Influence of CYP3A5 genotype on the pharmacokinetics and pharmacodynamics of the cytochrome P4503A probes alfentanil and midazolam.

The hepatic and first-pass cytochrome P4503A (CYP3A) probe alfentanil (ALF) is also metabolized in vitro by CYP3A5. Human hepatic microsomal ALF metabolism is higher in livers with at least one CYP3A5*1 allele and higher CYP3A5 protein content, compared with CYP3A5*3 homozygotes with little CYP3A5. The influence of CYP3A5 genotype on ALF pharmacokinetics and pharmacodynamics was studied, and compared to midazolam (MDZ), another CYP3A probe. Healthy volunteers (58 men, 41 women) were genotyped for CYP3A5 *1, *3, *6, and *7 alleles. They received intravenous MDZ then ALF, and oral MDZ and ALF the next day. Plasma MDZ and ALF concentrations were determined by mass spectrometry. Dark-adapted pupil diameters were determined coincident with blood sampling. In CYP3A5(*)3/(*)3 (n=62), (*)1/(*)3 (n=28), and (*)1/(*)1 (n=8) genotypes, systemic clearances of ALF were 4.6+/-1.8, 4.8+/-1.7, and 3.9+/-1.7 ml/kg/min and those of MDZ were 7.8+/-2.3, 7.7+/-2.3, and 6.0+/-1.4 ml/kg/min, respectively (not significant), and apparent oral clearances were 11.8+/-7.2, 13.3+/-6.1, and 12.6+/-8.2 ml/kg/min for ALF and 35.2+/-19.0, 36.4+/-15.7, and 29.4+/-9.3 ml/kg/min for MDZ (not significant). Clearances were not different between African Americans (n=25) and Whites (n=68), or between CYP3A5 genotypes within African Americans. ALF pharmacodynamics was not different between CYP3A5 genotypes. There was consistent concordance between ALF and MDZ, in clearances and extraction ratios. Thus, in a relatively large cohort of healthy subjects with constitutive CYP3A activity, CYP3A5 genotype had no effect on the systemic or apparent oral clearances, or pharmacodynamics, of the CYP3A probes ALF and MDZ, despite affecting their hepatic microsomal metabolism.

Pharmacokinetics and metabolism of the novel anticonvulsant agent N-(2,6-dimethylphenyl)-5-methyl-3-isoxazolecarboxamide (D2624) in rats and humans.

N-(2,6-dimethylphenyl)-5-methyl-3-isoxazolecarboxamide (D2624) belongs to a new series of experimental anticonvulsants related to lidocaine. This study was undertaken to understand the pharmacokinetics and metabolism of D2624 in rats and humans, with emphasis on the possible formation of 2,6-dimethylaniline (2,6-DMA). After oral administration of stable isotope-labeled parent drug to rats and GC/MS analysis of plasma samples, two metabolites were identified: D3017, which is the primary alcohol, and 2,6-DMA, formed by amide bond hydrolysis of either D2624 or D3017. In urine, three metabolites of D2624 were identified: namely D3017,2,6-DMA, and D3270 (which is the carboxylic acid derivative of D3017). Based on plasma AUC analysis, D3017 and 2,6-DMA accounted for > 90% of the dose of D2624. After oral administration, D2624 was found to be well absorbed (93%), but underwent extensive first-pass metabolism in the rat, thus resulting in 5.3% bioavailability. Rat and human liver microsomal preparations were capable of metabolizing D2624 to D3017 and 2,6-DMA. The formation of D3017 was NADPH-dependent, whereas 2,6-DMA formation was NADPH-independent and probably was catalyzed by amidase(s) enzymes. In a single-dose (25-225 mg) human volunteer study, the parent drug (D2624) was not detected in plasma at any dose, whereas 2,6-DMA was detected only at the two highest doses (150 and 225 mg). D3017 was detected after all doses of parent drug, with approximate dose proportionality in AUC and a half-life of 1.3-2.2 hr. The metabolic behavior observed in humans suggests there is a marked species difference in the oxidative and hydrolytic pathways of D2624.

CYP4 isozyme specificity and the relationship between omega-hydroxylation and terminal desaturation of valproic acid.

The cytochrome P450-dependent terminal desaturation of valproic acid (VPA) is of both toxicological and mechanistic interest because the product, 4-ene-VPA, is a more potent hepatotoxin than the parent compound and its generation represents a rather novel metabolic reaction for the cytochrome P450 system. In the present study, lung microsomes from rabbits were identified as a rich source of VPA desaturase activity. Monospecific polyclonal antibodies directed against CYP4B1 (anti-4B) inhibited 82% of 4-ene-VPA formation, whereas monospecific polyclonal antibodies directed against CYP2B4 (anti-2B) inhibited only 15% of 4-ene-VPA formation. Anti-4B also inhibited 95% of the 5-hydroxy-VPA formation, but only 42% of 4-hydroxy-VPA formation. These data suggest that CYP4B1 accounts for more than 80% of the 4-ene- and 5-hydroxy-VPA metabolites generated by rabbit lung microsomes. CYP4B1 expressed in HepG2 cells metabolized VPA with a turnover number of 35 min-1 and formed the 5-hydroxy-, 4-hydroxy-, and 4-ene-VPA metabolites in a ratio of 110:2:1, respectively. In contrast, the lauric acid omega-hydroxylases, CYP4A1 and CYP4A3, did not give rise to detectable levels of any of these VPA metabolites. Therefore, these studies demonstrate a new functional role for CYP4B1 in the terminal desaturation and omega-hydroxylation of this short, branched-chain fatty acid.(ABSTRACT TRUNCATED AT 250 WORDS)

Deuterium isotope effect on the toxicokinetics of monomethylamine in the rat.

The single-dose toxicokinetics of monomethylamine has been characterized in the rat by HPLC assay of serial blood samples. Biphasic first-order elimination was observed following an iv bolus dose of 19 mumol/kg with a terminal half-life of 19.1 +/- 1.3 min (mean +/- SE, N = 4). The apparent steady state volume of distribution, systemic blood clearance, and renal blood clearance were 1.21 +/- 0.09 liter/kg, 53.4 +/- 3.5 ml/min/kg, and 5.72 +/- 0.53 ml/min/kg, respectively. The administration of an intragastric dose permitted the calculation of the systemic bioavailability of monomethylamine as 69 +/- 3%. Duplicate experiments using the structural analogue with deuterium atoms substituted for hydrogens on the methyl group revealed a much slower elimination of the compound, although ultimately, 5 times as much was excreted unchanged in the urine. Isotope effects calculated as the ratios of terminal half-life, systemic blood clearance, and systemic bioavailability were 1.9, 2.2, and 1.8, respectively.

Metabolic denitrosation of N-nitrosodimethylamine in vivo in the rat.

Enzymatic denitrosation is a potentially inactivating metabolic route that has been shown to convert carcinogenic N-nitrosodimethylamine (NDMA) to methylamine (MA) in vitro. To investigate its quantitative course in vivo, groups of 8-week-old male Fischer rats have been given small (8-15 mumol/kg) p.o. or i.v. bolus doses of 14C-labeled NDMA and the subsequent formation of radioactive MA has been monitored by high performance liquid chromatographic analysis of serially collected blood samples from each individual. Adjusting the [14C]MA fluxes observed for the previously measured rates at which MA is itself eliminated from the system after intragastric administration, denitrosation was calculated to represent a rather uniform 21.3 +/- 1.3% (SE) of total NDMA elimination in the four animals studied. By contrast, repetition of the experiment with fully deuterated NDMA (NDMA-d6) revealed a significantly wider variance in the results (39.8 +/- 8.9%). An alternative calculation using values for elimination of i.v. doses of MA and its trideuteromethyl analogue gave an even larger difference for MA formation between NDMA and NDMA-d6, the estimated extents of in vivo denitrosation in this case being 14.5 +/- 0.9% and 48.3 +/- 10.8%, respectively. The results indicate that denitrosation is a major metabolic pathway for NDMA elimination and suggest that deuteration of the carcinogen induces a shift in its metabolism toward increasing denitrosation at the expense of the competing activation pathway. Consequently, denitrosation may be the previously undefined in vivo metabolic route, the existence of which was suggested by the findings that deuteration of NDMA lowered its hepatocarcinogenicity and liver DNA alkylating ability in rats.

Decomposition and quality control considerations in biological work with fecapentaene preparations.

Solutions of synthetic fecapentaene 12 (FP-12) intended for carcinogenicity studies were found to decompose extremely rapidly during customary dosage procedures. Apparent half-lives as short as 15 min were observed. While rates and even the qualitative course of decomposition were surprisingly variable in replicate experiments, high concentration and exposure to air were confirmed to be especially important destabilizing influences. The results suggested a primary role for a radical decomposition mechanism in the presence of atmospheric oxygen. Consistent with this hypothesis, FP-12 solutions were significantly stabilized by the radical chain-breaking antioxidant vitamin E. On the other hand, dithiothreitol greatly destabilized FP-12, presumably because of its nucleophilicity. The diacetyl diester of FP-12 was more soluble than the parent diol, but its decomposition rates in the presence and absence of vitamin E were similar to those of unesterified FP-12. Ultraviolet irradiation of an all-trans-FP-12 solution decreased its concentration by 70% in 0.5 min. The mutagenicities of the decomposition/isomerization products of FP-12, as studied in Salmonella typhimurium tester strain TA 100, ranged from negligible to comparable with all-trans-FP-12 itself. It is concluded that unchecked decomposition of fecapentaene preparations can profoundly affect biological tests therewith. While this can be largely controlled through the use of rigorous precautions, including protection from air, light, nucleophiles, and acids as well as selection of the lowest concentration compatible with the application at hand, the data argue strongly for inclusion of appropriate quality control measures in all future dosing operations to prove that the biological activity reported is that of the fecapentaene itself rather than that of a decomposed dosing solution.