Cytochrome P450 3A-Dependent Metabolism of a Potent and Selective γ-Aminobutyric Acid_Aα2/3 Receptor Agonist in Vitro: Involvement of Cytochrome P450 3A5 Displaying Biphasic Kinetics

Abstract

ABSTRACT:In vitro metabolism studies were conducted to determine the human cytochrome P450 enzyme(s) involved in the biotransformation of 7-(1,1-dimethylethyl)-6-(2-ethyl-2H-1,2,4-triazol-3-ylmethoxy)-3-(2-fluorophenyl)-1,2,4-triazolo[4,3b]pyridazine (TPA023), a selective agonist of human ␥-aminobutyric acid A receptor ␣2 and ␣3 subunits. Incubation of TPA023 with NADPH-fortified human liver microsomes resulted in the formation of t-butyl hydroxy TPA023, N-desethyl TPA023, and three minor metabolites. Both t-butyl hydroxylation and N-deethylation reactions were greatly inhibited (>85%) in the presence of CYP3A-selective inhibitory antibodies and chemical inhibitors, indicating that members of the CYP3A subfamily play an important role in TPA023 metabolism. EadieHofstee plots of t-butyl hydroxylation and N-deethylation in pooled CYP3A5-rich human liver microsomes revealed a low K m (3.4 and 4.5 M, respectively) and a high K m (12.7 and 40.0 M, respectively) component. For both metabolites, the high K m component was not observed with a pool of microsomal preparations containing minimal levels of CYP3A5. Preincubation of liver microsomes with mifepristone (selectivity for CYP3A4 > CYP3A5) greatly inhibited both t-butyl hydroxylation and N-deethylation (>75%); however, the residual activities were significantly higher in the pooled CYP3A5-rich liver microsomes (p < 0.0005). In addition, elevated levels of residual t-butyl hydroxylase and N-deethylase activities were observed in the presence of both CYP3A5-rich and CYP3A5-deficient preparations when the substrate concentration increased from 4 to 40 M. In agreement, metabolite formation catalyzed by recombinant CYP3A5 was described by a biphasic model. It is concluded that CYP3A4 plays a major role in TPA023 metabolism, and CYP3A5 may also contribute at higher concentrations of the compound.

GABAA Receptor Subtype‐Selective Efficacy: TPA023, an α2/α3 Selective Non‐sedating Anxiolytic and α5IA, an α5 Selective Cognition Enhancer

“…3). Both the t ‐butyl hydroxylation and the N ‐deethylation reactions in human liver microsomes were greatly inhibited by CYP3A‐specific antibodies and inhibitors, with CYP3A4 rather than CYP3A5 playing the major role in TPA023 metabolism (Ma et al 2007). The M1 and M2 metabolites of TPA023 were also found in vivo in rat as well as human but are not considered to play a pharmacological role based on their poor blood‐brain barrier penetration as well as, in the case of the M2 metabolite, lower affinity for GABA A receptors than the parent.…”

“… The major metabolic pathways of TPA023 and α5IA. Metabolism of TPA023 proceeds via the predominantly CYP3A4‐mediated t ‐butyl hydroxylation and N ‐deethylation (Ma et al 2007) whereas the single major metabolite of α5IA is the hydroxymethyl isoxazole. …”

Comparison of the Contributions of Cytochromes P450 3A4 and 3A5 in Drug Oxidation Rates and Substrate Inhibition

“…20) In addition, it has been reported that whether cytochrome b 5 is added by supplementation or coexpression also affects the metabolic activity. Ma et al 53) reported that the t-butyl hydroxylation and N-demethylation activities of TPA023 obtained by cytochrome b 5 -coexpressed CYP3A5 are higher than those of cytochrome b 5 -supplemented CYP3A5. Similarly, CYP3A4 coexpressed with cytochrome b 5 results in higher catalytic activity toward ifosfamide Ndechloroethylation than that of cytochrome b 5 -supplemented CYP3A4; this effect was greater than that for CYP3A5.…”

“…In contrast, most of the V max and V max /K m values for dehydrogenation of many of the dihydropyridine calcium antagonists, such as benidipine, felodipine, and nifedipine, and steroid hydroxylation, such as testosterone 6β-hydroxylation and estradiol 2-or 4-hydroxylation, mediated by CYP3A5, are lower than those mediated by CYP3A4, whereas the K m values for CYP3A5 are higher than those for CYP3A4. 20,[27][28][29]41) Similarly, the CYP3A5- 53) Bold: The ratio of the parameters for CYP3A5 and CYP3A4 was less than 0.5 times or more than 2 times. a) Benidipine, felodipine, nifedipine, and nitrendipine.…”

Preliminary Investigation of the Contribution of CYP2A6, CYP2B6, and UGT1A9 Polymorphisms on Artesunate-Mefloquine Treatment Response in Burmese Patients with Plasmodium falciparum Malaria

“…CYP2B6 functional polymorphisms are highly prevalent in several African [32][33][34][35] and Asian countries. 35,36 In most of these countries, artemisinin-based combination therapy has been used as first-line treatment for acute uncomplicated P. falciparum malaria. The CYP2B6*9 allele is commonly found in all ethnic groups, but more frequently in Hispanics compared with African-Americans, Caucasians, and Asians.…”