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The New Zealand Medical Journal

 Journal of the New Zealand Medical Association, 25-July-2003, Vol 116 No 1178

6-thioguanine nucleotides and thiopurine methyltransferase activity: important factors determining response to treatment and incidence of adverse effects from azathioprine and 6-MP
The thiopurine drugs, azathioprine and its metabolite 6-mercaptopurine (6-MP), are among the most effective agents for maintaining remission of the inflammatory bowel diseases, Crohn’s disease and ulcerative colitis.1,2 They are also used to treat a number of other conditions including acute lymphoblastic leukaemia, psoriasis and rheumatoid arthritis. They require metabolism to 6-thioguanine nucleotides (6-TGNs) for clinical effect (efficacy and toxicity). Unfortunately, the metabolism of these drugs is complex (Figure 1) and patients have highly variable 6-TGN concentrations for a given dose.3 For example, a standard dose of azathioprine might result in extremely high 6-TGN concentrations and profound myelosuppression in some patients, whereas other individuals might fail to respond to the same dose because of subtherapeutic 6-TGN concentrations.

Figure 1. Metabolism of thiopurine medications (click here to view)

In a recent study, 6-TGN concentrations greater than 235 pmol/8x108 were shown to correlate with disease remission for patients with inflammatory bowel disease (measured by clinical end points).4 Elevated concentrations (>5700 pmol/8x108) of another metabolite, 6-methylmercaptopurine (6-MMP) have been associated with hepatotoxicity.5
The clinical application of measuring 6-TGN and 6-MMP concentrations is in its early stages. However, there are a number of situations in which monitoring may be indicated. If a patient is not responding to an adequate trial of a thiopurine drug, a ‘therapeutic’ 6-TGN concentration (ie, >235 pmol/8x108) suggests that further dose escalation is unlikely to result in improved efficacy. This allows the drug to be stopped and alternate therapies to be trialled. On the other hand, if the 6-TGN concentration is low in such a patient, this may suggest non-compliance (especially if combined with a low 6-MMP concentration), under-dosing (if combined with an appropriate 6-MMP concentration) or drug resistance (if combined with a high 6-MMP concentration). This allows the clinician to educate the patient to improve compliance, increase the dose or cease the drug respectively.5,6
Traditionally, the dose of the thiopurine drug is titrated against mean cell volume, total white cell count or neutrophil count. Often the dose would be increased until leucopenia was encountered. Data concerning this approach are conflicting and suggest that it is less precise than metabolite monitoring.7,8
While a therapeutic range for 6-TGN concentration has been proposed for patients with inflammatory bowel disease, this is not the case for other conditions for which thiopurine drugs are used, except in childhood leukaemia where dosing of 6-MP may be adjusted according to 6-TGN concentration.9
The Departments of Clinical Pharmacology and Toxicology at Christchurch Hospital have developed assays for measuring the concentrations of 6-TGN and 6-MMP. These complement the thiopurine methyltransferase (TPMT) phenotyping and genotyping testing that are also available.
We advocate use of therapeutic drug monitoring for patients with inflammatory bowel disease taking azathioprine or 6-MP who are not responding appropriately despite an adequate duration of treatment. These tests may help to guide clinicians regarding the choice of dose escalation, drug cessation, or in discussing issues of compliance with the patient.
Richard Gearry
Department of Gastroenterology
Murray Barclay
Departments of Gastroenterology and Clinical Pharmacology
Sharon Gardiner
Department of Clinical Pharmacology
Christchurch Hospital
Mei Zhang
Department of Clinical Pharmacology, Christchurch Hospital
Canterbury Health Laboratories

References:
  1. George J, Present JH, Pou R, et al The long-term outcome of ulcerative colitis treated with 6-mercaptopurine. Am J Gastroenterol 1996;91:1711–4.
  2. Pearson DC, May GR, Fick G, Sutherland LR. Azathioprine for maintenance of remission in Crohn’s disease. Cochrane Database of Syst Rev 2001, CD000067.
  3. Gardiner SJ, Begg EJ, Barclay ML, Kirkpatrick CMJ. Genetic polymorphism and outcomes with azathioprine and 6-mercaptopurine. Adverse Drug React Toxicol Rev 2000;19:293–312.
  4. Dubinsky MC, Lamothe S, Yang HY, et al. Pharmacogenomics and metabolite measurement for 6-mercaptopurine therapy in inflammatory bowel disease. Gastroenterology 2000;118:705–13.
  5. Dubinsky MC, Yang H, Hassard PV, et al. 6-MP metabolite profiles provide a biochemical explanation for 6-MP resistance in patients with inflammatory bowel disease. Gastroenterology 2002;122:904–15.
  6. Cuffari C, Hunt S, Bayless T. Utilisation of erythrocyte 6-thioguanine metabolite levels to optimise azathioprine therapy in patients with inflammatory bowel disease. Gut 2001;48:642–6.
  7. Decaux G, Propert F, Horsmans Y, Desager JP. Relationship between red cell mean corpuscular volume and 6-thioguanine nucleotides in patients treated with azathioprine. J Lab Clin Med 2000;135:256–62.
  8. Sandborn WJ, Tremaine WJ, Wolf DC, et al. Lack of effect of intravenous administration on time to respond to azathioprine for steroid-treated Crohn’s disease. North American Azathioprine Group .Gastroenterology 1999;117:527–35.
  9. Lilleyman JS, Lennard L. Mercaptopurine metabolism and risk of relapse in childhood lymphoblastic leukaemia. Lancet 1994;343:1188–90.


Azathioprine, 6-mercaptopurine (6-MP) and thioguanine are cytotoxic agents, collectively known as thiopurines. These drugs are used in fields such as oncology, gastroenterology, organ transplantation, rheumatology and dermatology.1,2 Although very effective medications, they are also highly toxic, with significant side effects occurring in many patients.
Thiopurine methyltransferase (TPMT) is an enzyme that is partly responsible for the metabolism of thiopurine drugs (Figure 1, click here to view). TPMT shows trimodal variation in the Caucasian population, with approximately 90% of people having enzyme activity in the normal range, 10% having reduced activity and 1/300 no activity. This variation is due to the co-dominant inheritance of inactive mutant TPMT alleles.2
Low TPMT activity leads to excessive production of 6-thioguanine nucleotides, as more 6-MP is processed by hypoxanthine-guanine phosphoribosyltransferase (HGPRT) (Figure 1, click here to view). Consequently, the frequency of thiopurine myelosuppressive side effects is increased in individuals heterozygous for inactive TPMT alleles, and is very high in homozygotes.2 This risk has led many investigators to suggest mandatory testing for TPMT activity, prior to commencing thiopurine treatment.3–5 If a patient has no enzyme activity, thiopurines should be avoided or given at a much lower dose (10% of standard dose has been suggested). If a patient has intermediate activity (ie, is heterozygous for an inactivating mutation), the thiopurine starting dose should be lowered (50–60% of standard dose has been suggested) and white cell count carefully monitored. White cell count monitoring must continue as usual in individuals with TPMT levels in the normal range, as TPMT deficiency is not the sole cause of myelosuppression associated with these drugs.5
Canterbury Health Laboratories has developed a combined activity/genotype assay for TPMT. Activity level is tested on all samples and those with low TPMT activities are genotyped for the two most common mutant alleles. Genotyping provides confirmation of phenotype (85–95% of TPMT deficiency results from the two alleles assayed) and allows testing of other family members.
The importance of TPMT in clinical practice is illustrated by a recent case from Christchurch Hospital (personal communication, R Gearry, 2003). A standard dose of azathioprine was used to treat a patient with inflammatory bowel disease; the patient then went on to develop severe myelosuppression and spent three days recovering in the Bone Marrow Transplant Unit. Subsequent TPMT testing revealed that the patient in question was one of the 1/300 people who have no TPMT activity due to homozygosity for a mutant allele. In addition to the medicolegal risk and the cost to the patient, the economic burden of treating neutropenic patients is considerable.3 Early economic analyses have indicated that prevention of severe myelosuppression in TPMT-deficient patients by screening each patient prior to initiating treatment would have a favourable cost-benefit ratio.1,3 As evidence continues to accumulate in support of the determination of TPMT activity prior to thiopurine treatment, clinicians in a variety of fields will need to consider including routine TPMT testing in their practice.4,5
James Harraway
Peter George
Canterbury Health Laboratories
Rebecca Roberts
Martin Kennedy
Christchurch School of Medicine
Linda Pike
Canterbury Health Laboratories

References:
  1. Holme SA, Duley JA, Sanderson J, et al. Erythrocyte thiopurine methyl transferase assessment prior to azathioprine use in the UK. QJM 2002;95:439–44.
  2. Evans WE, Hon YY, Bomgaars L, et al. Preponderance of thiopurine S-methyltransferase deficiency and heterozygosity among patients intolerant to mercaptopurine or azathioprine. J Clin Oncol 2001;19:2293–301.
  3. Tavadia SM, Mydlarski PR, Reis MD, et al. Screening for azathioprine toxicity: a pharmacoeconomic analysis based on a target case. J Am Acad Dermatol 2000;42:628–32.
  4. Lennard L. TPMT in the treatment of Chron’s disease with azathioprine. Gut 2002;51:143–6.
  5. Dubinsky M. Maximising thiopurine therapy in inflammatory bowel disease. Clinical Perspectives in Gastroenterology: 343–6, Nov/Dec 2002.


     
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