A Study of the Roles of CYP3A4 and CYP2C19 In The Human Liver Biotransformation Of Bortezomib

Abstract

The dipeptidyl boronic acid, bortezomib (Velcade) is the first-in-class inhibitor of the ubiquitin-proteasome system (UPS). The ubiquitin-proteasome system (UPS) controls the degradation of signalling molecules, cell-cycle regulators, transcription factors, tumour suppressors and apoptosis regulatory proteins important in tumour cell proliferation. Bortezomib is currently approved by FDA for treatment of multiple myeloma and mantle cell lymphoma, and is also showing efficacy in other haematological malignancies as well as solid tumours such as prostate cancer. A number of studies have shown that bortezomib undergoes hepatic biotransformation to a pair of deboronated diastereomeric metabolites (M1 and M2) primarily through the action of CYP2C19 and CYP3A4. However detailed characterisation of this reaction was incomplete. The objectives of the research presented in this thesis were to (a) establish and validate the previously published method for characterising bortezomib metabolism in human liver microsomes, and (b) clarify the role of CYP2C19 relative to CYP3A4 in the inter-individual variation in the biotransformation of bortezomib. Up to 22-fold variation in biotransformation was observed in human liver microsomes (n=7). Both CYP3A4 and CYP2C19 Supersomes© could catalyse the deboronation of bortezomib. In pooled microsomes, ketoconazole inhibited (P<0.01) bortezomib deboronation whereas omeprazole had no effect. Across individual livers (n=7), there was no correlation between CYP3A4 protein and deboronation of bortezomib (Rs= -0.32; P>0.05). In contrast there was a negative correlation between bortezomib metabolism and CYP2C19 expression (Rs= -0.964; P<0.001). The homozygous null CYP2C19 liver had the highest rate of bortezomib biotransformation. In four livers with similar CYP3A4 expression there was an increase (P= 0.0006) in bortezomib biotransformation relative to decreasing CYP2C19 content. LC/MS analysis of the products of biotransformation indicate that CYP2C19 catalyses the formation of a putative oxidised tetrahedral intermediate. This metabolite was not observed with CYP3A4. In contrast CYP3A4 catalysed the formation of a product with a mass consistent with an imine amide. The results are consistent with the literature, in that both CYP2C19 and CYP3A4 can catalyse the deboronation of bortezomib. However, CYP2C19 may produce a reactive product which may inhibit catalysis by CYP3A4 or other CYPs. The combined expression of both CYP3A4 and CYP2C19 may play a complex role in the overall rate of biotransformation of bortezomib.