A Rapid and Reliable Absorbance Assay to Identify Drug-Drug Interactions with Thiopurine Drugs.

BACKGROUND

Thiopurine methyltransferase (TPMT) plays a crucial role in the detoxification of thiopurine drugs, including the antimetabolites azathioprine and 6-mercaptopurine (6-MP) used to treat autoimmune diseases and various cancers. These drugs interfere with DNA synthesis by inhibiting the production of purine-containing nucleotides, leading to the death of rapidly dividing cells. TPMT inactivates thiopurine drugs by methylating at the thiol group. The activity of TPMT can vary significantly between individuals, and its activity is impacted by co-administered drugs, altering the effectiveness and toxicity of thiopurine drugs. TPMT is inhibited by many drugs that are co-administered to treat symptoms associated with diseases treated with thiopurines. For example, aspirin and other anti-inflammatory drugs, including olsalazine, sulfasalazine, and balsalazide, inhibit TPMT. The impact of TPMT genotypes on its methylating activity is well defined, and genotyping patients to identify TPMT metabolizer status is common clinical practice. Unfortunately, there has been no concerted effort to comprehensively identify drugs on the market that impact TPMT activity. The inhibition of TPMT by co-administered drugs could in part be responsible for idiosyncratic toxicities associated with thiopurine drug therapy.

METHODS

Here, we report a facile approach to produce large quantities of recombinant TPMT and a high-throughput assay that utilizes the shift in absorbance due to the methylation of thiopurines to report on TPMT activity.

RESULTS AND CONCLUSIONS

With purified TPMT on hand and the absorbance activity assay, we confirmed several compounds that inhibit TPMT, and the results were comparable to a mass spectral assay that measured 6-MP methylation. Understanding the impact of co-administered drugs on TPMT activity will improve the safety and efficacy of thiopurine-based treatment regimens.