Pedro R. Chocair

THE IMPORTANCE OF THIOPURINE METHYLTRANSFERASE ACTIVITY FOR THE USE OF AZATHIOPRINE IN TRANSPLANT RECIPIENTS

The immunosuppressive efficacy of azathioprine is related to its rapid metabolism in vivo to 6-mercaptopurine (6MP), with subsequent conversion to thioguanine nucleotides by an anabolic route involving hypoxanthine-guanine phosphoribosyltransferase. Two alternative catabolic routes exist: oxidation to 6-thiouric acid via xanthine oxidase and methylation to 6-methylmercaptopurine via the enzyme thiopurine methyltransferase (TPMT). Catabolism via either route would restrict formation of the active metabolites.We analyzed TPMT activity in erythrocyte lysates of 25 controls, 25 uremic patients on dialysis, and 68 transplanted patients. Median activity was lower in controls (31.0 pmol/hr/mg Hb, range 16.2–43.0) and transplanted patients receiving only cyclosporine and prednisolone (31.7 pmol/hr/mg Hb, range 12.7–43.5) than in the azathioprine treated group, (36.1 pmol/hr/mg Hb, range 16.1–71.3), or the uremic group on dialysis, (35.5 pmol/hr/mg Hb, range 18.6–62.6) suggesting that both azathioprine and uremia induce the enzyme, but CsA does not.

Long‐term outcome of using allopurinol co‐therapy as a strategy for overcoming thiopurine hepatotoxicity in treating inflammatory bowel disease

Background  Hepatotoxicity results in the withdrawal of thiopurines drugs, azathioprine (AZA) and mercaptopurine (MP), in up to 10% of patients with inflammatory bowel disease. Our group previously demonstrated that allopurinol with AZA/ciclosporin/steroid ‘triple therapy’ improved renal graft survival.

Observations on the use of allopurinol in combination with azathioprine or mercaptopurine

Sirs, The report by Sparrow et al. of a manoeuvre using allopurinol to increase tioguanine (thioguanine) nucleotide (TGN) levels in inflammatory bowel disease (IBD) patients unresponsive to thiopurine drugs is important. In 1993, we published in The Lancet the results of the first trial of thiopurine/allopurinol combination therapy, in renal transplant patients. The combination was well tolerated. We thus applaud the work of Sparrow et al., as there has been no follow-up of this drug combination until now, in large part because the literature says the combination is unsafe. We now draw attention to several important points. The synergistic effect of allopurinol is enigmatic, for the following two reasons. First, patients who are genetically thiopurine methyl-transferase (TPMT)-deficient respond well and accumulate high red cell 6-TGN levels on doses of about 5% of normal, indicating that as much as 95% of a normal thiopurine dose is inactivated by this methylation pathway. Secondly, renal transplant patients who have clinical gout are often started on a reduced thiopurine dose, and the rule of thumb is to reduce the normal dose to about a third. This experience corresponds with the dose reduction reported by Sparrow et al., but implies that about two-thirds of a normal thiopurine dose is inactivated by the oxidation pathway of xanthine oxidase. Thus, the inactivation pathways do not add up. Importantly, Sparrow et al. have additionally shown that methylated metabolites of mercaptopurine (6-mercaptopurine; MP) are greatly reduced by the presence of allopurinol. Therefore, we are led inexorably to the unexpected conclusion that allopurinol inhibits TPMT. But what is the mechanism for this? As quoted in Sparrow et al. s report, unpublished work by Prometheus Laboratories found that allopurinol does not inhibit TPMT, presumably assayed as haemolysate activity in vitro. The first published assays of TPMT by Weinshilboum et al. also included allopurinol in the assay mixture, ostensibly to inhibit xanthine oxidase. We devised a simplified version of the TPMT assay, omitting the allopurinol (and an ion-exchange step), as it is a common error to use allopurinol as an in vitro inhibitor, particularly in cell-free assays. Allopurinol is an inactive prodrug. Its primary active form in vivo is oxypurinol, which is formed from allopurinol by aldehyde oxidase. Aldehyde oxidase and xanthine oxidase are effectively absent in human red cells, the major site of activity being liver. In addition, oxypurinol is converted in vivo to form the purine nucleotide analogue oxypurinol riboside monophosphate and its nucleoside, oxypurinol riboside – these are thought to be the active metabolites of allopurinol that inhibit the enzyme orotic phosphoribosyl transferase and provide the basis of the allopurinol load test for diagnosis of the metabolic disease ornithine transcarbamylase deficiency. Significantly, oxypurinol riboside monophosphate is a 6-oxo analogue of 6-thioinosine monophosphate, a known substrate for TPMT in vivo. Thus, there is a high probability that allopurinol provides oxypurinol metabolites in vivo that inhibit TPMT. This needs to be resolved. Sparrow et al. concluded by expressing their concern that there may be a heightened risk of nodular regenerative hyperplasia (NRH) with thiopurine plus allopurinol co-therapy. We suggest that this risk may actually be reduced, for the following two reasons. First, it has been shown that increased risk of hepatoxicity with MP or azathioprine (AZA) is associated with higher Aliment Pharmacol Ther 2005; 22: 1161–1166.

Does low-dose allopurinol, with azathioprine, cyclosporin and prednisolone, improve renal transplant immunosuppression?

This communication reports the results of two studies aimed at improving the immunosuppressive efficacy of azathioprine in renal transplant patients. Although azathioprine has been used in transplantation for over 30 years, the mechanism of its cytotoxicity is not yet completely defined. It is generally accepted that its principal mode of action involves rapid conversion in vivo to 6-mercaptopurine (6MP), which is further metabolised via 6-thioinosinic acid to cytotoxic thioguanine nucleotides (1). The efficacy of 6MP is reduced by catabolic pathways, one of them being via xanthine oxidase (XO), which oxidises 6MP to thiouric acid. Thus the therapeutic combination of azathioprine with allopurinol, which inhibits the XO path, is usually contra-indicated, having been associated with bone marrow toxicity (2). The importance of this catabolic route is illustrated by the severe myelotoxicity reported in an XO-deficient patient treated with azathioprine (3). Azathioprine and 6MP catabolism may also occur via aldehyde oxidase (AO) and thiopurine methyltransferase (TPMT) (Figure 1).

Thiopurine Methyltransferase Activity and Efficacy of Azathioprine Immunosuppression in Transplant Recipients

Azathioprine (AZA) has been given to transplanted patients since 1963 (1), but despite extensive use its metabolism is still not completely understood. After oral administration, it is converted rapidly to 6-mercaptopurine (6MP), mainly in the liver and the gut (2). 6MP is metabolised by three competing pathways (Figure 1): A) conversion to 6-thioinosinic acid by hypoxanthine guanine phosphoribosyltransferase (HPRT) and thence thioguanine nucleotides which exert their cytotoxicity by incorporation into DNA and RNA (2,3); B) catabolism by xanthine oxidase (XOD) to 6-thiouric acid; C) conversion to 6-methyl mercaptopurine (6MeMP) by the enzyme thiopurine methyl transferase (TPMT), which has a wide range of activity in the normal population and is inherited in a autosomal codominant fashion (3,4).

Monitoração terapêutica da azatioprina: uma revisão

Thioguanine nucleotides (6-TGN), active metabolites of azathioprine (AZA) and 6-mercaptopurine (6-MP), act as purine antagonists, inhibiting DNA, RNA, and protein synthesis and inducing cytotoxicity and immunosuppression. The genetically determined thiopurine methyltransferase enzyme (TPMT) is involved in the metabolism of these agents and, theoretically, determines the clinical response to thiopurines. Low activity of this enzyme decreases the methylation of thiopurines, what results in potential overdosing, whereas high TPMT status leads to overproduction of toxic metabolite 6-methilmercaptopurine (6-MMP) and ineffectiveness of AZA and 6-MP. Several mutations in the TPMT gene have been identified and correlated with low activity phenotypes. In this study, we also discuss the therapeutic monitoring of these drugs by means of red blood cell 6-TGN levels, which correlate with immunosuppression and mielotoxicity. 6-MMP is directly connected with hepatotoxicity. These metabolites assays are associated with the use of appropriate doses of this drug, what results in a better control of the disease and a decreased use of corticosteroids.

Comparação entre diagnósticos clínicos e histológicos no pós-transplante renal

PURPOSE: To assess the agreement between clinical and histopathological diagnosis in a renal transplantation center, 40 episodes of acute renal failure were studied. METHODS: Kidney biopsies were performed at the moment that a clinical diagnosis was made by the staff. RESULTS: Nineteen episodes of acute tubular necrosis (ATN), eighteen episodes of acute cellular rejection (ACR), 2 humoral rejections and 1 acute cyclosporin nephrotoxicity episodes were diagnosed. ATN episodes were confirmed by renal biopsy in 84.21%, ACR episodes in 83.33%, humoral rejections in 100%. Renal biopsy showed ATN in the occurrence of clinical cyclosporin nephrotoxicity. Total agreement was 82.5%. CONCLUSION: There is a good relationship between clinical and histopathological diagnosis in the post-transplantation period. Diagnostic mistakes occurred mainly when oliguria was present.

Mycophenolate or azathioprine maintenance in lupus nephritis

Intravenous (IV) cyclophosphamide has been first-line treatment for inducing disease remission in lupus nephritis. The comparative efficacy and toxicity of newer agents such as mycophenolate mofetil (MMF) and calcineurin inhibitors are uncertain.