in vivo activity, unless the compound has good bioavailability and desirable duration of its effect. The advance in natural products research requires a pharmacokinetic preclinical assessment to justify a therapeutic indication. Thereby, this present work aims to predict the CAS X in vivo bioavailability, by the in vitro characterization of the metabolism and intestinal absorption. The rat and human hepatic microsomal model was used for in vitro metabolism studies. An analytical method for quantification of CAS X in microsomes was developed, employing protein precipitation with acetonitrile for sample preparation and High Performance Liquid Chromatography for analysis. This method was validated in according to Agência Nacional de Vigilância Sanitária and European Medicine Agency guidelines (EMA). CAS X demonstrated to be substrate for carboxylesterases (CES) by hydrolysis reaction, with a Michaelis-Menten kinetic profile. The parameters Vmax and KM was estimated and the intrinsic clearance was 1.7-fold higher in humans than rats. The hepatic clearance was estimated by in vitro-in vivo extrapolation, resulting in more than 90% of the hepatic blood flow for both species. A qualitative study was carried out for the metabolite identification, using Mass Spectrometry, suggesting the formation of casearin X dialdehyde as metabolism product. Monolayer of Caco-2 cells was used for the in vitro intestinal absorption studies. An analytical method by Liquid Chromatography coupled to Mass Spectrometry was developed and validated, according to EMA, for the quantification of CAS X in cells systems. The apparent permeability was estimated in both, absorptive and secretory directions, using cells monolayers with inhibited CES hydrolysis. CAS X was able to cross the Caco-2 cells monolayer, probably by active transport, with no significant efflux, but with a high retention of the compound inside the cells. These findings demonstrated that CAS X is susceptible to first-pass metabolism, as substrate for specific CES expressed in both, liver and intestine.

Two specific goals were addressed for this dissertation. First to investigate and identify the mechanistic profile of ketoconazole (KT)-induced hepatotoxicity by utilizing in vivo and in vitro approaches determining the mechanism of action for the hepatotoxicity incurred. To date, there has not been a mechanistic determination of the hepatotoxicity associated with KT in vivo. This dissertation evaluates the possible metabolic bioactivation of KT by cytochrome-P450 (CYP) or flavin-containing monooxygenases (FMO) resulting in covalent binding with hepatic macromolecules. The hypothesis of this study was to reveal whether covalent binding by the parent compound, KT, and/or reactive metabolites produces hepatic damage associated with increased serum alanine aminotransaminase (ALT) release and decreased hepatic glutathione (GSH). The first objective was determination of in vivo covalent binding in a dose-time response comparison in Sprague-Dawley (SD) rat ALT and GSH levels. Increased ALT and reduced hepatic GSH levels occurred. The second objective was an in vitro comparison of covalent binding with GSH levels utilizing SD microsomal protein with incubations of KT. Covalent binding decreased with added GSH to microsomal incubations. Thirdly, correlate in vivo with in vitro findings. Covalent binding of KT in vivo and in vitro occurred with increased doses and time. The final objective was to determine the bioactivation pathway utilizing heat inactivation and no NADPH in vitro. Covalent binding of KT decreased in the absence of NADPH and deactivation of FMO. The second goal was to determine and quantitate in vitro the presence of FMO isozymes in microsomes of the human intestinal duodenum, jejunum, ileum, and colon as well as the Caco-2 (HTB-37), epithelial intestinal (CCL-241) and colon (CRL1790) cell lines. The presence of FMO could result in a first-pass effect decreasing the bioavailability of soft nucleophiles or a toxicity effect due to inhibition or modulation of the enzyme from co-administration. To date, this is the first evaluation of FMO isoforms in human intestine and cell lines. Western blot techniques were utilized for detection of human FMO1, FMO3, and FMO5 using human FMO-expressed recombinant cDNA from a baculovirus system. / Graduation date: 2003

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Verificou-se que microssomos de fígado apresentam uma atividade azo-redutásica completamente dependente de NADPH, usando DAB como substrato. Ao contrário de outros sistemas já descritos, a atividade azo-redutásica não pode ser identificada com a NADPH-citocromo c redutase, embora nossos resultados indiquem que esta enzima participa da reação. Não se pode excluir definitivamente a participação do citocromo b5 apesar de uma série de observações afastando essa possibilidade. Verificou-se que a atividade azo-redutásica pode ser induzida por tratamento com 3-MC, paralelamente à indução do citocromo P-450 tipo II. Isto sugere fortemente a participação desse citocromo na reação apesar desta não ser inibida por CO. O citocromo P-450 induzido por tratamento com 3-MC era funcionalmente ativo. A atividade azo-redutásica foi inibida especificamente pelo tratamento oral comoDAB, possibilitando a formulação de uma hipótese sobre a ação carcinogênica deste composto. Uma série de resultados mostra que a azo-redutase estudada é altamente específica podendo ser inibida por KCN e por mersalil. Face aos fatos expostos acima, as perspectivas do sistema parecem muito interessantes. A possibilidade do uso de novos métodos de fracionamento dos microssomos pode levar à resolução do sistema da azo-redutase. As semelhanças com a dessaturação de ácidos graxos, aliadas às indicações da existência de um novo tipo de citocromo P-450 que se combina com KCN, permitem descortinar maior amplitude para os limites do sistema da azo-redutase. Enfim, trata-se de uma reação de redução de um composto exógeno, com características até agora não relatadas, envolvendo processos biológicos de importância e cujo estudo terá prosseguimento. / Not available

O miclobutanil é fungicida quiral da família dos triazóis, comercializado como mistura racêmica. Apesar dos enantiômeros apresentarem as mesmas propriedades físico-químicas, estes podem diferir em termos de atividade, metabolismo, excreção e toxicidade. No presente trabalho, foram realizados estudos in vitro enantiosseletivos de metabolismo empregando microssomas hepáticos de humanos cujos objetivos foram determinar os parâmetros cinéticos das enzimas do citocromo P450 (CYP450) após metabolismo do miclobutanil (na forma de racemato e enantiômeros isolados), determinar quais isoformas do CYP450 são responsáveis pelo metabolismo do praguicida e também a capacidade deste praguicida em inibir as principais enzimas do CYP450. Os estudos foram realizados empregando a mistura racêmica e também os enantiômeros isolados. Para tanto, foi desenvolvido e validado um método para análise enantiosseletiva do miclobutanil em meio microssomal empregando a cromatografia líquida de alta eficiência acoplada a espectrometria de massas. A separação dos enantiômeros foi realizada na coluna Chiralpak AD® empregando metanol (100%) como fase móvel. Após a validação do método, os parâmetros cinéticos foram determinados, com valores de Vmáx, Km e CLint de 66,06 + 4,59 nmol min-1 mg-1, 3,61 + 0,88 ?mol L-1 e 18,30 mL min-1 mg-1 respectivamente, quando o substrato foi o racemato e de 305,50 + 18,39 nmol min-1 mg-1, 6,85 + 1,29 ?mol L-1 e 44,60 mL min-1 mg-1 respectivamente, quando o (+)-miclobutanil foi empregado como substrato. O (?)-miclobutanil não foi metabolizado pelas enzimas presentes nos microssomas hepáticos de humanos. As isoformas responsáveis pelo metabolismo do miclobutanil foram a CYP2C19 e a CYP3A4. Os estudos in vitro de inibição mostraram que o miclobutanil é um inibidor moderado das enzimas CYP2D6 e CYP2C9 um inibidor forte das enzimas CYP3A4/5 e CYP2C19. / Myclobutanil is a chiral triazole fungicide, sold as a racemic mixture. Although the enantiomers have the same physico-chemical properties, they may exhibit different bioactivity, metabolism, excretion and toxicity. In the present work, in vitro enantioselective metabolism studies were carried out by using human liver microsomes, aiming to determine the kinetic parameters of cytochrome P450 (CYP450) enzymes after myclobutanil metabolism and the main CYP450 isoforms involved in the metabolism. In addition, the myclobutanil inhibition capacity over the main CYP450 enzymes was evaluated. The studies were carried out with rac-myclobutanil as well as with the isolated enantiomers. To accomplish that, an enantioselective method for myclobutanil analysis was developed and validated by using high performance liquid chromatography coupled with mass spectrometry. The separation of enantiomers was realized on a Chiralpak AD® column and methanol (100%) was used as mobile phase. The enzymatic kinetics, Vmáx, Km and CLint, were: 66.06 + 4.59 nmol min-1 mg-1, 3.61 + 0.88 ?mol L-1 and 18.30 mL min-1 mg-1, respectively, for rac-myclobutanil and 305.50 + 18.39 nmol min-1 mg-1, 6.85 + 1.29 ?mol L-1 and 44.60 mL min-1 mg-1, respectively, for the (+)-myclobutanil. The (?)-myclobutanil was not metabolized by CYP450 enzymes. The isoforms involved in myclobutanil metabolism were CYP2C19 and CYP3A4. In vitro inhibition studies showed that myclobutanil is a medium inhibitor of CYP2D6 and CYP2C9 enzymes and a strong inhibitor of CYP3A4/A5 and CYP2C19 enzymes.