Mechanism of allopurinol induced TPMT inhibition
Graphical abstract
Introduction
The thiopurines azathioprine (AZA) and mercaptopurine (MP) are first line immunomodulators used in the management of inflammatory bowel disease (IBD). These drugs are of proven efficacy for the induction and maintenance of corticosteroid-free remission, and in addition improve the response to anti-TNFα antibody therapy, promote fistula healing and reduce post-operative recurrence of Crohn's disease (CD) [1], [2], [3], [4], [5]. These agents also remain important as immunosuppressive therapy in other chronic inflammatory conditions and in haem-oncology [6], [7], [8]. Whilst the majority of patients with IBD tolerate these agents well, 9% are resistant to thiopurines and between 15 and 28% experience adverse reactions, which often necessitates drug withdrawal [9], [10]. Opportunities to improve the efficacy and tolerability of AZA/MP are therefore of particular importance in IBD treatment strategies.
AZA and MP are pro-drugs with a complex metabolism (Fig. 1) [11]. After ingestion, AZA is broken down following conjugation with glutathione to release MP. MP is a synthetic analogue of the purine base hypoxanthine, which is metabolized in nucleated cells via enzymes of the purine salvage pathway to form thioguanine nucleotides (TGN). TGNs are believed to mediate the main therapeutic effects but at high concentrations (>450 pmol/8 × 108 RBCs) have been associated with myelotoxicity [12].
MP may also undergo oxidation by xanthine oxidase/dehydrogenase (XDH) and aldehyde oxidase (AO) via the intermediates 8-hydroxymercaptopurine and thioxanthine (TX; 2-hydroxymercaptopurine) to form thiouric acid (TUA). Alternatively, MP can be deactivated by thiopurine-S-methyltransferase (TPMT) to produce methylmercaptopurine (MeMP). TPMT is additionally responsible for the S-methylation of thioinosine nucleotides including the monophosphate nucleotide (thioinosine monophosphate, TIMP), forming methylmercaptopurine ribonucleotide (MeMPR), which is a potent inhibitor of de novo purine synthesis [13], [14]. TPMT activity demonstrates a tri-modal distribution according to genetic polymorphism and is inversely correlated with TGN concentrations and thereby the risk of myelotoxicity. Pre-treatment measurement of TPMT activity is therefore recommended to guide initial thiopurine dosing [15].
Metabolite monitoring, of patients with IBD established on thiopurine therapy, provides a rational basis for dose adjustment in order to maximize clinical response, limit toxicity and prevent treatment failure. In this regard TGN concentrations >235–260 pmol/8 × 108 RBCs have been associated with disease remission, whereas MeMP levels >5300–5700 pmol/8 × 108 RBCs have been correlated with hepatotoxicity, which is typically characterized by a transaminitis [9], [16], [17]. Approximately 15–20% of patients demonstrate a skewed drug metabolism, preferentially metabolizing MP to MeMP instead of TGN, a situation referred to as thiopurine hypermethylation. This phenotype is associated with non-response to treatment, hepatotoxicity and other side effects [18], [19], [20]. Furthermore, thiopurine dose escalation in this group often leads to a disproportionate rise in MeMP with a paradoxical reduction in TGN levels [9], [21]. Importantly, thiopurine hypermethylation is not predicted by high TPMT activity, suggesting an influence of other factors in the thiopurine pathway [22].
Allopurinol, an inhibitor of XDH, was originally designed by Gertrude Elion for use alongside MP to potentiate its therapeutic index in patients with leukaemia [23]. Whilst the original studies of combination therapy in man showed an increase in anti-tumour activity, it was associated with a proportionate rise in toxicity [24]. This strategy was therefore abandoned and allopurinol monotherapy subsequently found its niche in the management of gout and tumour lysis syndrome. Interest in co-prescription was renewed in the 1990s with the discovery that low dose AZA and allopurinol improved renal allograft survival, which occurred through optimization of thiopurine metabolite profiles [25], [26], [27]. In this regard the addition of allopurinol leads to a significant reduction in methylated metabolites with a concomitant rise in TGN levels [28], [29], [30].
In patients with IBD, combination treatment with low dose AZA/MP (25–33% standard dose) and allopurinol provides an opportunity to circumvent thiopurine hypermethylation and thereby reduce drug toxicity and recapture treatment response [20], [28], [29]. It has additionally been used to ameliorate other adverse drug reactions unrelated to preferential methylation [20], [30].
The mechanism by which allopurinol leads to a reduction in methylated thiopurine metabolites remains unclear. Inhibition of TPMT by allopurinol would explain these results; however both in vitro and in vivo studies have shown a lack of inhibition of erythrocyte and human liver cytosolic TPMT activity following the addition of both allopurinol and its active metabolite, oxypurinol (alloxanthine) [29]. Therefore the aim of this study was to resolve the biochemical mechanism underlying the interaction between allopurinol and AZA/MP.
A previous study in patients with non-Hodgkin's lymphoma demonstrated a rise in plasma thioxanthine (TX) levels and a reduction in red blood cell (RBC) MeMPR following treatment with intravenous MP and allopurinol [31]. We therefore hypothesized that the thiopurine intermediate TX acts as a direct TPMT inhibitor, and secondly that levels of TX will be elevated in IBD patients receiving combination therapy with oral low dose AZA and allopurinol.
Section snippets
Materials
All reagents and chemicals were of analytical grade and were supplied by Sigma–Aldrich Company Ltd. (Gillingham, UK) or Merck Chemicals Ltd. (Nottingham, UK).
Metabolism of MP, TX and oxypurinol in intact red blood cells (RBCs)
1.5 mL of EDTA whole blood, from a healthy volunteer with wild-type TPMT activity (38 pmol MeMP/h/mgHb), was centrifuged at 12,000 × g for 30 s, the plasma and top fifth containing the buffy coat, platelets and reticulocytes was removed and the red cells washed twice with a 0.9% NaCl solution. 100 μL aliquots of packed RBCs were transferred
The effect of TX and oxypurinol on the production of MeMP in intact RBCs exposed to MP
For intact RBCs incubated with 250 μM MP, the concentration of MeMP in both the protein free red cell extract and cell free supernatant media increased with time (Fig. 2). The concentration of MeMP in the cell free supernatant media was approximately 5-fold lower than that of the protein free red cell extracts. In comparison, MeMP was not detected in either the protein free red cell extract or cell free supernatant media following incubation with 250 μM TX.
Intact RBCs pre-incubated with 250 μM MP
Discussion
Our results show that TX is an inhibitor of TPMT activity. We have also demonstrated that levels of TX are increased in the urine of patients with IBD prescribed combination treatment with oral AZA and allopurinol. We propose that elevated TX levels, leading to inhibition of TPMT in patients receiving combination therapy, are therefore likely to contribute to the dramatic reduction in methylated MP metabolites that we and others have observed in patients treated with combination therapy [20],
Acknowledgements
This work was supported by charitable grants from Guy's and St Thomas’ Charity, for Crohn's Charity and Crohn's and Colitis UK (CCUK). We declare no other conflicts of interest.
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