Reaction phenotyping: current industry efforts to identify enzymes responsible for metabolizing drug candidates

Abstract

Reaction phenotyping studies to identify specific enzymes involved in the metabolism of drug candidates are increasingly important in drug discovery efforts. Experimental approaches used for CYP reaction phenotyping include incubations with cDNA expressed CYP enzyme systems and incubations containing specific CYP enzyme inhibitors. Since both types of experiments present specific advantages as well as known drawbacks, these studies are generally viewed as complementary approaches. Although glucuronidation pathways are also known to present potential drug-drug interaction issues as well as challenges related to their polymorphic expression, reaction phenotyping approaches for glucuronidation are generally limited to cDNA expressed systems due to lack of availability of specific UGT inhibitors. This article presents a limited review of current approaches to reaction phenotyping studies used within the pharmaceutical industry.

Fig. 1

Concentration vs time curve for disappearance of parent from initial concentrations of 10 and 1 μM. Linear regression fit was used to estimate initial slopes from first three data points (no more than 20% metabolism). Dividing slope by initial concentration yields an estimate of the rate constant (0.011 and 0.014 min⁻¹ for 10 and 1 μM starting concentrations, respectively)

Fig. 2

Concentration vs time curve for disappearance of parent from initial concentrations of 10 μM and 1 μM. Nonlinear regression fitting was used to estimate the rate constant (0.012 and 0.015 min⁻¹ for 10 and 1 μM starting concentrations, respectively)

Fig. 3

Concentration vs time curve for disappearance of parent from expressed CYP enzyme incubations with initial substrate concentrations of 1 μM. No turnover was observed in incubations containing CYP2B6, CYP2C9, CYP2C19 or CP2C8. Nonlinear regression fitting was used to estimate the rate constants for CYP1A2 (0.0002 min⁻¹) and CYP3A4 (0.0065 min⁻¹)

Fig. 4

Flowchart for typical reaction phenotyping evaluation of a lead drug candidate. In all cases where results indicate a heightened risk of untoward effects, a decision to advance or terminate a compound must be made in the context of the risk–benefit balance and include consideration of anticipated in vivo drug concentrations at efficacious doses, nature of the untoward effect, alternatives to the lead compound and nature of the disease target