Validation of an HPLC Method for the Simultaneous Quantification of Metabolic Reaction Products Catalysed by CYP2C11 Enzymes in Rat Liver Microsomes: In Vitro Inhibitory Effect of Salicylic Acid on CYP2C11 Enzyme.

The inhibitory effect of new chemical entities on rat liver P450 marker activities was investigated in a functional approach towards drug development. Treatment of colorectal cancer (CRC) and chemoprevention using salicylic acid has gained a lot of attention, mainly in the prevention of the onset of colon cancer. Thus, an in vitro inhibitory effect of salicylic acid on rat CYP2C11 activity was examined by using high performance liquid chromatography (HPLC). High performance liquid chromatography analysis of a CYP2C11 assay was developed on a reversed phase C18 column (SUPELCO 25 cm × 4.6 mm × 5 µm) at 243 nm using 32% phosphate buffer (pH 3.36) and 68% methanol as a mobile phase. The CYP2C11 assay showed good linearity for all components (R2 > 0.999). Substrates and metabolites were found to be stable for up to 72 hours. Additionally, the method demonstrated good reproducibility, intra- and inter-day precision (<15%), acceptable recovery and accuracy (80%-120%), and low detection (1.3501 µM and 3.2757 µM) and quantitation limit values (4.914 µM and 9.927 µM) for 16α-hydroxytestosterone and testosterone, respectively. Salicylic acid acts reversibly as a noncompetitive (weak) inhibitor with Ki = 84.582 ± 2.67 µM (concentration of inhibitor to cause 50% inhibition of original enzyme activity (IC50) = 82.70 ± 2.67 µM) for CYP2C11 enzyme activity. This indicates a low potential to cause toxicity and drug-drug interactions.

Structural and energetic analysis to provide insight residues of CYP2C9, 2C11 and 2E1 involved in valproic acid dehydrogenation selectivity.

Docking and molecular dynamics (MD) simulation have been two computational techniques used to gain insight about the substrate orientation within protein active sites, allowing to identify potential residues involved in the binding and catalytic mechanisms. In this study, both methods were combined to predict the regioselectivity in the binding mode of valproic acid (VPA) on three cytochrome P-450 (CYP) isoforms CYP2C9, CYP2C11, and CYP2E1, which are involved in the biotransformation of VPA yielding reactive hepatotoxic intermediate 2-n-propyl-4-pentenoic acid (4nVPA). There are experimental data about hydrogen atom abstraction of the C4-position of VPA to yield 4nVPA, however, there are not structural evidence about the binding mode of VPA and 4nVPA on CYPs. Therefore, the complexes between these CYP isoforms and VPA or 4nVPA were studied to explore their differences in binding and energetic stabilization. Docking results showed that VPA and 4nVPA are coupled into CYPs binding site in a similar conformation, but it does not explain the VPA hydrogen atom abstraction. On the other hand, MD simulations showed a set of energetic states that reorient VPA at the first ns, then making it susceptible to a dehydrogenation reaction. For 4nVPA, multiple binding modes were observed in which the different states could favor either undergo other reaction mechanism or ligand expulsion from the binding site. Otherwise, the energetic and entropic contribution point out a similar behavior for the three CYP complexes, showing as expected a more energetically favorable binding free energy for the complexes between CYPs and VPA than with 4nVPA.

Bioactivation of nevirapine to a reactive quinone methide: implications for liver injury.

Nevirapine (NVP) treatment is associated with a significant incidence of liver injury. We developed an anti-NVP antiserum to determine the presence and pattern of covalent binding of NVP to mouse, rat, and human hepatic tissues. Covalent binding to hepatic microsomes from male C57BL/6 mice and male Brown Norway rats was detected on Western blots; the major protein had a mass of ~55 kDa. Incubation of NVP with rat CYP3A1 and 2C11 or human CYP3A4 also led to covalent binding. Treatment of female Brown Norway rats or C57BL/6 mice with NVP led to extensive covalent binding to a wide range of proteins. Co-treatment with 1-aminobenzotriazole dramatically changed the pattern of binding. The covalent binding of 12-hydroxy-NVP, the pathway that leads to a skin rash, was much less than that of NVP, both in vitro and in vivo. An analogue of NVP in which the methyl hydrogens were replaced by deuterium also produced less covalent binding than NVP. These data provide strong evidence that covalent binding of NVP in the liver is due to a quinone methide formed by oxidation of the methyl group. Attempts were made to develop an animal model of NVP-induced liver injury in mice. There was a small increase in ALT in some NVP-treated male C57BL/6 mice at 3 weeks that resolved despite continued treatment. Male Cbl-b(-/-) mice dosed with NVP had an increase in ALT of >200 U/L, which also resolved despite continued treatment. Liver histology in these animals showed focal areas of complete necrosis, while most of the liver appeared normal. This is a different pattern from the histology of NVP-induced liver injury in humans. This is the first study to report hepatic covalent binding of NVP and also liver injury in mice. It is likely that the quinone methide metabolite is responsible for NVP-induced liver injury.

CYP2C76 non-synonymous variants in cynomolgus and rhesus macaques.

Cynomolgus CYP2C76, not orthologous to any human cytochrome P450, partly accounts for species differences in drug metabolism between cynomolgus macaques and humans. To discover the CYP2C76 variants, we previously surveyed cynomolgus macaque genomes and found several non-synonymous variants, including a null allele. However, the analysis was limited to cynomolgus macaques, and the number of genomes was relatively small. In this study, therefore, further screening was conducted using 74 cynomolgus and 30 rhesus macaques. A total of 18 non-synonymous variants was found, among which 7 were in substrate recognition sites, important for protein function, and 14 (74%) were shared by both macaque lineages. In cynomolgus macaques, 3 (16%) non-synonymous variants were unique to Indochinese animals, whereas all the variants found in Indonesian animals were shared by Indochinese animals. Among the 18 variants, as compared with the wild type, in progesterone 16α-hydroxylation, L65F, M310L, and N364S variants showed lower metabolic activity and lower intrinsic clearance by kinetic analysis. Molecular modeling indicated that the reduced catalytic activity of the L65F variant in progesterone 16α-hydroxylation possibly resulted from a longer distance of progesterone to the heme in the active site of the CYP2C76 protein. L65F, M310L, and N364S variants might partly influence inter-animal variations of CYP2C76 metabolic activities.

Evaluation of the binding orientations of testosterone in the active site of homology models for CYP2C11 and CYP2C13.

Cytochromes P-450 2C11 and 2C13 are the major CYPs in rat liver microsomes. Despite a high degree of sequence identity, these two isozymes display different positional and regio-specific metabolism of steroid hormones, such as testosterone. CYP2C11 converts testosterone to 2alpha-hydroxyl and 16alpha-hydroxyl metabolites, while CYP2C13 produces primarily the 6beta-hydroxyl metabolite. Using a human CYP2C9 crystal structure as the template, homology models were generated for CYP2C11 and CYP2C13. Despite similar volume of the binding pockets for CYP2C11 and CYP2C13, the models for these two CYPs showed a substantial difference in the shape of the substrate-binding sites. Substrate docking using rigid and induced-fit methods showed that testosterone fits into the substrate-binding sites of both CYP2C11 and CYP2C13 without the need of added constraints. These docking exercises appear to support testosterone binding in both CYP2C11 and CYP2C13. A constrained docking using energy minimization is required to position testosterone for more precise positional and regio-specificity in supporting the observed metabolism. These results demonstrate the complexity of using modeling for understanding the binding of substrate to CYPs, and suggest that, as a complement to the metabolism data, modeling and docking may yield reliable structural information for the molecular interaction between the substrate and the CYPs.