Cytochrome P450 2C19 (CYP2C19, EC 1.14.14.1) is a member of the cytochrome P450 monooxidase (CYP) family of microsomal xenobiotic metabolism enzymes. CYPs are membrane-bound hemeproteins responsible for Phase I biotransformation reactions, in which lipophilic drugs and other xenobiotic compounds are converted to more hydrophilic products to facilitate excretion from the body. CYP2C19 is primarily expressed in liver and intestinal tissue and catalyzes oxidation of neutral or weakly basic lipophilic molecules with 2-3 hydrogen bond donor/acceptor sites. Isoforms of the CYP2C subfamily are responsible for metabolism of nearly 20% of all small molecule drugs commonly used by humans. Polymorphisms in the human CYP2C19 gene have been implicated in clinical drug/drug interactions involving widely-prescribed drugs, including proton pump inhibitors, antiplatelet agents and anticonvulsants. In addition, for drugs whose pharmacological activity requires metabolism from a pro-drug form, CYP2C19 inhibition or allelic deficiency can lead to decreased drug efficacy. Assay Genie's CYP2C19 Activity Assay Kit enables rapid measurement of native or recombinant CYP2C19 activity in biological samples such as liver microsomes. The assay utilizes a non-fluorescent CYP2C19 substrate that is converted into a highly fluorescent metabolite detected in the visible range (Ex/Em = 406/468 nm), ensuring a high signal-to-background ratio with little interference by autofluorescence. A highly-selective CYP2C19 inhibitor is provided for determination of CYP2C19 activity in heterogeneous biological samples, where other CYP isozymes may contribute to substrate metabolism. The inhibitor displays greater than 100-fold selectivity for CYP2C19 over other CYPs, ensuring targeted inhibition. CYP2C19 specific activity is calculated by running parallel reactions in the presence and absence of the selective inhibitor and subtracting any residual activity detected with the inhibitor present. The kit contains a complete set of reagents sufficient for performing 50 sets of paired reactions (in the presence and absence of inhibitor).

Figure: (a) 3-CHC standard curve. One mole of 3-CHC corresponds to the metabolism of one mole of CYP2C19 substrate. (b) Reaction kinetics of fluorogenic substrate metabolism in human liver microsomes (0.25 mg/mL) at 37°C in the presence and absence of the selective CYP2C19 inhibitor (+)-N-3-benzylnirvanol (the solvent control contained assay buffer with 0.5% acetonitrile). (c) Specific activity of CYP2C19 in pooled human liver microsome sample (0.25 mg/mL). Assays were performed according to the kit protocol.