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We implement reaction phenotyping studies to identify the CYP450 isoforms (1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 3A4/5) that may be involved in loss of parent compound. Results from our studies inform CYP induction studies.
In general, we identify CYPs using non-radiolabeled test compound in pooled human liver microsomes (HLM) in the absence and presence of CYP isoform selective chemical inhibitors. In order to utilize chemical inhibitors and HLM, there must be sufficient turnover of the test compound to permit detection of the loss of parent if the metabolites are not known. If the test article meets this criterion, the isoforms can be identified and the contribution towards the metabolism of the test article can be quantified. As a confirmation, incubation studies are then conducted with recombinant human CYP isoforms (baculovirus/insect cells). Alternatively, if the metabolism of the test compound is slow in HLM, as observed for low clearance drugs, then CYP reaction phenotyping is conducted using only recombinant CYPs and a relative activity factor (Crespi et. al. (1999) Pharmacology & Therapeutics 84: 121-131) is utilized to identify the contribution of the CYP isoform.
As part of our program we determine the solubility limits of the test compound so that appropriate concentrations can be evaluated. We optimize the test system to determine appropriate concentrations and time points for the study. We conduct the assay in HLM and specific inhibitors using one concentration in the presence and absence of inhibitors at 5 time points. Then we conduct the assay in eight (rCYPs (1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 3A4, and 3A5) at one concentration and a single time point. Our study report provides the estimate % contribution of each CYP towards metabolism of the test compound.