Aminobenzotriazole inhibits and induces several key drug metabolizing enzymes complicating its utility as a pan CYP inhibitor for reaction phenotyping

Abstract

Aminobenzotriazole (ABT) is commonly used as a non-selective inhibitor of cytochrome P450 (CYP) enzymes to assign contributions of CYP versus non-CYP pathways to the metabolism of new chemical entities. Despite widespread use, a systematic review of the drug-drug interaction (DDI) potential for ABT has not been published nor have the implications for using it in plated hepatocyte models for low clearance reaction phenotyping. The goal being to investigate the utility of ABT as a pan-CYP inhibitor for reaction phenotyping of low clearance compounds by evaluating stability over the incubation period, inhibition potential against UGT and sulfotransferase enzymes, and interaction with nuclear receptors involved in the regulation of drug metabolizing enzymes and transporters. Induction potential for additional inhibitors used to ascribe fraction metabolism (f_m ), pathway including erythromycin, ketoconazole, azamulin, atipamezole, ZY12201, and quinidine was also investigated. ABT significantly inhibited the clearance of a non-selective UGT substrate 4-methylumbelliferone, with several UGTs shown to be inhibited using selective probe substrates in human hepatocytes and rUGTs. The inhibitors screened in the induction assay were shown to induce enzymes regulated through Aryl Hydrocarbon Receptor, Constitutive Androstane Receptor, and Pregnane X Receptor. Lastly, a case study identifying the mechanisms of a clinical DDI between Palbociclib and ARV-471 is provided as an example of the potential consequences of using ABT to derive f_m . This work demonstrates that ABT is not an ideal pan-CYP inhibitor for reaction phenotyping of low clearance compounds and establishes a workflow that can be used to enable robust characterization of other prospective inhibitors.

FIGURE 1

Evaluation of mRNA level changes for UGT1A1 (pink squares), CYP1A2 (teal triangles), CYP2B6 (black circles), CYP2C8 (pink triangles), CYP2C9 (teal upside down triangles), or CYP3A4 (black squares), UGT1A6 (data not shown) following administration of ABT, erythromycin, azamulin, or rifampin (a, b, c, d, respectively) to Hμrel co‐culture model or ABT, ketoconazole, atipamezole, quinidine, or ZY12201 to sandwich cultured hepatocytes (e, f, g, h, i, respectively). Note studies conducted with Hμrel (blue background) looked at all mRNA end points, whereas studies conducted in sandwich cultured hepatocytes (yellow background) focused on sensitive markers of AhR, CAR, and PXR activation (CYP1A2, CYP2B6, and CYP3A4, respectively). Each data point is representative of two with error, dashed line represents twofold change from vehicle control. Induction potential for ABT and other commonly used inhibitors in the Hμrel co‐culture model (a, b, c, d) or sandwich cultured human hepatocytes (e, f, g, i) Dashed line represents twofold increase in mRNA levels.

FIGURE 2

Impact of ARV‐471 on palbociclib intrinsic clearance (a) mean of two, derivation of ARV‐471 inhibition parameters (b) mean of two. Impact of ARV‐471 on palbociclib intrinsic clearance (a), derivation of ARV‐471 inhibition parameters (b).

FIGURE 3

Impact of ARV‐471 on DHEAS formation in recombinant SULT2A1 mean of three (a), Hμrel (b) mean of three, and the formation of palbociclib sulfate in Hμrel (c) mean of two. Impact of ARV‐471 on DHEAS formation in recombinant SULT2A1 (a), Hμrel (b), and the formation of palbociclib sulfate in Hμrel (c).

FIGURE 4

Impact of ARV‐471 on CYP3A activity as measured by 1'Ohmidazolam, direct inhibition (a) mean of three, time‐dependent inhibition (b) mean of three. The 0 h, 120 h, and 168 h refer to the time of exposure to the test article prior to determining the enzyme activity Impact of ARV‐471 on CYP3A activity as measured by 1'Ohmidazolam, direct inhibition (a), time‐dependent inhibition (b).

FIGURE 5

Metabolic scheme for palbociclib in co‐culture human hepatocyte models (H = Hμrel, S = stromal cells in H, TV = TruVivo, F = feeder cells in TV) pooled samples from three replicates, over a timecourse on two separate occasions.