Use of Specific Inhibitory Antibodies for P450 Reaction Phenotyping of Investigational Drugs

Abstract

Identifying the CYP450 enzymes underlying metabolism of investigational drugs (i.e., P450 reaction phenotyping; PRP) is critical to the development of effective therapeutics. Information gained from such studies can: a) provide insight into structural modifications of test drugs in order to improve metabolic stability and; b) specify an estimate of drug‐drug interaction potential as well as that of interindividual and species differences in oxidative metabolism. CYP450 chemical inhibitors (e.g., a‐naphthoflavone and sulfaphenazole) are often used in reaction phenotyping studies but the outcome is sometimes equivocal due to the non‐specific effects of these chemical agents plus their capacity to undergo P450‐dependent catabolism. In contrast, inhibitory P450 antibodies offer high specificity and irreversibility, and may thus represent a superior approach for characterizing individual P450 enzyme involvement in drug metabolism. We recently developed a compilation of highly‐specific polyclonal and monoclonal antibodies to the CYP450s comprising the major oxidative drug‐metabolizing enzymes in human liver, including CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 (aka CYP ImmunoInhibit antibodies). As shown here, each of these antibodies proved capable of eliciting > 80% inhibition of probe drug metabolism by the cognate P450 antigens in human liver microsomes (HLMx), while giving insignificant effects on different CYP450s (Figure 1). The inhibition observed with each of the six CYP450 antibodies was not only highly‐specific but was also dose‐dependent, with maximal inhibition (80–90%) achieved at IgG:microsomal protein ratios ranging from 0.1 mg/mg to 3 mg/mg (Figure 2). Control (pre‐immune) antibodies had no effect on substrate metabolism. Such results show that inhibitory antibodies coupled with HLMx represent an alternative and powerful approach to chemical inhibitors for evaluating involvement of specific P450 enzymes in the hepatic metabolism of investigational drugs. Interestingly, studies ongoing with CYP ImmunoInhibit antibodies and MetMax™ cryopreserved human hepatocytes, which are liver cells that have been permeabilized and cryopreserved, have given analogous results to those achieved with HLMx. The utilization of inhibitory antibodies with HLMx represents an accurate model for PRP of new therapeutics due to the specificity of these immunoreagents, and the irreversible nature of the inhibition achieved, an important benefit when working with metabolically‐stable test agents.Specific Inhibition of Microsomal Drug Metabolism by CYP ImmunoInhibit AntibodiesFigure 1Dose‐Response Inhibition of Microsomal Drug Metabolism by CYP ImmunoInhibit AntibodiesFigure 2