Utility of thiopurine methyltransferase genotyping and phenotyping, and measurement of azathioprine metabolites in the management of patients with autoimmune hepatitis

Background/Aims

Azathioprine is a key drug in the management of autoimmune hepatitis (AIH), with effects mediated via conversion to 6-thioguanine (6-TG) and 6-methylmercaptopurine (6-MMP), the latter controlled by thiopurine methyltransferase (TPMT). Our aims were to evaluate the role of TPMT genotyping and phenotyping and to examine 6-TG and 6-MMP metabolite levels in patients with AIH.

Methods

TPMT genotyping and phenotyping was performed on 86 patients with AIH, and metabolites evaluated in assessable patients.

Results

Eighty-six patients with AIH received azathioprine; 22 developed toxicity and 4/22 were heterozygous for TPMT alleles. Cirrhosis was more common amongst patients who developed toxicity (12/22 (54.5%) versus 19/64 (29.6%), P = 0.043). Patients who required persistent prednisone at equivalent azathioprine doses had a higher mean fibrosis stage (P = 0.044). TPMT activity, but not metabolites, was lower in patients with stage III/IV fibrosis versus stage I/II fibrosis (30 ± 1.92 versus 35.2 ± 1.93, P = 0.044). Azathioprine dose significantly correlated with measured 6-TG levels (r = 0.409, P < 0.0001) and 6-MMP levels (r = 0.387, P < 0.001).

Conclusions

Advanced fibrosis but not TPMT genotype or activity predicts azathioprine toxicity in AIH. Overlap in 6-TG and 6-MMP metabolite levels is noted whether or not steroid therapy is used to maintain remission.

Introduction

Corticosteroid therapy alone or in conjunction with azathioprine results in disease remission in over 80% of patients with autoimmune hepatitis (AIH) [1], [2], [3], [4]. Moreover, azathioprine monotherapy has been established as the immunosuppressant of choice in the maintenance of remission in patients with AIH [5], [6]. Despite the promising results obtained using azathioprine as a steroid sparing agent, a proportion of patients with AIH remain on corticosteroid therapy either as a consequence of azathioprine toxicity or as a result of resistant disease [4], [6], [7], [8], [9].

The principal determinant of intolerance to azathioprine is the complex metabolism of the drug [10], [11]. Following oral administration, azathioprine is rapidly converted to 6-mercaptopurine (6-MP) through a non-enzymatic pathway. Following this conversion, 6-MP undergoes enzyme-dependent metabolism through one of three pathways (Fig. 1) [11]. This transformation of 6-MP into its active metabolites occurs intracellularly along the competing routes catalysed by thiopurine methyltransferase (TPMT) and hypoxanthine phosphoribosyltransferase (HPRT), giving rise to 6-methyl mercaptopurine (6-MMP), 6-methyl-thioinosine 5′-monophosphate, and 6-thioguanine nucleotides (6-TGNs), respectively. 6-TG is considered the active metabolite [11]. Incorporation of 6-TGNs into lymphocyte DNA influences cellular function and causes cytotoxicity and immunosuppression. The direct lymphocytotoxic properties of 6-MP form the basis of its use in the treatment of lymphoma and leukemia. In such patients, low erythrocyte 6-TG levels have been associated with a low 6-MP dose and an increased risk of disease relapse and conversely, high levels of 6-TG were associated with reduced risk of disease relapse but an increased risk of neutropenia [12], [13].

As illustrated in Fig. 1, 6-MP is metabolised by the enzyme TPMT to 6-MMP, and it appears that the most important determinant in disparity in metabolism is TPMT activity. A number of genetic polymorphisms have been observed in the gene encoding TPMT activity such that a trimodal distribution of TPMT activity has been observed [11]. As a result of the heterogeneity in TPMT activity, 0.3% of individuals may have either negligible or absent activity (TPMT^L/TPMT^L), 11% have intermediate activity (TPMT^L/TPMT^H), and 89% inherit normal to high levels of enzyme activity (TPMT^H/TPMT^H) [11], [14]. Several non-functional variants of the gene have been characterised and major mutations causing loss of function of alleles have been recognised. Identification of mutations has led to the development of technology combining simple DNA-based assays with phenotypic analysis to identify patients at higher risk of drug toxicity and treatment failure. Moreover, it has been possible in other disease states such as ulcerative colitis and Crohn’s disease to designate a therapeutic window for metabolites of azathioprine or 6-MP and identify a target range for 6-TGNs in these populations [15], [16].

A target range for azathioprine and 6-MP metabolites has been suggested in children with AIH and it has been suggested that measurement of 6-TGN levels in conjunction with 6-MMP levels might differentiate azathioprine-induced hepatotoxicity from flares in disease activity [17]. A separate study examined the role of TPMT genotyping and phenotyping in the prediction of azathioprine-induced adverse events in patients with AIH and established that neither approach could universally predict in whom these events would occur [18]. For these reasons, we have performed a comprehensive evaluation of the utility of erythrocyte 6-TG and 6-MMP metabolite monitoring in adult patients with AIH. In addition, we have examined the role of TPMT genotyping and phenotyping in predicting adverse reactions to azathioprine in the management of AIH.