Selective Mechanism-Based Inactivation of CYP3A4 by CYP3cide (PF-04981517) and Its Utility as an In Vitro Tool for Delineating the Relative Roles of CYP3A4 versus CYP3A5 in the Metabolism of Drugs

Abstract

CYP3cide (PF-4981517; 1-methyl-3-[1-methyl-5-(4-methylphenyl)-1H-pyrazol-4-yl]-4-[(3S)-3-piperidin-1-ylpyrrolidin-1-yl]-1H-pyrazolo[3,4-d]pyrimidine) is a potent, efficient, and specific time-dependent inactivator of human CYP3A4. When investigating its inhibitory properties, an extreme metabolic inactivation efficiency (k inact /K I ) of 3300 to 3800 ml ⅐ min ؊1 ⅐ mol ؊1 was observed using human liver microsomes from donors of nonfunctioning CYP3A5 (CYP3A5 *3/*3). This observed efficiency equated to an apparent K I between 420 and 480 nM with a maximal inactivation rate (k inact ) equal to 1.6 min ؊1 . Similar results were achieved with testosterone, another CYP3A substrate, and other sources of the CYP3A4 enzyme. To further illustrate the abilities of CYP3cide, its partition ratio of inactivation was determined with recombinant CYP3A4. These studies produced a partition ratio approaching unity, thus underscoring the inactivation capacity of CYP3cide. WhenCYP3cide was tested at a concentration and preincubation time to completely inhibit CYP3A4 in a library of genotyped polymorphic CYP3A5 microsomes, the correlation of the remaining midazolam 1-hydroxylase activity to CYP3A5 abundance was significant (R 2 value equal to 0.51, p value of <0.0001). The work presented here supports these findings by fully characterizing the inhibitory properties and exploring CYP3cide's mechanism of action. To aid the researcher, multiple commercially available sources of CYP3cide were established, and a protocol was developed to quantitatively determine CYP3A4 contribution to the metabolism of an investigational compound. Through the establishment of this protocol and the evidence provided here, we believe that CYP3cide is a very useful tool for understanding the relative roles of CYP3A4 versus CYP3A5 and the impact of CYP3A5 genetic polymorphism on a compound's pharmacokinetics.

Characterization of Phase I Metabolism of Resibufogenin and Evaluation of the Metabolic Effects on Its Antitumor Activity and Toxicity

“…Among the selective inhibitors tested, ketoconazole (CYP3A inhibitor) strongly inhibited the catalytic activity of HLM, further implying the prominent role of CYP3A in RB hydroxylation. Furthermore, CYP3cide, a potent and specific time-dependent inhibitor of CYP3A4 (Walsky et al, 2012), exhibited similar inhibitory effects on RB 5b-hydroxlation in HLM and rhCYP3A4 (Supplemental Fig. 1).…”

“…The selective inhibitors and their concentrations were as follows (Bjornsson et al, 2003): montelukast (2 mM) for CYP2C8 (Walsky et al, 2005), sulfaphenazole (10 mM) for CYP2C9, omeprazole (20 mM) for CYP2C19, quinidine (10 mM) for CYP2D6, clomethiazole (50 mM) for CYP2E1, ketoconazole (1 mM) for CYP3A. Inhibition by furafylline (10 mM) for CYP1A2, 8-methoxypsoralen (2.5 mM) for CYP2A6, TEPA (50 mM) for CYP2B6 (Rae et al, 2002), CYP3cide (2 mM) for CYP3A4 (Walsky et al, 2012) and ABT (500 mM) for broad P450s (Emoto et al, 2003) were examined by adding RB after preincubation with NADPH-generating system at 37°C for 40 minutes.…”

In Vitro Kinetic Characterization of Axitinib Metabolism

“…Clinical results indicated no clear correlation of CYP3A5 polymorphisms with variability in axitinib pharmacokinetics (Brennan et al, 2012), indicating CYP3A5 may have a rather small clinical contribution to overall oral clearance. Recently, in vitro tools to confirm or refute the contribution of CYP3A5 have been made available via the use of CYP3cide, a specific CYP3A4 inhibitor (Walsky et al, 2012b). When axitinib was assessed using CYP3cide, in tandem with CYP3A5-genotyped donor pools of microsomes (CYP3A5 *1/*1 and *3/*3) and axitinib substrate loss was followed over time.…”

“…These CYP3cide substrate depletion studies were not conducted with a dilution step, which if diluted would minimize any time-dependent effects of CYP3cide on CYP3A5 activity. The absence of the dilution step method has been shown to be an effective method for delineating CYP3A5 contribution from CYP3A4 (Walsky et al, 2012b), especially when combined with quantifying the metabolite formation in the presence and absence of inhibitors at a time point where relatively little loss of the parent compound is observed. When comparing the data from the formation of the sulfoxide metabolite, a similar contribution of CYP3A5 (18%) was noted to that of the parent loss method.…”

“…Axitinib (1.0 mM) was incubated, in triplicate, with pooled lots of HLM CYP3A5 *1/*1 (from five donors, see the Materials section) and HLM CYP3A5 *3/*3 (from six donors, see the Materials section) at a protein concentration of 1 mg/ml for measurement of intrinsic clearance (Cl int ) in the presence and absence of chemical inhibitors, essentially as previously described (Walsky et al, 2012b;Tseng et al, 2014). Incubations were carried out in triplicate in 100 mM KH 2 PO 4 (pH 7.4) containing MgCl 2 (3.3 mM), ketoconazole (2 mM), or CYP3cide (1 mM) and NADPH (1.3 mM) in a total volume of 0.3 ml.…”