Yes / Epidemiological studies have shown that head and neck cancer (HNC) is a complex multistage process that in part involves exposure to a combination of carcinogens and the capacity of certain drug-metabolising enzymes including cytochrome P450 (CYP) to detoxify or activate such carcinogens. In this study, CYP1A1, CYP1B1 and CYP2W1 expression in HNC was correlated with potential as target for duocarmycin prodrug activation and selective therapy. In the HNC cell lines, elevated expression was shown at the gene level for CYP1A1 and CYP1B1 whereas CYP2W1 was hardly detected. However, CYP2W1 was expressed in FaDu and Detroit-562 xenografts and in a cohort of human HNC samples. Functional activity was measured in Fadu and Detroit-562 cells using P450-Glo™ assay. Antiproliferative results of duocarmycin prodrugs ICT2700 and ICT2706 revealed FaDu and Detroit-562 as the most sensitive HNC cell lines. Administration of ICT2700 in vivo using a single dose of ICT2700 (150 mg/kg) showed preferential inhibition of small tumour growth (mean size of 60 mm3) in mice bearing FaDu xenografts. Significantly, our findings suggest a potential targeted therapeutic approach to manage HNCs by exploiting intratumoural CYP expression for metabolic activation of duocarmycin-based prodrugs such as ICT2700. / The authors would like to thank Bradford Institute for Health Research for funding a PhD studentship to DP through a competitive scheme and Yorkshire Cancer Research programme Grant (B381PA) for supporting our cytochrome P450-focused drug discovery research.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

The cytochromes P450 are enzymes participating in metabolism of endogenous and exogenous compounds. Their substrates include also carcinogens which may initiate carcinogenesis after activation by CYP450. Inductors of these enzymes are also chemopreventive compounds which are very popular and recommended in current time. Thus, studying of the effect of the chemopreventive compounds on cytochromes P450 induction and cancer development is of a high clinical importance. The CYPs are most commonly found in the liver. However, there are forms that have not been detected in any human healthy tissue but their overexpression was observed in tumors. For this reason, they could serve for diagnosis and prognosis of cancer. Among these cytochromes are CYP2S1 and 2W1 which can be prognostic markers of colorectal cancer. Therefore, it would be opportune to have some tools for these enzyme detection. One option is immunodetection of cytochromes P450 by Western blot using the specific antibodies. Today mammalian antibodies (IgG) are the most widely used but antibodies isolated from egg yolk (IgY) become popular mainly due to the large number of undisputed advantages. For the preparation of the peptide immunogen, suitable peptide sequences were selected from CYP2S1 and 2W1 primary structure. The synthesized peptides...

In recent years, tyrosine kinase inhibitors have been widely used for the treatment of certain tumors as so-called targeted therapy. Many studies are concerned with their metabolism and the role of enzymes in the biotransformation process, but very little is known about the impact of tyrosine kinase inhibitors on the expression and activity of biotransformation enzymes. Nevertheless modification of the expression and activity of enzymes may cause adverse interactions of co-administered drugs and their negative impact on the human body. This diploma thesis studies the effect of tyrosine kinase inhibitors vandetanib and lenvatinib and cytotoxic alkaloid ellipticine on biotransformation enzymes in a rat model organism in vivo. The aim was to characterize the effect of the investigated compounds on gene expression, protein expression and activity of cytochromes P450 (CYP) 1A1, 1A2 and 1B1 and flavin-containing monooxygenases FMO1 and FMO3 in renal and hepatic microsomes. Microsomes and RNA were isolated from kidneys of control rats and the pretreated rats. Western blot and immunodetection was used to compare the protein expression levels of studied enzymes in kidney and liver. By reverse transcription, cDNA was prepared from isolated RNA and used as a template for quantitative PCR to compare the...

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Adéla Janoušková Supervisor: RNDr. Jakub Hofman, Ph.D. Title of diploma thesis: Study on impact of selected protein kinase inhibitors on drug resistance mediated by cytochromes P450 Pharmacokinetic drug resistance often leads to failure of an anticancer therapy. One of the mechanisms is increased efflux of drugs from tumour cells, whereas some studies suggest that increased drug conversion to an inactive metabolite might be another contributing mechanism. The aim of this work was to define the possible role of CYP3A4 and CYP2C8 enzymes in the phenomenon of pharmacokinetic resistance and to investigate the possibility of its modulation by new targeted drugs. In the first part, we used the MTT proliferation method together with HepG2 cells stably transduced with particular human enzymes and demonstrated significant involvement of CYP3A4 in docetaxel resistance. In the following part, we examined the inhibitory effects of four selected tyrosine kinase inhibitors on the CYP3A4 activity in intact cells using a commercial kit. Cobimetinib and dabrafenib showed significant inhibitory activity, while osimertinib and brivanib did not. In the final part, we demonstrated the ability of the first two...

O desenvolvimento testicular e a manutenção da espermatogênese são controlados por gonadotrofinas e testosterona, cujos efeitos são modulados por uma rede complexa de fatores produzidos localmente e, entre eles, os estrógenos estão em causa. Uma compreensão da dinâmica dos hormônios esteroides sexuais mostra-se importante para revelar as funções durante o desenvolvimento testicular. Sendo assim, este trabalho teve como objetivo aprofundar os conhecimentos sobre a espermatogênese do Galea spixii, associando a atuação das enzimas do complexo citocromo P450: P450 aromatase e P450c17 (17-α-hidroxilase/17,20-liase) importantes para a biossíntese de hormônios ligados à reprodução durante o desenvolvimento sexual pós-natal. Fragmentos de testículos de preás machos nas fases impúbere, pré-púbere, púbere e pós-púbere foram coletados no Centro de Multiplicação da Universidade Federal Rural do Semiárido, Mossoró, RN, fixados em Paraformoaldeido 4% e RNAlater, e processados para Imunohistoquímica e PCR em tempo real. A expressão gênica das enzimas esteroidogênicas foram cruciais da prépuberdade para a puberdade. Durante as fases do desenvolvimento sexual a enzima P450c17 apresentou imunomarcação positiva apenas nas células de Leydig. A imunomarcação da enzima P450 aromatase foi positiva em diferentes tipos celulares ao longo do desenvolvimento sexual. A síntese de estrógenos no parênquima testicular não ficou restrita às células somáticas, as células germinativas também mostraram capacidade de converter andrógenos em estrógenos / The testis development and maintenance of spermatogenesis are controlled by gonadotropins and testosterone, whose effects are modulated by a complex factor locally produced, and the estrogens are involved. An understanding of the dynamics of sex steroid hormones shown to be important to reveal the functions during testicular development. Thus, the aimed was study the spermatogenesis of Galea spixii, associating the performance of cytochrome P450 complex: P450 aromatase and P450c17 (17-α-hydroxylase / 17,20-lyase) important for the biosynthesis of hormones related to reproduction during postnatal sexual development. Fragments of testes of immature, prepubertal, pubertal and post-pubertal were collected at Centro de Multiplicação da Universidade Federal Rural do Semiárido, Mossoró, RN, fixed in Paraformaldehyde 4% and RNAlater, processed for immunohistochemistry and real time PCR. The steroidogenic enzymes gene expression were significant from prepubertal to pubertal stage. Cytochrome P450c17 expression in testicular parenchyma showed a positive reaction only in Leydig cell clusters. The expression of cytochrome P450 aromatase in testicular parenchyma were different during the sexual development of Galea spixii. During sexual development was observed that estrogen synthesis was not restricted to somatic cells (Leydig cells / Sertoli cells), the germ cells have also shown to be capable to convert androgens into estrogens via aromatase

Na avicultura industrial, diferenças no desempenho zootecnico de machos e femeas de linhagens pesadas forcam o setor a alojar separadamente os sexos com o propósito de facilitar manejo, uniformizar lotes, reduzir custos e otimizar a producao. Desse modo, meios que reduzam ou eliminem a disparidade entre sexos no ambito zootecnico podem resultar em ganhos gerenciais e econômicos no mercado avicola. A masculinizacao de femeas objetivando aproximar sua performance aquela manifestada por machos pode ser considerado um recurso interessante. Sendo assim a aplicacao de inibidores ou bloqueadores da aromatase P450 durante o desenvolvimento embrionario pode induzir graus variados de masculinizacao das femeas sem a utilizacao de protocolos hormonais, em um fenomeno chamado reversao sexual. Assim, os objetivos desse projeto foram: (1) comparar a acao de inibidores de aromatase de terceira geracao (Droga B e droga C) em relacao ao fadrozole (farmaco amplamente citado em literatura) sobre a taxa e o grau de reversao sexual em frangas de corte; e (2) estimar o impacto desses tratamentos sobre parametros de incubacao (mortalidade embrionaria e eclodibilidade) e indices zootecnicos (peso ao nascer, ganho de peso, conversao alimentar e peso final aos 42 dias). Os resultados, deste estudo, mostraram que o inibidor de aromatase droga B obteve maior proporção machos quando comparado aos outros IAs testados. Contudo, quando avaliamos as aves tratadas por este fármaco, em relação aos índices zootécnicos, estas apresentaram resultados similares ao grupo misto e inferior quando comparadas aos machos genéticos. Na sexagem morfológica aos 42 dias de idade foi observado que no grupo tratado com a droga B, 58% das aves apresentavam ovário ou ovostestis ao invés de testículos o que impactou negativamente no ganho de peso deste grupo. / Performance diferences between male and female broilers compel the poultry industry to rear sexes separately in order to favor management, uniform flocks, reduce costs and optimize production. Notwithstanding, this practice has logistical implications that create additional expenses, and in some cases, broiler companies encounter producer preferences for a particular sex due to its productive traits. Thus, methods that reduce or eliminate gender disparity in meat production efficiency can result in operational and economical benefits to poultry market. Masculinization of females aiming to bring performance closer to those expressed by males may be viewed as an interesting alternative. Therefore, the application of aromatase inhibitors or blockers during embryonic development can induce varying degrees of female masculinization without the use of hormonal protocols, in a process called sex reversal. Therefore, our objectives herein were: (1) to compare the action of third generation aromatase inhibitors (Drug B and Drug D) in relation to fadrozole (a drug widely used in literature) on the rate and degree of sexual reversal in broiler pullets; and (2) to estimate the impact of these treatments on incubation (embryonic mortality and hatchability) and performance parameters (birth weight, weight gain, feed conversion and final weight at 42 days). The results, from this study, showed that the aromatase inhibitor drug B obtained a higher rate of sexual reversion when compared to the other AIs tested. However, when we evaluated the birds treated by this drug, in relation to the zootechnical indexes, these presented similar results to the mixed and inferior group when compared to the genetic males. In the morphological sexing at 42 days of age, it was observed that in the group treated with AI, 58% of the birds presented ovary or ovostestis instead of testicles, which impacted the weight gain of this group.

O mutante redA de Dictyostelium discoideum, obtido por inativação gênica aleatória, tem crescimento aparentemente normal, porém seu ciclo de desenvolvimento não progride além do estágio de agregados compactos. Neste trabalho relatamos a caracterização deste mutante, cujo gene defeituoso codifica a enzima NADPH citocromo P450 redutase (NCPR). O principal papel desta enzima é transportar elétrons do NADPH para as várias isoformas do citocromo P450. Um cDNA de 2094 pb que codifica a NCPR de D. discoideum (DdNCPR) foi isolado através da varredura de uma biblioteca de cDNA com uma sonda derivada de um fragmento do gene inativado no mutante redA. A análise da seqüência de aminoácidos deduzida do cDNA DdNCPR revelou que esta codifica uma proteína de 631 aminoácidos com 31% de identidade e 50% de similaridade com a NCPR humana. Verificamos o acúmulo do mRNA da DdNCPR durante fase de crescimento mas durante as fases iniciais do desenvolvimento ocorre significativa diminuição em seus níveis até a formação dos agregados compactos onde o mRNA da NCPR não é detectável. Demonstramos que o gene que codifica a NCPR aparentemente está presente em uma única cópia no genoma de Dictyostelium. Ademais, a análise de outras linhagens mutantes nocautes do gene da NCPR confirmaram que a inativação deste gene está diretamente relacionada ao fenótipo exibido pelo mutante redA-. Contudo, é provável que um ou mais produtos gênicos possam complementar a ausência desta enzima, uma vez que nem a linhagem redA nem as outras linhagens nocautes do gene da NCPR apresentaram alteração na taxa de crescimento e, em algumas circunstâncias experimentais, não exibiram qualquer alteração no ciclo de desenvolvimento. Nossos resultados sugerem, ainda que o bloqueio do desenvolvimento eventualmente observado no mutante redA pode ser devido a um provável papel da NCPR no metabolismo de DIF-1 (fator indutor de diferenciação-1), que parece desempenhar um papel primordial no controle da diferenciação de células pré-talo e células pré-esporo durante o desenvolvimento de D. discoideum. / The Dictyostelium discoideum redA mutant, obtained by random gene inactivation, exhibits normal growth but has its developmental cycle impaired at tight mound stage. In this study we describe the characterization of this mutant whose defective gene encodes the enzyme NADPH cytochrome P450 reductase (NCPR). NCPR is known to play an essential role in the transfer of reducing equivalents from NADPH to various cytochrome P450 isoforms. We isolated a 2094 bp cDNA that encodes D. discoideum NCPR (DdNCPR) by screening a cDNA library using as probe the mutated gene fragment rescued from redA cells. Analysis of the deduced aminoacid sequence of DdNCPR cDNA shows that it encodes a 631 aminoacid protein with 31% of identity and 50% of similarity with human NCPR. Northern blot analysis showed that DdNCPR mRNA levels is maximum during growth phase and decreases at early stages of the development. After slug stage this mRNA is not detectable. D. discoideum has a single copy of NCPR gene and, as shown by analysis of other NCPR knockout mutants, inactivation of this gene is strongly correlated to the redA phenotype. However, redA, as well as the other NCPR knockout strains, do have growth alterations and in some circumstances they do not show the described developmental defects. Thus, it is possible that one or more proteins be able to compensate for the lack of NCPR in these mutants. Our results also suggest that the redA developmental phenotype might play a role of NCPR on the metabolism of DIF-1, a prime candidate for controlling prestalk and prespore cell differentiation during D. discoideum development.

Na etapa inicial do desenvolvimento de novos fármacos, a avaliação do metabolismo e da toxicidade é fundamental para definir seu potencial emprego como candidato a fármaco. Nestes estudos, diversos modelos in vitro são empregados, dentre eles linhagens de hepatoma humano. Entretanto, uma grande limitação ao uso deste modelo in vitro é a baixa expressão das enzimas do sistema citocromo P450. O carotenóide bixina, componente majoritário do anato (urucum), apresentou em estudos in vivo, a capacidade de induzir algumas isoformas do sistema citocromo P450, com a vantagem de apresentar baixa toxicidade. Neste trabalho, a fração lipossolúvel do anato (bixina) e hidrossolúvel (norbixina) foram avaliadas como indutores do sistema citocromo P450 em linhagens de hepatoma humano. Ensaios de MTT, empregando as linhagens HepG2, C3A e SK-HEP-1 indicaram que bixina e norbixina em concentrações abaixo de 0,22 mM são seguras quanto à citotoxicidade. A expressão dos genes CYP 1A1, 1A2, 2C9, 2B6, 2E1 e 3A4 foi avaliada, através de ensaios de RT-PCR em tempo real, em linhagens de hepatoma humano submetidas a tratamento com os compostos bixina e norbixina. Os resultados mostraram que células HepG2 e C3A tratadas com bixina nas concentrações de 0,05 e 0,1 mM, por períodos de 24 e 48 horas, apresentaram aumento de expressão da CYP 1A1 e CYP 1A2. Porém, a exposição de células HepG2 e C3A ao composto norbixina não resultou em aumento de expressão das isoformas avaliadas neste estudo. Os resultados deste trabalho indicaram o potencial emprego de bixina como agente indutor das CYPs 1A1 e 1A2, em linhagens de hepatoma humano utilizadas como modelo in vitro, para estudo de compostos cuja metabolização envolva uma destas vias, entretanto, estudos adicionais são fundamentais, a fim de avaliar a ação deste composto sobre outras isoformas do sistema citocromo P-450, bem como outros sistemas enzimáticos. / In the early development stage of the new drugs, the pharmacological and toxicological properties are critical to define the potential use of the candidate drug. During this stage, several in vitro models systems are employed, including human hepatoma cell lines. However, the main limitation of the use of cell lines as in vitro model is the low expression level of cytochrome P450 enzymes. A carotenoid knowed as bixin, the main pigment in the annatto (urucum), it has been reported to induce some isoforms of cytochrome P450 in rats, with the advantage of its low toxicity. In this work, the oil-soluble (bixin) and aqueous soluble extracts (norbixin) were evaluated as inducers of the cytochrome P450 system in human hepatoma cell lines (HepG2, C3A, SK-HEP-1). The results of MTT assays showed that bixin concentrations below 0.22 mM were not cytotoxic in HepG2, C3A and SK-HEP-1 cell lines. Expression changes in CYP 1A1, 1A2, 2C9, 2B6, 2E1 and 3A4 were evaluated, by real time RT-PCR and the results showed that the exposition to 0,05 mM and 0,1 mM bixin, for 24 and 48 hours of treatment, lead to an increase in CYP 1A1 and CYP 1A2 expression level. By contrast, the cytochrome P450 isoforms were not affected by the exposition to norbixin. In conclusion, this work indicated the potential use of bixin induced hepatoma cell lines as in vitro model for studies of biotransformation and toxicity of drugs involving CYP 1A, however, further studies are necessary to evaluate the effect of bixin on the other cytochrome P450 isoforms as well as other enzymatic systems.

Neste trabalho foram investigados sistemas modelos do citocromo P450 para o estudo do metabolismo da monensina A empregando três estratégias de abordagem: a) utilização de metaloporfirinas e complexos salen como catalisadores para a oxidação da monensina A por diferentes oxidantes e meios reacionais; b) utilização de fungos de diferentes cepas para estudos de biotransformação deste antibiótico e c) emprego de microssomas de fígado de ratos e humanos para o estudo do metabolismo in vitro da monensina A. Os produtos obtidos nestes três sistemas foram comparados com os metabólitos formados em estudos in vivo relatados na literatura. Os resultados obtidos com os sistemas envolvendo os catalisadores mostraram que a formação dos produtos é dependente da escolha do meio reacional e do oxidante empregado. Os estudos de biotransformação da monensina A empregando microssomas de fígado e os fungos Aspergillus awamori, Beauveria bassianna, Cunninghamella echinulata, Cunninghamella elegans, Fusarium oxysporum, M61, Mucor rouxii e Penicillium brevicompactum mostraram que estes sistemas são viáveis nos processos de biotransformação deste fármaco nas condições empregadas. Os produtos obtidos nas reações e/ou meios de cultura com os diferentes sistemas foram identificados por espectrometria de massas sequencial e também por comparação com padrões obtidos anteriormente. Foram obtidos três principais metabólitos: (i) 3-O-desmetil-monensina A, (ii) 12-hidroxi-monensina A e (iii) 12-hidroxi-3-O-desmetil-monensina A, os quais coincidem com os principais metabólitos obtidos em estudos in vivo. Assim, os resultados mostraram que os modelos estudados podem ser usados para predizer o metabolismo da monensina A. Os metabólitos 3-O-desmetil-monensina A e 12-hidroxi-monensina A puderam ser produzidos e isolados dos sistemas catalíticos envolvendo a metaloporfirina e o catalisador de Jacobsen. Os ensaios biológicos de atividade tóxica em mitocôndrias, bem como a atividade antimicrobiana da monensina A e de seus metabólitos 3-O-desmetil-monensina A e 12-hidroxi-monensina A mostraram que estes metabólitos possuem menor ou nenhuma atividade nos parâmetros biológicos testados quando comparados à monensina A. Assim, pode-se inferir que o metabolismo da monensina A corresponde a uma via de detoxicação clássica, através da qual as moléculas produzidas são mais polares, dificultando o transporte de complexos catiônicos através das membranas, diminuindo suas propriedades biológicas e facilitando a sua eliminação. / This study used model systems to investigate monensin A metabolism. More specifically, this work employed three strategies: (i) use of biomimetic systems, involving metalloporphyrins and salen complexes, to catalyze monensin A oxidation by different oxidants in distinct reaction media; (ii) application of different fungal strains to conduct biotransformation studies of this antibiotic; and (iii) use of rat and human liver microsomes as a cytochrome P450 model to monitor the in vitro metabolism of monensin A and compare the products with the metabolites generated in in vivo studies reported in the literature. Studies involving chemical catalysts showed that product formation depended on the choice of reaction medium and oxidant. Monensin A biotransformation studies employing fungi revealed that Aspergillus awamori, Beauveria bassianna, Cunninghamella echinulata, Cunninghamella elegans, Fusarium oxysporum, Marine M61, Mucor rouxii, and Penicillium brevicompactum successfully biotransformed the drug under the employed conditions. Liver microsomes also effectively transformed the target compound. Spectrometric analysis of the evaluated models attested to the formation of three main metabolites: (i) 3-O-demethyl monensin A, (ii) 12-hydroxy monensin A, and (iii) 12-hydroxy-3-O-demethyl-monensin A as the main monensin A derivatives. The products were identified by tandem mass spectrometry as well as by comparison with standards obtained in other studies. Taken together, the results demonstrated that the models studied herein could help to predict monensin A metabolismthey produced the main metabolites obtained in in vivo studies. Toxicity tests performed on mitochondria and antimicrobial assays revealed that the metabolites 3-O-demethyl-monensin A and 12-hydroxy-monensin A isolated from the reactions that employed chemical catalysts were less active or inactive as compared with monensin A. Therefore, it was possible to infer that monensin A metabolism is a classical detoxification pathway that generates polar molecules. The transport of such cationic molecules through the membrane is more difficult, decreasing their biological properties and facilitating their elimination.