Azamulin

Early determination of CYP3A4/5 contribution to the clearance of new chemical entities is critical to inform on the risk of drug-drug interactions with CYP3A inhibitors and inducers. Several in vitro approaches (recombinant P450 enzymes, correlation analysis, chemical and antibody inhibition in human liver microsomes) are available, but they are usually labor-intensive and/or suffer from specific limitations. In the present study, we have validated the use of azamulin as a specific CYP3A inhibitor in human hepatocytes. Azamulin (3 µM) was found to significantly inhibit CYP3A4/5 (>90%), whereas other P450 enzymes were not affected (less than 20% inhibition). Because human hepatocytes were used as a test system, the effect of azamulin on other key drug-metabolizing enzymes (aldehyde oxidase, carboxylesterase, UGT, flavin monooxygenase, and sulfotransferase) was also investigated. Apart from some UGTs showing minor inhibition (~20%-30%), none of these non-P450 enzymes were inhibited by azamulin. Use of CYP3A5-genotyped human hepatocyte batches in combination with CYP3cide demonstrated that azamulin (at 3 µM) inhibits both CYP3A4 and CYP3A5 enzymes. Finally, 11 compounds with known in vivo CYP3A4/5 contribution have been evaluated in this human hepatocyte assay. Results showed that the effect of azamulin on the in vitro intrinsic clearance of these known CYP3A4/5 substrates was predictive of the in vivo CYP3A4/5 contribution. Overall, the study showed that human hepatocytes treated with azamulin provide a fast and accurate estimation of CYP3A4/5 contribution in metabolic clearance of new chemical entities. SIGNIFICANCE STATEMENT: Accurate estimation of CYP3A4/5 contribution in drug clearance is essential to anticipate risk of drug-drug interactions and select the appropriate candidate for clinical development. The present study validated the use of azamulin as selective CYP3A4/5 inhibitor in suspended human hepatocytes and demonstrated that this novel approach provides a direct and accurate determination of the contribution of CYP3A4/5 (fraction metabolized by CYP3A4/5) in the metabolic clearance of new chemical entities.

Recognizing the challenges of determining the relative contribution of different drug metabolizing enzymes to the metabolism of slowly metabolized compounds, a cytochrome P450 reaction phenotyping (CRP) method using cocultured human hepatocytes (HEPATOPAC) has been established. In this study, the emphasis on the relative contribution of different cytochrome P450 (P450) isoforms was assessed by persistently inhibiting P450 isoforms over 7 days with human HEPATOPAC. P450 isoform-selective inhibition was achieved with the chemical inhibitors furafylline (CYP1A2), tienilic acid (CYP2C9), (+)-N-3-benzylnirvanol (CYP2C19), paroxetine (CYP2D6), azamulin (CYP3A), and a combination of 1-aminobenzotriazole and tienilic acid (broad spectrum inhibition of P450s). We executed this CRP method using HEPATOPAC by optimizing for the choice of P450 inhibitors, their selectivity, and the temporal effect of inhibitor concentrations on maintaining selectivity of inhibition. In general, the established CRP method using potent and selective chemical inhibitors allows to measure the relative contribution of P450s and to calculate the fraction of metabolism (f m) of low-turnover compounds. Several low-turnover compounds were used to validate this CRP method by determining their hepatic intrinsic clearance and f m, with comparison with literature values. We established the foundation of a robust CRP for low-turnover compound test system which can be expanded to include inhibition of other drug metabolizing enzymes. This generic CRP assay, using human long-term hepatocyte cultures, will be an essential tool in drug development for new chemical entities in the quantitative assessment of the risk as a victim of drug-drug interactions. SIGNIFICANCE STATEMENT: An ongoing trend is to develop drug candidates which have limited metabolic clearance. The current studies report a generic approach to conducting reaction phenotyping studies with human HEPATOPAC, focusing on P450 metabolism of low-turnover compounds. Potent and selective chemical inhibitors were used to assess the relative contribution of the major human P450s. Validation was achieved by confirming hepatic intrinsic clearance and fraction of metabolism for previously reported low-turnover compounds. This approach is adaptable for assessment of all drug metabolizing enzymes.

Cytochrome P450 3A4 and 3A5 catalyze the metabolic clearance of a large portion of therapeutic drugs. Azamulin is used as a selective inhibitor for 3A4 and 3A5 to define their roles in metabolism of new chemical entities during drug development. In contrast to 3A4, 3A5 exhibits homotropic cooperativity for the sequential binding of two azamulin molecules at concentrations used for inhibition. To define the underlying sites and mechanisms for cooperativity, an X-ray crystal structure of 3A5 was determined with two azamulin molecules in the active site that are stacked in an antiparallel orientation. One azamulin resides proximal to the heme in a pose similar to the 3A4-azamulin complex. Comparison to the 3A5 apo structure indicates that the distal azamulin in 3A5 ternary complex causes a significant induced fit that excludes water from the hydrophobic surfaces of binding cavity and the distal azamulin, which is augmented by the stacking interaction with the proximal azamulin. Homotropic cooperativity was not observed for the binding of related pleuromutilin antibiotics, tiamulin, retapamulin, and lefamulin, to 3A5, which are larger and unlikely to bind in the distal site in a stacked orientation. Formation of the 3A5 complex with two azamulin molecules may prevent time-dependent inhibition that is seen for 3A4 by restricting alternate product formation and/or access of reactive intermediates to vulnerable protein sites. These results also contribute to a better understanding of sites for cooperative binding and the differential structural plasticity of 3A5 and 3A4 that contribute to differential substrate and inhibitor binding.