Elsevier

Biochemical Pharmacology

Volume 86, Issue 4, 15 August 2013, Pages 539-547
Biochemical Pharmacology

Mechanism of allopurinol induced TPMT inhibition

https://doi.org/10.1016/j.bcp.2013.06.002Get rights and content

Abstract

Up to 1/5 of patients with wildtype thiopurine-S-methyltransferase (TPMT) activity prescribed azathioprine (AZA) or mercaptopurine (MP) demonstrate a skewed drug metabolism in which MP is preferentially methylated to yield methylmercaptopurine (MeMP). This is known as thiopurine hypermethylation and is associated with drug toxicity and treatment non-response. Co-prescription of allopurinol with low dose AZA/MP (25–33%) circumvents this phenotype and leads to a dramatic reduction in methylated metabolites; however, the biochemical mechanism remains unclear. Using intact and lysate red cell models we propose a novel pathway of allopurinol mediated TPMT inhibition, through the production of thioxanthine (TX, 2-hydroxymercaptopurine). In red blood cells pre-incubated with 250 μM MP for 2 h prior to the addition of 250 μM TX or an equivalent volume of Earle's balanced salt solution, there was a significant reduction in the concentration of MeMP detected at 4 h and 6 h in cells exposed to TX (4 h, 1.68, p = 0.0005, t-test). TX acts as a direct TPMT inhibitor with an apparent Ki of 0.329 mM. In addition we have confirmed that the mechanism is relevant to in vivo metabolism by demonstrating raised urinary TX levels in patients receiving combination therapy. We conclude that the formation of TX in patients receiving combination therapy with AZA/MP and allopurinol, likely explains the significant reduction of methylated metabolites due to direct TPMT inhibition.

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.

References (60)

  • M.P. Sparrow et al.

    Effect of allopurinol on clinical outcomes in inflammatory bowel disease nonresponders to azathioprine or 6-mercaptopurine

    Clin Gastroenterol Hepatol

    (2007)
  • M. Deininger et al.

    Purine substrates for human thiopurine methyltransferase

    Biochem Pharmacol

    (1994)
  • E. Weir et al.

    The effect of allopurinol on the excretion of oxypurines by the chick

    Biochim Biophys Acta

    (1970)
  • M.R. Rashidi et al.

    In vitro study of 6-mercaptopurine oxidation catalysed by aldehyde oxidase and xanthine oxidase

    Drug Metab Pharmacokinet

    (2007)
  • T.A. Krenitsky et al.

    A comparison of the specificities of xanthine oxidase and aldehyde oxidase

    Arch Biochem Biophys

    (1972)
  • T.A. Krenitsky et al.

    Ribonucleosides of allopurinol and oxoallopurinol. Isolation from human urine, enzymatic synthesis, and characterization

    J Biol Chem

    (1967)
  • U.Z. Malik et al.

    Febuxostat inhibition of endothelial-bound XO: implications for targeting vascular ROS production

    Free Radic Biol Med

    (2011)
  • L.C. Woodson et al.

    Human kidney thiopurine methyltransferase. Purification and biochemical properties

    Biochem Pharmacol

    (1983)
  • R.M. Weinshilboum et al.

    Human erythrocyte thiopurine methyltransferase: radiochemical microassay and biochemical properties

    Clin Chim Acta

    (1978)
  • T. Spector et al.

    Monophosphates of formycin B and allopurinol riboside. Interactions with leishmanial and mammalian succino-AMP synthetase and GMP reductase

    Biochem Pharmacol

    (1984)
  • E. Petit et al.

    Differential toxic effects of azathioprine, 6-mercaptopurine and 6-thioguanine on human hepatocytes

    Toxicology In Vitro

    (2008)
  • D.H. Present et al.

    Treatment of Crohn's disease with 6-mercaptopurine. A long-term, randomized, double-blind study

    N Engl J Med

    (1980)
  • S. Ardizzone et al.

    Azathioprine in steroid-resistant and steroid-dependent ulcerative colitis

    J Clin Gastroenterol

    (1997)
  • W. Sandborn et al.

    Azathioprine or 6-mercaptopurine for inducing remission of Crohn's disease

    Cochrane Database Syst Rev

    (2000)
  • A.D. Askanase et al.

    Use of pharmacogenetics, enzymatic phenotyping, and metabolite monitoring to guide treatment with azathioprine in patients with systemic lupus erythematosus

    J Rheumatol

    (2009)
  • G.P. Clunie et al.

    Relevance of thiopurine methyltransferase status in rheumatology patients receiving azathioprine

    Rheumatology

    (2004)
  • M. Schwab et al.

    Azathioprine therapy and adverse drug reactions in patients with inflammatory bowel disease: impact of thiopurine S-methyltransferase polymorphism

    Pharmacogenetics

    (2002)
  • P.A. Blaker et al.

    The pharmacogenetic basis of individual variation in thiopurine metabolism

    Personal Med

    (2012)
  • T. Dervieux et al.

    Differing contribution of thiopurine methyltransferase to mercaptopurine versus thioguanine effects in human leukemic cells

    Cancer Res

    (2001)
  • S.A. Coulthard et al.

    The effect of thiopurine methyltransferase expression on sensitivity to thiopurine drugs

    Mol Pharmacol

    (2002)
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