recombinant polymerases in vitro. Antimicrob. Agents Chemother.2002; 46: 2525–32.

10 Tenney DJ, Levine SM, Rose RE et al. Clinical emergence ofentecavir-resistant hepatitis B virus requires additional substitutionsin virus already resistant to Lamivudine. Antimicrob. AgentsChemother. 2004; 48: 3498–507.

11 Tenney DJ, Rose RE, Baldick CJ et al. Two-year assessment ofentecavir resistance in Lamivudine-refractory hepatitis B viruspatients reveals different clinical outcomes depending on theresistance substitutions present. Antimicrob. Agents Chemother. 2007;51: 902–11.

12 Colonno R, Rose R, Pokornowski K et al. Four year assessment ofentecavir resistance in nucleoside naive and lamivudine refractorypatients. J. Hepatol. 2007; 46 (Suppl. 1): S294.

13 Lampertico P, Vigano M, Manenti E, Iavarone M, Sablon E,Colombo M. Low resistance to adefovir combined with lamivudine:a 3-year study of 145 lamivudine-resistant hepatitis B patients.Gastroenterology 2007; 133: 1445–51.

14 Lee YS, Suh DJ, Lim YS et al. Increased risk of adefovir resistancein patients with lamivudine-resistant chronic hepatitis B after 48weeks of adefovir dipivoxil monotherapy. Hepatology 2006; 43:1385–91.

15 Chen CH, Wang JH, Lee CM et al. Virological response andincidence of adefovir resistance in lamivudine-resistant patientstreated with adefovir dipivoxil. Antivir. Ther. 2006; 11: 771–8.

16 Lampertico P, Vigano M, Manenti E, Iavarone M, Lunghi G,Colombo M. Adefovir rapidly suppresses hepatitis B inHBeAg-negative patients developing genotypic resistance tolamivudine. Hepatology 2005; 42: 1414–9.

17 van Bommel F, Zollner B, Sarrazin C et al. Tenofovir for patientswith lamivudine-resistant hepatitis B virus (HBV) infection and highHBV DNA level during adefovir therapy. Hepatology 2006; 44:318–25.

18 Lai CL, Gane E, Liaw YF et al. Telbivudine versus lamivudine inpatients with chronic hepatitis B. N. Engl. J. Med. 2007; 357:2576–88.

19 Han SH, Lai CL, Gane E et al. Telbivudine GLOBE Trial at YearTwo: Efficacy, Safety, and Predictors of Outcome in Patients withChronic Hepatitis B. Gastroenterology 2007; 132: A–765. [S1777].

Azathioprine: When less ismoreJohn A Duley*,† and Timothy HJ Florin*,‡

*University of Queensland, †Pathology Department and‡Gastroenterology Unit, Mater Health Services Adult Hospital,Brisbane, Australia

See article in J. Gastroenterol. Hepatol. 2008; 23: 1373–1377.

Thiopurine pharmacogeneticsThe thiopurine pro-drugs—azathioprine (AZA), mercaptopurine(6MP) and, to a lesser extent, thioguanine (6TG)—have provided

one of the classic ‘pharmacogenetic’ models, arising from twoaspects of metabolism of the drugs. The first was the role ofgenetic variation of thiopurine methyltransferase (TPMT). In Cau-casian populations, approximately one in 300 individuals is com-pletely TPMT deficient and approximately 11% have intermediateactivity. The second aspect of thiopurine pharmacogenetics wasthe development of an assay for red cell 6-thioguanine nucleotide(TGN), the immunosuppressive form of all thiopurine drugs and,later, for methylthiopurine metabolites as the therapeutically inac-tive but hepatotoxic products of TPMT. Eventually, TPMT geno-typing married TGN monitoring, thereby providing a metaboliccorrelation with genetic status, and thiopurine pharmacogeneticswas born.1

Following on from their developmental use for leukemia andorgan transplantation, thiopurines have also been a great successstory for inflammatory bowel disease (IBD). The success rate forinducing remission in Crohn’s disease in the West is presently40–66% for AZA/6MP.2 The response rate of AZA/6MP is higherfor TPMT heterozygotes, typically 70–90%.3,4

TPMT: Optimising thiopurine dosingand safetyTwo decades of experience with measuring TGN led to an ‘optimaltherapeutic range’ of approximately 250–450 units (pmol/8 ¥ 108

red cells), using red cells as surrogates for the target, activated Tlymphocytes. This TGN range, combined with a suggested rangefor methylthiopurines of less than approximately 6000 units, hasbeen adopted as a useful guide—and subject of a disputed patentin the USA—when prescribing AZA/6MP for IBD and otherdiseases.4

In the West, the typical AZA dose is 2–2.5 mg/kg per day forpatients with normal TPMT. Prospective TPMT genotyping orphenotyping identifies TPMT-deficient patients who are at highrisk for developing potentially fatal bone marrow toxicity on thi-opurines. However, use of the TPMT test must be tempered by itspoor predictive capability, as patients with zero or intermediateTPMT activity constitute only one in four cases of clinically sig-nificant leukopenia.5

This Western experience provides a backdrop to an article byAndoh and coworkers in this issue of Journal of Gastroenterologyand Hepatology,6 which reports, for the first time, on the Japaneseusage of AZA/6MP for IBD. There are major differences with theWest. First and foremost, determining TPMT status appears irrel-evant in Japan, because of the low incidence of its polymorphism.A survey of 32 Brazilian-residing Japanese in 1993 failed to find alow activity variant,7 implying a mutation frequency of approxi-mately 3% in the cohort. More recently, TPMT allelic mutation inJapan was estimated to be approximately 1%,8 and this fits with thereport of Andoh and coworkers, where no TPMT mutations werefound among 83 patients. The Hardy–Weinberg distribution wouldthus predict complete TPMT deficiency in Japan to be approxi-mately one in 40 000, which would not make routine screeningeconomic.

The low risk of TPMT deficiency might be considered a reasonto adopt an aggressive approach to thiopurine prescribing, but theauthors point out that Japanese prescribing of AZA/6MP is con-servative, generally using low doses of 1 mg/kg per day AZA or

Accepted for publication 25 June 2008.

Correspondence

Dr John Duley, Mater Pathology Services, Mater Hospital, Brisbane,Qld 4101, Australia. Email: john.duley@mater.org.au

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Journal compilation © 2008 Journal of Gastroenterology and Hepatology Foundation and Blackwell Publishing Asia Pty Ltd

0.6 mg/kg per day 6MP, ostensibly to avoid toxicity, but alsobecause thiopurine efficacy in Japanese IBD patients has beenperceived as being higher than in Caucasians.9 Andoh and cowork-ers report a remission rate of 50%.6 This efficacy, which should beassessed in the context of the clinical severity of the disease in theirpatients, seems comparable with rates in Western studies, yet it isachieved with half of the usual Western dose of AZA/6MP. Whileall of the Japanese patients were taking 5-aminosalicylates, knownto have some in vitro inhibitory activity against TPMT, the effectof these drugs on TGN is, at best, modest.10

The authors did not comment on whether thiopurine toxicityamong patients manifests as dose-dependent side-effects, such asbone marrow suppression or hepatotoxicity, or dose-independentside-effects. It would have been beneficial to have methylthiopu-rine measurements in the Japanese patients, as these metaboliteswould provide some insight into toxicity mechanisms and TPMTactivity.

TGN levels in Japanese IBD patientsA novel finding is that the lower thiopurine doses used in Andoh’sstudy produce red cell TGN concentrations similar to thoseexpected in Caucasians on higher doses: 11 of the Japanesepatients had red cell TGN in excess of 500 units.

There are difficulties, however, with interpreting TGN levelsmeasured this way.11 First, there may be a problem with the reli-ance on the red cell as a surrogate for the target cells in IBDimmunosuppression. Red cells are peripheral circulating cells,whereas inflammation in IBD is amplified by resident non-circulating activated T lymphocytes and macrophages. Second, redcells are unable to synthesize TGN directly from AZA/6MP andmust rely on an unknown ‘third-party’ tissue. Finally, the com-monly used assay does not measure TGN directly, but as thehydrolysis product 6TG, so these measurements do not provideinformation on the relative contributions of the different forms ofTGN nor would they be informative in the context of the faster-acting 6TG.

One theoretical explanation for higher TGN levels (and perhapsgreater susceptibility to AZA toxicity) in the Japanese may bereduced TPMT activity in vivo resulting from lower levels of theessential TPMT cofactor, S-adenosylmethionine. An adequatesupply of S-adenosylmethionine is dependent upon the folate meta-bolic pathway, which is regulated by the key enzyme, methylenetetrahydrofolate reductase (MTHFR). Dose-dependent side-effectsin Caucasian IBD patients on AZA have been associated with acommon MTHFR mutation, 677T.12 But the frequencies of this, anda second MTHFR mutation 1298C, are relatively similar for Cau-casian and Japanese populations, which would appear to negate arole for MTHFR in low TPMT/high TGN levels among Japanese.

Another explanation of the relatively high TGN levels in Japa-nese may be ethnic differences in transporter genes regulatingthiopurine uptake. This is a relatively new area of research and onegroup has begun to approach this question, but with negativeresults so far.13

Dose-independent AZA toxicityThere are potentially other genetic differences influencing thiopu-rine response in Japanese and Caucasian IBD patients. Thecause(s) of thiopurine allergic toxicity, principally flu-like

symptoms and pancreatitis, remains to be resolved,9 but one poly-morphic gene that has been implicated is inosine triphosphatehydrolase (ITPA).

Interest in ITPA deficiency arose from a study in British IBDpatients on AZA, which found a significantly increased risk ofallergic-type toxicity associated with a common mutation, C94A.This mutation, which is responsible for profound ITPA deficiency,has a frequency of approximately 6% among Caucasians, but, inJapan, it is much higher, at approximately 15%.14 We would there-fore predict any association between AZA/6MP allergic toxicityand ITPA deficiency to be more pronounced in Japan.

Future directionsThe ability to use 6TG without the dreaded side-effect of veno-occlusive disease would provide a major therapeutic advance,because this drug works much faster and is associated with fewerallergic effects, such as pancreatitis.15

Another therapeutic advance comes with allopurinol/thiopurinecotherapy. This is commonly used in renal transplants, where therule of thumb is to reduce the thiopurine to approximately 25–33%of the normal dose. This cotherapy is enjoying a renaissance inIBD, with Sparrow and coworkers demonstrating that this combi-nation boosts red cell TGN and reduces hepatotoxic methylthiopu-rine metabolites.16 This paradoxical effect, which is not predictedby the classical pathways of AZA/6MP metabolism, results inincreased therapeutic efficacy with less medication. The combina-tion of allopurinol and thiopurines in IBD is being investigatedprospectively for safety and efficacy (M Sparrow, pers. comm.,2008). The effect of allopurinol on thiopurine metabolism isexerted by inhibition of xanthine oxidase. The latter enzyme is amajor catabolic pathway for thiopurines which is strongly affectedby dietary intake of purines. In this respect, our group has recentlyshed new light on the role of xanthine oxidase in thiopurinemetabolism with respect to dietary as well as genetic influences17

and these may be relevant to differences in the Japanese response.In summary, the apparently normal red cell TGN levels on

low-dose AZA/6MP in the Japanese population raises the possi-bility of genetic and environmental differences. Thus, furtherstudy of thiopurine pharmacokinetics among Japanese people iswarranted as a means towards unravelling genetic differences inthiopurine metabolism and predicting individual therapeuticresponse. The findings may facilitate more effective application ofthiopurines to control IBD, thereby benefiting those affected bythese chronic inflammatory diseases around the world.

References1 Lennard L, Van Loon JA, Lilleyman JS et al. Thiopurine

pharmacogenetics in leukemia: correlation of erythrocyte thiopurinemethyltransferase activity and 6-thioguanine nucleotideconcentrations. Clin. Pharmacol. Ther. 1987; 4: 18–25.

2 Bebb JR, Scott BB. How effective are the usual treatments forCrohn’s disease? Aliment. Pharmacol. Ther. 2004; 20: 151–9.

3 Ansari A, Hassan C, Duley J et al. Thiopurine methyltransferaseactivity and the use of azathioprine in inflammatory bowel disease.Aliment. Pharmacol. Ther. 2002; 16: 1743–50.

4 Seidman EG. Clinical use and practical application of TPMT enzymeand 6-mercaptopurine metabolite monitoring in IBD. Rev.Gastroenterol. Disord. 2003; 3 (Suppl. 1): S30–8.

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5 Colombel JF, Ferrari N, Debuysere H et al. Genotypic analysis ofthiopurine S-methyltransferase in patients with Crohn’s disease andsevere myelosuppression during azathioprine therapy.Gastroenterology 2000; 118: 1025–30.

6 Andoh A, Tsujikawa T, Ban H et al. Monitoring 6-thioguaninenucleotide concentrations in Japanese patients withinflammatory bowel disease. J. Gastroenterol. Hepatol.2008; 23: 1373–7.

7 Chocair PR, Duley JA, Sabbaga E et al. Fast and slow methylators:do racial differences influence risk of allograft rejection? Quart. J.Med. 1993; 86: 359–63.

8 Kubota T, Nishida A, Takeuchi K et al. Frequency distribution ofthiopurine S-methyltransferase activity in red blood cells ofa healthy Japanese population. Ther. Drug Monit. 2004; 26:319–21.

9 Hibi T, Inoue N, Ogata H et al. Introduction and overview: recentadvances in the immunotherapy of inflammatory bowel disease. J.Gastroenterol. 2003; 38 (Suppl. 15): 36–42.

10 Teml A, Schaeffeler E, Herrlinger KR et al. Thiopurine treatment ininflammatory bowel disease. Clinical pharmacology and implicationof pharmacogenetically guided dosing. Clin. Pharmacokinet. 2007;46: 187–208.

11 Duley JA, Florin THJ. Thiopurine therapies: problems, complexities,

and progress with monitoring thioguanine nucleotides. Ther. DrugMonit. 2005; 27: 647–54.

12 Arenas M, Simpson G, Lewis CM et al. Genetic variation in theMTHFR gene influences thiopurine methyltransferase activity. Clin.Chem. 2005; 51: 2371–4.

13 Conklin LS, Cuffari C, Saatian B et al. Inherent differences in 6MPtransport in human lymphocytes: correlation with drug-inducedcytotoxicity (DDW abstract). Gastroenterology 2008; 134 (Suppl.):A500.

14 Sumi S, Ueta A, Maeda T, Ito T et al. A Japanese case with inosinetriphosphate pyrophosphohydrolase deficiency attributable to anenzymatic defect in white blood cells. J. Inherit. Metab. Dis. 2004;27: 277–8.

15 Cheung TK, Florin THJ. 6-thioguanine: a new old drug to procureremission in inflammatory bowel disease. Intern. Med. J. 2003; 33:44–6.

16 Sparrow MP, Hande SA, Friedman S et al. Effect of allopurinol onclinical outcomes in inflammatory bowel disease nonresponders toazathioprine or 6-mercaptopurine. Clin. Gastroenterol. Hepatol.2007; 5: 209–14.

17 Ansari A, Aslam Z, De Sica A et al. The influence of xanthineoxidase on thiopurine metabolism in Crohn’s disease. Aliment.Pharmacol. Ther. 2008; in press.

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Journal compilation © 2008 Journal of Gastroenterology and Hepatology Foundation and Blackwell Publishing Asia Pty Ltd