Investigating the contribution of CYP2J2 to ritonavir metabolism in vitro and in vivo.

Ritonavir, an HIV protease inhibitor, is successfully used for the prevention and treatment of HIV infections. Ritonavir pharmacokinetics are complicated by inhibition, induction and pharmacogenetics of cytochrome P450 (CYP) enzymes mediating its clearance. This investigation revealed that CYP2J2, along with CYP3A4/5 and CYP2D6, efficiently metabolizes ritonavir, and to a CYP2J2-specific (minor) metabolite. Chemical inhibition of ritonavir metabolism, clearance, KI/kinact and abundance of CYP2J2 in liver microsomes were evaluated and then applied to an in vitro-in vivo static scaling model to estimate the contribution of each isozyme, as a function of CYP abundance, activity, and genotype. Disposition of the CYP2J2-specific metabolite was also evaluated in vivo. In plasma, metabolite abundance was well above previously reported levels with circulating concentrations measured at 2 µM for the main hydroxylisopropyl metabolite. Ritonavir and metabolite plasma profiles were simulated using Simcyp(®). A modest (2-6%) contribution of CYP2J2 to ritonavir clearance is predicted which increases to more than 20% in subjects carrying CYP2D6 poor metabolizer polymorphisms and CYP3A4 irreversible inhibition. These results indicate that minor drug metabolizing enzymes could become quantitatively important in RTV clearance if main metabolic pathways are impeded.

Cytochrome P450BM-3 reduces aldehydes to alcohols through a direct hydride transfer.

Cytochrome P450BM-3 catalyzed the reduction of lipophilic aldehydes to alcohols efficiently. A k(cat) of ∼25 min(-1) was obtained for the reduction of methoxy benzaldehyde with wild type P450BM-3 protein which was higher than in the isolated reductase domain (BMR) alone and increased in specific P450-domain variants. The reduction was caused by a direct hydride transfer from preferentially R-NADP(2)H to the carbonyl moiety of the substrate. Weak substrate-P450-binding of the aldehyde, turnover with the reductase domain alone, a deuterium incorporation in the product from NADP(2)H but not D(2)O, and no inhibition by imidazole suggests the reductase domain of P450BM-3 as the potential catalytic site. However, increased aldehyde reduction by P450 domain variants (P450BM-3 F87A T268A) may involve allosteric or redox mechanistic interactions between heme and reductase domains. This is a novel reduction of aldehydes by P450BM-3 involving a direct hydride transfer and could have implications for the metabolism of endogenous substrates or xenobiotics.

Cerivastatin in vitro metabolism by CYP2C8 variants found in patients experiencing rhabdomyolysis.

OBJECTIVES: Cerivastatin, a 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor withdrawn from the market because of serious adverse effects, is metabolized primarily by CYP2C8. The occurrence of associated myotoxicity and rhabdomyolysis were attributed to altered cerivastatin pharmacokinetics on account of gemfibrozil-inhibition or genetic variations in CYP2C8 and drug transporters involved in cerivastatin clearance. However, the effect of CYP2C8 genetic variation on cerivastatin metabolism has not been fully elucidated. METHODS: In this study, patients (n=126) with confirmed cases of rhabdomyolysis after cerivastatin administration had their CYP2C8 gene resequenced and the metabolism of cerivastatin by the discovered CYP2C8 variants was assessed in proteins expressed in Escherichia coli. RESULTS: In this unique patient population, 12 novel single nucleotide polymorphisms were discovered of which six were exclusively found in patients not using gemfibrozil. Three rare exonic variants resulted in amino acid substitutions and a frame shift deletion (V472fsL494 generating a defective mostly heme-free CYP2C8 protein). A particular promoter located deletion (-635_-634delTA) was tightly linked to CYP2C8*3. Heterologously expressed CYP2C8.3 and CYP2C8.4 displayed an increase in cerivastatin metabolic clearance of up to six-fold compared with the wild-type enzyme. Similarly, an independent sample of microsomes from human livers carrying the CYP2C8*3 and CYP2C8*4 alleles exhibited a 2-fold to 14-fold increase in normalized cerivastatin intrinsic clearance, compared with microsomes from livers carrying only the wild type allele. CONCLUSION: Gain or loss of catalytic function found in the CYP2C8 gene could certainly alter cerivastatin pharmacokinetics and may influence, at least in part, susceptibility to the development of myotoxicity.