In the treatment of arrhythmias, amiodarone is a primary therapeutic agent. Cytochromes P450 (CYP) 1A1 and 1A2 facilitate biotransformation of amiodarone to the biologically active desethylamiodarone. Side effects have been reported during therapy and some are correlated with increased desethylamiodarone levels. Exposure to polycyclic aromatic hydrocarbons (PAH) like -naphthoflavone induces CYP1A1 and CYP1A2 and therefore can increase desethylamiodarone levels. Desethylamiodarone, however, was reported to inactivate human CYP1A1 and therefore can conceivably inhibit its CYP1A1-mediated formation. Our primary objective was to investigate the effect of -naphthoflavone on amiodarone disposition. Since rats were used, CYP isoenzymes involved in desethylamiodarone formation in human were compared to their rat counterparts. The effect of ketoconazole on desethylamiodarone formation, the inactivating potential of desethylamiodarone on CYP1A1 and the mechanism of -naphthoflavone-amiodarone interaction were assessed. Human CYP1A1 and rat CYP2D1 had the highest intrinsic clearance (Clint) for desethylamiodarone. Human and rat CYP1A2 had the lowest Clint. Ketoconazole (18.8 M) inhibited all isoforms except for rat CYP1A2; it potently inhibited human CYP1A1 and CYP3A4 and rat CYP2D2 and CYP1A1. After a single amiodarone dose was administered to control and -naphthoflavone pretreated rats, the plasma area under concentration-time curve (AUC) of desethylamiodarone increased. With multiple doses, amiodarone AUC(0-24h) decreased in -naphthoflavone plasma (30%), lung (35%), liver (48%), kidney (52%), heart (34%), and intestine (43%). Desethylamiodarone AUC(0-24h) increased in -naphthoflavone plasma (36%), lung (56%), liver (101%), kidney (65%), and heart (73%). Desethylamiodarone caused no inactivation of CYP1A1 when preincubations were diluted and nicotinamide adenine dinucleotide phosphate (NADPH) was added in the probe incubation samples. Evidence for reversible mixed-competitive inhibition was apparent. Addition and/or replenishment of NADPH were important factors in maintaining control activity. -naphthoflavone increased desethylamiodarone formation only in lung and kidney microsomes. Desethylamiodarone formation in liver, intestine and heart microsomes was not altered. Body-weight-normalized liver mass was significantly increased (27%) by -naphthoflavone. In conclusion, human CYP1A1 was more efficient in forming desethylamiodarone than rat isoenzyme. Exposure to PAH increased desethylamiodarone levels in vivo. Increased desethylamiodarone levels were partly caused by CYP1A1 induction, and by increased liver mass. Desethylamiodarone did not inactivate CYP1A1 activity. / Pharmaceutical sciences

No description available.

Cyclophilins (Cyp) are proteins that catalyze the interconversion of trans/cis isomers of proline belonging to the peptidyl-prolyl isomerases family (PPIase). In addition to their PPIase activity, Cyps have diverse biological roles and have been implicated in a number of different diseases such as HIV-1 and HCV. Although several Cyp inhibitors have been reported in the literature, none are able to inhibit with high specificity various Cyp isoforms. To facilitate the development of isoform-specific Cyp ligands, we have pursued detailed studies of Cyp dynamics and ligand binding thermodynamics using molecular simulations, biophysical assays and protein X-ray crystallography. Research efforts were focussed on the identification of novel Cyp inhibitors using X-ray crystallographic studies and Surface Plasmon Resonance (SPR) experiments on fragments from an in-house bespoke library of small compounds. These biophysical studies revealed a number of fragments that are able to bind to diverse Cyp isoforms with high micromolar – low millimolar activity. To further examine the binding of these fragments to cyclophilins, identify interactions with the proteins and explain specificity trends from SPR and X-ray results, molecular dynamics (MD) simulations and free energy calculations were pursued. Models of apo and holo Cyps in complex with fragments that we had experimentally tested were set up using the Amber, AmberTools and FESetup software. Free energy calculations were performed using the thermodynamic integration (TI) technique with the Sire/OpenMM software. The results were analysed with custom scripts. Correlations between computed and measured binding energies, and calculated and observed binding modes were analysed to help develop guidelines for the development of isoform specific cyclophilin ligands. A detailed comparison of the merits and drawbacks of the experimental and computational techniques used in this work has also been made, and strategies for effective combination of the methodologies in structure-based projects are outlined.

Infantile exposure to xenobiotics, e.g. from breastfeeding, poses a serious toxicity risk. Since the toxicokinetic mechanisms that principally determine exposure outcomes undergo a significant developmental maturation, infants may respond to exposures in a different way than adults. Hence, suitable model systems are required to provide risk relevant information in pediatric populations. This dissertations primary goal was to provide a critical evaluation of two such model systems; first, a pharmacokinetic model that may predict an infants capacity to eliminate toxicants by cytochrome P-450 (CYP) mechanisms and second, the developing rat as a model of human CYP2E1 and CYP1A2 ontogeny.<p>The first objective was to evaluate underlying assumptions of a pharmacokinetic model that describes the ontogeny of hepatic CYP activity using the rat. The study recognized some discrepancies with the stated assumptions. The impact of these discrepancies on the potential applicability of the model is discussed. As proof-of-concept, the observed data were fit to a model describing rat CYP2E1 and CYP1A2 ontogeny. A reasonable correlation (r = 0.75) was observed between observed and predicted oral clearance values of a CYP2E1 substrate indicating the potential applicability of such a model in risk assessment. <p>The second objective was to conduct an extensive characterization of rat hepatic CYP2E1 and CYP1A2 ontogeny at mRNA, protein, activity and intrahepatic expression levels. The results were compared to available human data to determine the appropriateness of the rat for assessment of toxicokinetic mechanisms underlying age-dependent differences in susceptibility to toxicity. Similarities in age-dependent changes in mRNA, activity and zonal hepatic expression patterns were noted between the rat and human prior to weaning. Unlike human data, rats show good correlation between changes in CYP2E1 and CYP1A2 activity and transcript levels, but not with the immunoquantifiable protein. Recognizing such similarities and differences between rats and human regarding onset, rate and pattern of CYP ontogeny will improve the accuracy of rat-to-human extrapolation of developmental toxicokinetic data. <p>Overall, the dissertation research provides mounting and supportive evidence for the use of such model systems in providing risk-relevant information in pediatric populations and to identify toxicokinetic mechanisms underlying age-dependent differences in susceptibility to toxicity.

Infantile exposure to xenobiotics, e.g. from breastfeeding, poses a serious toxicity risk. Since the toxicokinetic mechanisms that principally determine exposure outcomes undergo a significant developmental maturation, infants may respond to exposures in a different way than adults. Hence, suitable model systems are required to provide risk relevant information in pediatric populations. This dissertations primary goal was to provide a critical evaluation of two such model systems; first, a pharmacokinetic model that may predict an infants capacity to eliminate toxicants by cytochrome P-450 (CYP) mechanisms and second, the developing rat as a model of human CYP2E1 and CYP1A2 ontogeny.<p>The first objective was to evaluate underlying assumptions of a pharmacokinetic model that describes the ontogeny of hepatic CYP activity using the rat. The study recognized some discrepancies with the stated assumptions. The impact of these discrepancies on the potential applicability of the model is discussed. As proof-of-concept, the observed data were fit to a model describing rat CYP2E1 and CYP1A2 ontogeny. A reasonable correlation (r = 0.75) was observed between observed and predicted oral clearance values of a CYP2E1 substrate indicating the potential applicability of such a model in risk assessment. <p>The second objective was to conduct an extensive characterization of rat hepatic CYP2E1 and CYP1A2 ontogeny at mRNA, protein, activity and intrahepatic expression levels. The results were compared to available human data to determine the appropriateness of the rat for assessment of toxicokinetic mechanisms underlying age-dependent differences in susceptibility to toxicity. Similarities in age-dependent changes in mRNA, activity and zonal hepatic expression patterns were noted between the rat and human prior to weaning. Unlike human data, rats show good correlation between changes in CYP2E1 and CYP1A2 activity and transcript levels, but not with the immunoquantifiable protein. Recognizing such similarities and differences between rats and human regarding onset, rate and pattern of CYP ontogeny will improve the accuracy of rat-to-human extrapolation of developmental toxicokinetic data. <p>Overall, the dissertation research provides mounting and supportive evidence for the use of such model systems in providing risk-relevant information in pediatric populations and to identify toxicokinetic mechanisms underlying age-dependent differences in susceptibility to toxicity.

Ovarian toxicants 4-vinylcychlohexene (VCH) and 7,12-dimethylbenz[a]anthracene (DMBA) requires bioactivation to induce follicle loss. VCH is bioactivated to monoepoxides (1,2-VCM and 7,8-VCM), and subsequently to an ovotoxic diepoxide (VCD) by hepatic CYP 2A and CYP 2B. DMBA is sequentially bioactivated to the ovotoxicant DMBA-3,4-diol-1,2-epoxide by hepatic CYP 1B1, microsomal epoxide hydrolase (mEH), and CYP 1A1/1B1 enzymes. Even though the liver is the primary organ metabolizing VCH and DMBA to reactive intermediates, several studies suggest that the ovary can also metabolize these two compounds. Studies were designed to investigate the role of ovarian metabolism in the resulting ovotoxicity of these two compounds using a novel mouse ovarian culture system. The hypothesis was that the ovary can participate in bioactivation and detoxification of VCH/VCM and DMBA and thereby influence the resulting ovotoxicity.Postnatal day 4 CYP 2E1 wild-type, null and B6C3F1 mouse ovaries were incubated with 1,2-VCM, VCD or DMBA for various lengths of time. 28 day old female CYP 2E1 wild-type and null mice were dosed (15d, i.p) with VCH, 1,2-VCM, VCD, or sesame oil (control). Following incubations and dosing, ovaries were prepared for histological evaluation of follicle numbers, mEH mRNA level, or mEH protein level. Medium from cultures were analyzed by LC/MS for VCD-GSH adducts.DMBA was found to be a potent ovotoxicant compared to VCH/VCM/VCD. In the ovarian culture system, VCM-induced toxicity required the CYP 2E1 enzyme. However, in vivo dosing studies indicated that in the presence of hepatic metabolism the ovary plays a minimal role in VCH/VCM-induced toxicity. Studies utilizing LC/MS showed that once bioactivated to VCD, this ovotoxic metabolite can be detoxified by glutathione conjugation in the ovary. Follicle loss induced by the ovotoxicant DMBA was found to involve mEH enzyme in culture.Collectively, these studies show that the ovary has the capacity to bioactivate and detoxify ovotoxicants. In the presence of hepatic metabolism ovarian effects might play only a minimal role in the resulting toxicity. The role of ovarian metabolism in the whole animal needs to be further investigated, especially for potent toxicants such as DMBA that can induce ovotoxicity at nanomolar concentrations.

Tuberculosis (TB) is a disease that the World Health Organisation (WHO) regards as a global pandemic. There is a great need for new drugs to combat this threat. Drug resistant strains of the causative agent, Mycobacterium tuberculosis (Mtb), have increased the urgency of this quest for novel anti-mycobacterial medicines. Publication of the Mtb genome sequence revealed a large number of cytochrome P450 (CYP) enzymes [Cole, S. T. et al. 1998]. These mono-oxygenase enzymes have been studied for many years and are responsible for metabolic functions in every kingdom of life. Research on the Mtb P450s to date has highlighted several of them as having critcal roles within the organism. CYP121 and CYP128 have been implicated as essential through gene knockout studies. It has been demonstrated that CYP125 is not essential for viability. However, it is part of a gene cluster highly important for Mtb infectivity and virulence. Due to the prospective importance of P450s to Mtb, this group of enzymes is under investigation as a source of novel drug targets. CYP142 was discovered as a potential drug target after it was located to a gene cluster involved in cholesterol catabolism during Mtb dormancy. As part of this PhD project, it was demonstrated that CYP142 performs an almost identical role to that reported for CYP125. These enzymes both perform C27 hydroxylation and carboxylation of the cholesterol side chain. However, variations in the level of oxidation have been identified, dependent upon the redox system with which these P450s are associated. A crystal structure of CYP142 showing high similarity in active site architecture to CYP125 supports the physiological role of CYP142 in cholesterol catabolism. Combining this with in vitro data which demonstrates that CYP142 possesses high affinity for a range of azole anti-fungal agents [Ahmad, Z. et al. 2005, 2006] supports the suggestion that it is a candidate target for the next generation of anti-mycobacterial drugs. CYP144 was highlighted as being important during the latent phase of Mtb growth, a phase that is not targeted by any of the current antimycobacterials. Work performed as part of this PhD has shown that many characteristics of CYP144 are highly comparable to those reported for other MtbP450s. CYP144 shows high affinity and specificity towards many azole molecules. Econazole, clotrimazole and miconazole have repeatedly been shown to bind to MtbP450s, including CYP144 and CYP142, with high affinity and are excellent potential candidates as novel anti-mycobacterial agents. An N-terminally truncated form of CYP144, CYP144-T, has been investigated in the pursuit of a CYP144 crystal structure. It is hoped that this will enable the elucidation of a physiological role for CYP144. Both CYP142 and CYP144 have demonstrated biochemical and biophysical characteristics that contribute to our knowledge of P450 enzymes. This PhD has established that CYP142 exhibits an equilibrium between P450 and P420 species in its CO-bound, ferrous form. A conversion from P420, and stabilisation of P450, upon substrate binding was also demonstrated. CYP144 displays unusual azole coordination characteristics when examined by EPR and removal of the CYP144 gene from Mtb increased sensitivity of the strain to clotrimazole. Studies of these enzymes has advanced knowledge of P450 and Mtb redox chemistry, established roles for the MtbP450 cohort and identified the potential of anti-mycobacterial drugs and associated targets.

TxtE is a protein related to cytochrome P450 enzymes, which catalyze a number of reactions that typically involve oxygen and not nitrogen. It has been discovered that TxtE can nitrate tryptophan through an unusual reaction in which it uses nitric oxide (NO) as a nitrogen donor to install the nitro group despite NO typically being considered toxic to bacteria. This project will determine if all cytochromes P450 can catalyze nitration as long as they are given NO. This will have an impact on understanding drug delivery and metabolism for which nitration is important.

With an increasing population suffering from obesity or Diabetes Mellitus (DM), it is more pertinent than ever to understand how physiological changes impact cellular processes. Patients with DM often suffer from obesity, hyperglycemia, altered fatty acids that contribute to vascular dysfunction, and increased risk to ischemia. Caenorhabditis elegans is a model system used to study the conserved insulin signaling pathway, cellular responses in whole organisms and the impact a glucose diet has on oxygen deprivation (anoxia) responses. RNA-sequencing (RNA-Seq) was used to analyze the expression of genes in the anoxia sensitive populations of N2 (wild-type) fed glucose and hyl-2(tm2031), a mutant with altered ceramide metabolism. Comparison of the altered transcripts in the anoxia sensitive populations revealed 199 common transcripts- 192 upregulated and 7 downregulated. One of the gene families that have altered expression in the anoxia sensitive populations encode for Cytochrome P450 (CYP). CYPs are located both in the mitochondria and endoplasmic reticulum (ER), but the CYPs of interest are all predicted to be mainly subcellularly localized to the ER. Here, I determined that knock-down of specific cyp genes, using RNA interference (RNAi), increased anoxia survival in N2 animals fed a standard diet. Anoxia sensitivity of the hyl-2(tm2031) animals was supressed by RNAi of cyp-25A1 or cyp-33C8 genes. These studies provide evidence that the CYP detoxification system impacts oxygen deprivation responses. using hsp-4::GFP animals, a transcriptional reporter for ER unfolded protein response (UPR), I further investigated the impact of cyp knock-down, glucose, and anoxia on ER UPR due to the prediction of CYP-33C8 localization to the ER. Glucose significantly increased ER UPR and cyp knock-down non-significantly increased ER UPR. Measurements of ER UPR due to anoxia were made difficult, but representative images show an increase in ER stress post 9-hour anoxia exposure. This study provides evidence that glucose affects ER stress and that ER stress is involved in oxygen deprivation responses.

A ciclofosfamida (CPA) é um agente alquilante da classe das oxazafosforinas amplamente usada no tratamento de múltiplas formas de câncer e de doenças autoimunes em adultos e crianças. A CPA está disponível na clínica como racemato, no entanto, dados pré-clínicos demonstraram diferenças na eficácia e toxicidade dos seus enantiômeros, sendo o enantiômero (S)-(-)-CPA o de maior índice terapêutico. O presente estudo investigou a enantiosseletividade e a influência do CYP2B6, CYP2C9, CYP2C19 e CYP3A na disposição cinética da ciclofosfamida (CPA) em pacientes portadoras de câncer de mama. Foram incluídas na investigação 15 pacientes previamente submetidas ao procedimento de retirada do tumor e tratadas com CPA racêmica (900-1000 mg) e epirrubicina. A atividade in vivo do CYP3A foi avaliada empregando o midazolam como fármaco marcador. As amostras seriadas de sangue foram coletadas até 24 horas após a administração do primeiro ciclo da CPA. Os enantiômeros da CPA foram extraídos do plasma, usando mistura de acetato de etila:clorofórmio (75:25, v/v) e separados na coluna Chiralcel® OD-R com fase móvel constituída por acetonitrila:água (25:75, v/v), contendo 0,2% de ácido fórmico. Os enantiômeros da CPA foram analisados por LC-MS-MS, sendo que os íons protonados e seus respectivos íons-produtos foram monitorados nas transições 261>141 para a CPA e 189>104 para o padrão interno (antipirina). A recuperação foi maior que 95% para ambos os enantiômeros da CPA e o limite de quantificação foi de 2,5 ng/mL de plasma para cada enantiômero. Os coeficientes de variação e os erros relativos obtidos na avaliação da precisão e exatidão intra e inter-ensaios foram menores que 10%. Os parâmetros farmacocinéticos foram calculados empregando o programa WinNonlin e utilizando modelo monocompartimental e cinética de primeira ordem. Os parâmetros farmacocinéticos com razões enantioméricas diferentes da unidade foram avaliados com base no teste de Wilcoxon (P0,05). A disposição cinética da CPA é enantiosseletiva em pacientes com câncer de mama, com acúmulo plasmático do enantiômero (S)-(-)-CPA (AUC 195,00 vs 174,80 g.h/mL) em função do clearance preferencial do enantiômero (R)- (+)-CPA (5,13 vs 5,99 L/h). Os clearances de ambos os enantiômeros da ciclofosfamida não diferem em função dos genótipos CYP2B6, CYP2C9 e CYP2C19 e da atividade in vivo do CYP3A avaliada pelo clearance do midazolam. / Cyclophosphamide (CPA) is an alkylating oxazaphosphorine agent widely used in the treatment of multiple forms of cancer and autoimmunes disesases in adults and children. CPA is used as a racemic mixture, although preclinical data have demonstrated differences in the efficacy and toxicity of its enantiomers, with the (S)-(- )-CPA exhibiting a higher therapeutic index. The present study investigated the enantioselectivity and influence of CYP2B6, CYP2C9, CYP2C19 and CYP3A on the kinetic disposition of cyclophosphamide (CPA) in patients with breast cancer. Fifteen patients previously submitted to removal of the tumor and treated with racemic CPA (900-1000 mg) and epirubicin were included in the study. The in vivo activity of CYP3A was evaluated using midazolam as a marker drug. Serial blood samples were collected up to 24 h after administration of the first cycle of CPA. The CPA enantiomers were extracted from plasma using a mixture of ethyl acetate:chloroform (75:25, v/v) and separated on a Chiralcel® OD-R column, with the mobile phase consisting of acetonitrile:water (25:75, v/v) and 0.2% formic acid. The CPA enantiomers were analyzed by LC-MS-MS, and the protonated ions and their respective ion products were monitored at transitions of 261>141 for CPA and of 189>104 for the internal standard (antipyrine). Recovery was higher than 95% for both CPA enantiomers and the quantification limit was 2.5 ng/mL plasma for each enantiomer. The coefficients of variation and relative errors obtained for the evaluation of the intra- and interassay precision and accuracy were less than 10%. The pharmacokinetic parameters were calculated with the WinNonlin program using a monocompartmental model and first-order kinetics. The pharmacokinetic parameters presenting enantiomer ratios different from one were evaluated using the Wilcoxon test (P0.05). The kinetic disposition of CPA was enantioselective in patients with breast cancer, with plasma accumulation of the (S)-(-)-CPA enantiomer (AUC 195.00 vs 174.80 g.h/mL) due to the preferential clearance of the (R)-(+)-CPA enantiomer (5.13 vs 5.99 L/h). Clearances of both CPA enantiomers did not differ between the CYP2B6, CYP2C9 and CYP2C19 genotypes or as a function of in vivo activity of CYP3A evaluated by the midazolam clearance.