Abstract
The effect of changes in microsomal incubation conditions (NADPH, Mg(2+), Cl(-), NADPH-regenerating system and pH) on the formation of the CYP3A4 metabolites AM1 and AM9 from CsA were studied by application of a fractional factorial design. Metabolism was studied in microsomes of transfected human liver epithelial (THLE) cells specifically expressing CYP3A4. Within the conditions tested, a 3-4-fold difference in formation of CsA metabolites was observed. Formation of both AM1 and AM9 was favoured by a low Mg(2+) concentration (0.5 mM) and no addition of Cl(-) to the incubation matrix. However, while a high NADPH concentration (1.75 mM) was the single most important factor for the formation of AM1, changes in NADPH concentration between 0.25 and 1.75 mM had no influence on AM9 formation. Formation of the two metabolites also differed in their influence by pH changes, as a change in pH from 7.2 to 7.5 significantly increased the formation of AM9, while formation of AM1 was unaffected by this change. The present study showed that relatively small changes in the incubation matrix had a significant influence on the microsomal CYP3A4-mediated metabolism of CsA. Systematic studies on microsomal incubation conditions could be a key to improve metabolic in vitro-in vivo extrapolations in drug development.
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