Complete Substrate Inhibition of Cytochrome P450 2C8 by AZD9496, an Oral Selective Estrogen Receptor Degrader

Abstract

AZD9496 ((E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid) is an oral selective estrogen receptor degrader currently in clinical development for treatment of estrogen receptor-positive breast cancer. In a first-in-human phase 1 study, AZD9496 exhibited dose nonlinear pharmacokinetics, the mechanistic basis of which was investigated in this study. The metabolism kinetics of AZD9496 were studied using human liver microsomes (HLMs), recombinant cytochrome P450s (rP450s), and hepatocytes. In addition, modeling approaches were used to gain further mechanistic insights. CYP2C8 was predominantly responsible for biotransformation of AZD9496 to its two main metabolites whose rate of formation with increasing AZD9496 concentrations exhibited complete substrate inhibition in HLM, rCYP2C8, and hepatocytes. Total inhibition by AZD9496 of amodiaquine -deethylation, a specific probe of CYP2C8 activity, confirmed the completeness of this inhibition. The commonly used substrate inhibition model analogous to uncompetitive inhibition fit poorly to the data. However, using the same model but without constraints on the number of molecules occupying the inhibitory binding site (i.e.,SES) provided a significantly better fit (F test, 0.005). With the improved model, up to three AZD9496 molecules were predicted to bind the inhibitory site of CYP2C8. In contrast to previous studies showing substrate inhibition of P450s to be partial, our results demonstrate complete substrate inhibition of CYP2C8 via binding of more than one molecule of AZD9496 to the inhibitory site. As CYP2C8 appears to be the sole isoform catalyzing formation of the main metabolites, the substrate inhibition might explain the observed dose nonlinearity in the clinic at higher doses.

Efavirenz Metabolism: Influence of Polymorphic CYP2B6 Variants and Stereochemistry

“…For R-efavirenz hydroxylation, only CYP2B6.4 showed substrate inhibition. Several models were evaluated for fitting positive cooperativity and substrate inhibition, including (i) a combination of the Hill equation with substrate inhibition (Müller et al, 2015), (ii) homotropic cooperativity with complete substrate inhibition and a single substrate molecule binding to an inhibitory site (a simplified version of the LiCata model, below) (Kapelyukh et al, 2008), (iii) cooperative catalysis and substrate inhibition (Pastra-Landis et al, 1978), (iv) substrate inhibition analogous to uncompetitive inhibition (Michaelis-Menten plus substrate inhibition) with facilitated sequential binding of two additional substrate (inhibitor) molecules (Bapiro et al, 2018), (v) uncompetitive inhibition (Michaelis-Menten plus substrate inhibition) assuming simultaneous binding of n molecules (determined from the data) of the substrate to the inhibitory site (Bapiro et al, 2018), and (vi) a modified Hill equation with cooperative substrate binding, substrate inhibition, and cooperative inhibitor binding (eq. 2) (LiCata and Allewell, 1997).…”

“…reported with P450s (Denisov et al, 2009), multiple inhibitor binding (Bapiro et al, 2018) and concomitant catalytic and inhibitory cooperativity are relatively uncommon (Müller et al, 2015). Comprehensive modeling of both catalytic and inhibitory cooperativity resulted in many parameters relative to the number of experimental observations, with a concern for an over-parameterized model.…”

In vitro and in vivo methods to assess pharmacokinetic drug– drug interactions in drug discovery and development

“…When the binding site activity increases, positive cooperativity has happened, whereas when the binding site activity decreases, negative cooperativity has occurred. In vitro enzyme allosteric effects on CYP3A and CYP2C have been reported (Bapiro et al, ; Galetin, Clarke, & Houston, ; Shou et al, ). However, literature information for in vivo or clinical activation of CYP enzymes is scarce (Blobaum et al, ; Mikus et al, ; Yang, Atkins, Isoherranen, Paine, & Thummel, ).…”

Styryl Group, a Friend or Foe in Medicinal Chemistry

“…It metabolizes via CYP P450s. [149] LZS102 ( 188) is an orally bioavailable benzothiophene that shows potent ERα antagonism and selective ER degradation. It is currently under phase I clinical trial to treat advanced or metastatic ERα + breast cancer.…”

“…It shows acceptable safety, tolerability, and prolonged stabilization in the advanced breast cancer patients pretreated with fulvestrant. It metabolizes via CYP P450s [149] . LZS102 ( 188 ) is an orally bioavailable benzothiophene that shows potent ERα antagonism and selective ER degradation.…”