Cyclosporine forms the cornerstone of therapy to prevent rejection after organ transplantation. However, the clinical use of the drug is compromised by a narrow therapeutic window and a wide inter- and intra-individual variation in metabolism. Cyclosporine is metabolised by the CYP3A4 isoenzymes in both the liver and intestine, while it has been reported that the metabolism of the drug can be inhibited by certain furocoumarin derivatives in grapefruit juice. Methoxsalen (8-methoxypsoralen) is a furocoumarin and a potent inhibitor of the cytochrome P450 system in both the liver and intestine. The study was conducted to investigate the possibility whether methoxsalen may inhibit the metabolism of cyclosporine and thereby increase the bioavailability of the drug. The interaction is of clinical relevance since both drugs are used in the treatment of psoriases. The study, conducted in 12 healthy male volunteers, was a three-way comparative bioavailability study with a wash out period of one week between treatments. The patients received 40 mg methoxsalen, 200 mg cyclosporine or a combination of the two on three separate occasions. Blood samples of 10 ml were collected by venupuncture at the following times: 0, 0.5, 1, 1.5, 2, 2.5, 3.4, 5,6, 8, 12 and 24 hours after drug administration. Methoxsalen was analysed by a high pressure liquid chromatograph method (HPLC) with UV detection (LOQ = 10 ng/ml), while cyclosporine was analysed using a fluorescence polarisation immunoassay (FPIA) technique. There was a statistical significant difference in AUCo-00 and Cmax ' for cyclosporine when methoxsalen was added to the drug regimen. When the methoxsalen levels were compared with those in the presence of cyclosporine, the levels were lower, although the difference was not statistical significant. We conclude that methoxsalen increase the levels of cyclosporine by inhibiting the P450 system enzymes in the liver and intestine. However, the absorption of methoxsalen is highly variable in the same individual which needs to be considered before this interaction can be regarded as being of any clinical relevance. / Thesis (M.Sc.(Pharmacology))--North-West University, Potchefstroom Campus, 2004.

Cyclosporine forms the cornerstone of therapy to prevent rejection after organ transplantation. However, the clinical use of the drug is compromised by a narrow therapeutic window and a wide inter- and intra-individual variation in metabolism. Cyclosporine is metabolised by the CYP3A4 isoenzymes in both the liver and intestine, while it has been reported that the metabolism of the drug can be inhibited by certain furocoumarin derivatives in grapefruit juice. Methoxsalen (8-methoxypsoralen) is a furocoumarin and a potent inhibitor of the cytochrome P450 system in both the liver and intestine. The study was conducted to investigate the possibility whether methoxsalen may inhibit the metabolism of cyclosporine and thereby increase the bioavailability of the drug. The interaction is of clinical relevance since both drugs are used in the treatment of psoriases. The study, conducted in 12 healthy male volunteers, was a three-way comparative bioavailability study with a wash out period of one week between treatments. The patients received 40 mg methoxsalen, 200 mg cyclosporine or a combination of the two on three separate occasions. Blood samples of 10 ml were collected by venupuncture at the following times: 0, 0.5, 1, 1.5, 2, 2.5, 3.4, 5,6, 8, 12 and 24 hours after drug administration. Methoxsalen was analysed by a high pressure liquid chromatograph method (HPLC) with UV detection (LOQ = 10 ng/ml), while cyclosporine was analysed using a fluorescence polarisation immunoassay (FPIA) technique. There was a statistical significant difference in AUCo-00 and Cmax ' for cyclosporine when methoxsalen was added to the drug regimen. When the methoxsalen levels were compared with those in the presence of cyclosporine, the levels were lower, although the difference was not statistical significant. We conclude that methoxsalen increase the levels of cyclosporine by inhibiting the P450 system enzymes in the liver and intestine. However, the absorption of methoxsalen is highly variable in the same individual which needs to be considered before this interaction can be regarded as being of any clinical relevance. / Thesis (M.Sc.(Pharmacology))--North-West University, Potchefstroom Campus, 2004.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2015-10-01T14:25:59Z No. of bitstreams: 1 Pietro Araujo dos Santos Efeito... 2015.pdf: 1429638 bytes, checksum: 830edcdf557efac1a5f1e22d8e0e6a23 (MD5) / Approved for entry into archive by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2015-10-01T14:26:17Z (GMT) No. of bitstreams: 1 Pietro Araujo dos Santos Efeito... 2015.pdf: 1429638 bytes, checksum: 830edcdf557efac1a5f1e22d8e0e6a23 (MD5) / Made available in DSpace on 2015-10-01T14:26:17Z (GMT). No. of bitstreams: 1 Pietro Araujo dos Santos Efeito... 2015.pdf: 1429638 bytes, checksum: 830edcdf557efac1a5f1e22d8e0e6a23 (MD5) Previous issue date: 2015 / Universidade Federal da Bahia. Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil / Doença de Parkinson (DP) é caracterizada por uma perda seletiva e profunda dos neurônios dopaminérgicos da substância nigra pars compacta (SNpc) do mesencéfalo, acompanhada pela espoliação de dopamina no corpo estriado. A maioria dos casos de DP apresenta etiologia multifatorial, com a presença de componentes genéticos e ambientais. Embora existam diferentes causas possíveis, a patogênese da desordem parece convergir para mecanismos relacionados à disfunção mitocondrial, estresse oxidativo e mau enovelamento proteico. Um modelo estabelecido na literatura para estudo desta doença, tanto in vitro quanto in vivo é a administração de rotenona, um pesticida derivado de plantas que inibe o complexo I mitocondrial e favorece a geração de espécies reativas de oxigênio (ERO), levando a uma espoliação de glutation reduzido (GSH) através do processo de detoxificação destes compostos eletrofílicos, catalisados por glutation S-transferases (GSTs). Sendo assim, a busca por novas substâncias com atividade neuroprotetora é atualmente o foco de estudos, e metabólitos isolados de plantas podem ser fontes destas moléculas. Dessa forma, o 8-metoxipsoraleno (8-MOP), uma furocumarina, foi testado como um possível agente protetor sobre a citotoxicidade causada pela rotenona em modelos in vitro de gliomas, considerando o papel do glutation neste processo. O estudo adotou uma abordagem que associa técnicas bioquímicas e de biologia celular. Ensaios de viabilidade celular foram realizados em células de glioma murino (C6) e glioblastoma multiforme humano (U251) através da redução do brometo de 3-(4,5-dimetiltiazol-2-il)-2,5-difeniltetrazolium (MTT), e visualização por microscopia de contraste de fase. O tipo de morte celular provocada pela rotenona nas células U251 foi realizado por marcação com anexina V e iodeto de propídeo (IP), seguido por quantificação por citometria de fluxo. A determinação do conteúdo de GSH intracelular após tratamento com rotenona e 8-MOP foi visualizado por marcação com monoclorobimano (MCB) nas linhagens C6 e U251. Os resultados demonstraram que a rotenona foi citotóxica em ambas as linhagens, reduzindo a viabilidade e alterando a morfologia celular, enquanto que o 8-MOP não apresentou atividade citotóxica. Contudo, o tratamento com o 8-MOP não foi capaz de proteger as células contra os efeitos deletérios da rotenona. No estudo do tipo de morte celular, a porcentagem de células marcadas com anexina V foi maior nos grupos tratados com rotenona, demonstrando que a morte celular ocorre principalmente por apoptose. A análise com MCB demonstrou que a rotenona espoliou GSH, porém o pré-tratamento com 8-MOP inibiu a espoliação. Embora o 8-MOP não tenha sido bem sucedido na proteção das células, a manutenção do conteúdo de GSH corrobora com estudos prévios que descrevem este composto como um potencial inibidor de GST, uma atividade farmacológica que deve ser testada para confirmar a sua eficácia como agente terapêutico. / Parkinson’s disease (PD) is characterized by a profound and selective loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) accompanied by midbrain dopamine depletion in the striatum. Most cases of PD present multifactorial etiologies, with the presence of genetic and environmental components. Although there are different possible causes, the pathogenesis of the disorder seems to converge to mechanisms related to mitochondrial dysfunction, oxidative stress and bad protein folding. An established model in the literature to study this disease, both in vitro and in vivo is rotenone administration, a pesticide derived from plants that inhibits the mitochondrial complex I and favors the generation of reactive oxygen species (ROS), leading to reduced glutathione (GSH) depletion through the detoxification process of this electrophilic compound catalyzed by glutathione S-transferases (GSTs). Thus, the search for new substances with neuroprotective activity is currently the focus of studies, and plant isolated metabolites can be sources of these molecules. Thus, 8-methoxypsoralen (8-MOP), a furocoumarin, was tested as a potential protective agent on the cytotoxicity caused by rotenone in glioma cells in vitro models, considering the role of glutathione in the process. The study adopted an approach that combines biochemical and cell biology techniques. Cell viability assays were performed in murine glioma cells (C6) and human glioblastoma (U251) cells through the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and visualization by phase contrast microscopy. The type of cell death caused by rotenone in U251 cells was performed by staining with annexin V and propidium iodide (PI) followed by flow cytometric quantitation. The determination of intracellular GSH content after treatment with rotenone and 8-MOP was visualized by staining with monochlorobimane (MCB) in the lineages U251 and C6. The results demonstrated that rotenone was cytotoxic to both cell lineages, reducing the viability and changing the cell morphology, whereas the 8-MOP did not show cytotoxic activity. However, the treatment with 8-MOP was not able to protect cells against the deleterious effects of rotenone. In the type of cell death studies, the percentage of cells stained with annexin V was higher in the groups treated with rotenone, demonstrating that cell death occurs primarily by apoptosis. The analysis with MCB has shown that rotenone depleted GSH, but pre-treatment with 8-MOP inhibited the depletion. Although the 8-MOP has not been successful in protecting cells, the maintenance of GSH content corroborates with previous studies that describe this compound as a potential inhibitor of GST, a pharmacological activity that should be tested to confirm its effectiveness as a therapeutic agent.

INTRODUCTION: Smoking is one of the single greatest causes of numerous preventable diseases. We were interested in developing an animal model of nicotine metabolism that can be used to examine the effects of potential CYP2A6 inhibitors on nicotine metabolism and nicotine-mediated behaviours. Pharmacogenetic studies have demonstrated that in humans, smoking behaviour is associated with rates of nicotine metabolism by the CYP2A6 enzyme. Mouse CYP2A5 shares structural and functional similarities to human CYP2A6 and has been implicated in nicotine self-administration behaviours in mice, therefore the mouse represents a potential animal model for studying nicotine metabolism. METHODS: We characterized nicotine and cotinine metabolism in two commonly used mouse strains (DBA/2 and C57Bl/6). We also examined the association between nicotine self-administration behaviours and nicotine metabolism, and the impact of direct manipulation (i.e. inhibition) of nicotine metabolism on nicotine pharmacodynamics (hot-plate and tail-flick tests) in mice. Finally, we studied the effect of selegiline (a known cytochrome P450 mechanism-based inhibitor) on nicotine metabolism in mice and in human CYP2A6. RESULTS: Nicotine metabolism in mice in vitro was mediated by CYP2A5, and this enzyme was responsible for over 70% and 90% of the metabolism of nicotine to cotinine and cotinine to 3-hydroxycotinine as shown by immuno-inhibition studies, respectively. A polymorphism in CYP2A5 between mouse strains, known to alter the probe substrate coumarin’s metabolism, did not affect nicotine metabolism but dramatically altered cotinine metabolism. Nicotine self-administration behaviour in mice was associated with level of hepatic CYP2A5 proteins and rates of nicotine metabolism in male mice. In inhibition studies, the CYP2A5/6 inhibitor methoxsalen inhibited both in vitro and in vivo nicotine metabolism in mice and substantially increased the anti-nociceptive effect of nicotine. Finally, selegiline was found to be an inhibitor of CYP2A5 decreasing nicotine metabolism in vitro and in vivo in mice. Moreover, we showed that selegiline is a mechanism-based inhibitor of CYP2A6 inhibiting nicotine metabolism irreversibly. CONCLUSION: The above data suggested that the mouse model may be suitable for examining the impact of inhibition (and genetic variation) on nicotine metabolism and nicotine-mediated behaviours and may potentially be used to screen for novel inhibitors of nicotine metabolism.

INTRODUCTION: Smoking is one of the single greatest causes of numerous preventable diseases. We were interested in developing an animal model of nicotine metabolism that can be used to examine the effects of potential CYP2A6 inhibitors on nicotine metabolism and nicotine-mediated behaviours. Pharmacogenetic studies have demonstrated that in humans, smoking behaviour is associated with rates of nicotine metabolism by the CYP2A6 enzyme. Mouse CYP2A5 shares structural and functional similarities to human CYP2A6 and has been implicated in nicotine self-administration behaviours in mice, therefore the mouse represents a potential animal model for studying nicotine metabolism. METHODS: We characterized nicotine and cotinine metabolism in two commonly used mouse strains (DBA/2 and C57Bl/6). We also examined the association between nicotine self-administration behaviours and nicotine metabolism, and the impact of direct manipulation (i.e. inhibition) of nicotine metabolism on nicotine pharmacodynamics (hot-plate and tail-flick tests) in mice. Finally, we studied the effect of selegiline (a known cytochrome P450 mechanism-based inhibitor) on nicotine metabolism in mice and in human CYP2A6. RESULTS: Nicotine metabolism in mice in vitro was mediated by CYP2A5, and this enzyme was responsible for over 70% and 90% of the metabolism of nicotine to cotinine and cotinine to 3-hydroxycotinine as shown by immuno-inhibition studies, respectively. A polymorphism in CYP2A5 between mouse strains, known to alter the probe substrate coumarin’s metabolism, did not affect nicotine metabolism but dramatically altered cotinine metabolism. Nicotine self-administration behaviour in mice was associated with level of hepatic CYP2A5 proteins and rates of nicotine metabolism in male mice. In inhibition studies, the CYP2A5/6 inhibitor methoxsalen inhibited both in vitro and in vivo nicotine metabolism in mice and substantially increased the anti-nociceptive effect of nicotine. Finally, selegiline was found to be an inhibitor of CYP2A5 decreasing nicotine metabolism in vitro and in vivo in mice. Moreover, we showed that selegiline is a mechanism-based inhibitor of CYP2A6 inhibiting nicotine metabolism irreversibly. CONCLUSION: The above data suggested that the mouse model may be suitable for examining the impact of inhibition (and genetic variation) on nicotine metabolism and nicotine-mediated behaviours and may potentially be used to screen for novel inhibitors of nicotine metabolism.