Analytical Pitfalls of Therapeutic Drug Monitoring of Thiopurines in Patients With Inflammatory Bowel Disease

Abstract

The use of thiopurines in the treatment of inflammatory bowel disease (IBD) can be optimized by the application of therapeutic drug monitoring. In this procedure, 6-thioguanine nucleotides (6-TGN) and 6-methylmercaptopurine (6-MMP) metabolites are monitored and related to therapeutic response and adverse events, respectively. Therapeutic drug monitoring of thiopurines, however, is hampered by several analytical limitations resulting in an impaired translation of metabolite levels to clinical outcome in IBD. Thiopurine metabolism is cell specific and requires nucleated cells and particular enzymes for 6-TGN formation. In the current therapeutic drug monitoring, metabolite levels are assessed in erythrocytes, whereas leukocytes are considered the main target cells of these drugs. Furthermore, currently used methods do not distinguish between active nucleotides and their unwanted residual products. Last, there is a lack of a standardized laboratorial procedure for metabolite assessment regarding the substantial instability of erythrocyte 6-TGN. To improve thiopurine therapy in patients with IBD, it is necessary to understand these limitations and recognize the general misconceptions in this procedure.

FIGURE 1.

Simplified metabolic pathway of thiopurines. Bold lines represent the purine salvage pathway in which the pharmacologically active metabolites [6-thioguanine nucleotides (6-TGN)] are formed, whereas dotted lines represent the competing pathways. Azathioprine (AZA) is converted into mercaptopurine (MP) by separating the imidazole group. Mercaptopurine is subsequently metabolized into 6-TGN through a multistep pathway, by the enzymes hypoxanthine–guanine phosphoribosyl transferase (HGPRT), inosine monophosphate dehydrogenase (IMPDH), and guanosine monophosphate synthetase (GMPS). Through competing pathways, MP is converted by xanthine oxidase (XO) into 6-thiouric acid (6-TUA) or by TPMT into 6-methylmercaptopurine (6-MMP) and 6-methylmercaptopurine ribonucleotides (6-MMPR). Thioguanine (TG) is converted into 6-TGN in 1 step through the purine salvage pathway for which only HGPRT is necessary. Thioguanine may also be transformed into 6-methylthioguanine (6-MTG) by TPMT or into 6-TUA by guanine deaminase (GD) and XO. 6-TGN consists of 6-thioguanine monophosphate (6-TGMP), 6-thioguanine diphosphate (6-TGDP), and 6-thioguanine triphosphate (6-TGTP). The 6-TGTP nucleotides target Rac1 and finally induce T-cell apoptosis.

FIGURE 2.

Therapeutic and toxicity reference ranges in therapeutic drug monitoring of thiopurines in IBD. The association of erythrocyte 6-thioguanine nucleotides (x-axis) and erythrocyte 6-methylmercaptopurine (y-axis) metabolites with therapeutic response and toxicity to azathioprine and mercaptopurine treatment in IBD. Reference values are depicted in the method by Lennard. RBC, red blood cell.