A Physician’s Guide to Azathioprine Metabolite Testing Carmen Cuffari, MD, FRCP
Dr. Cuffari is Associate Professor of Pediatrics at The Johns Hopkins School of Medicine, Division of Pediatric Gastroenterology, in Baltimore, Md.
Abstract: 6-Mercaptopurine (6-MP) and its prodrug azathioprine
Address correspondence to: C. Cuffari, MD, FRCP, The Johns Hopkins Hospital, Department of Pediatrics, Division of Pediatric Gastroenterology and Nutrition, 600 N. Wolfe Street, Brady 320, Baltimore, MD 21287-2631; Tel: 410-955-8769; Fax: 410-955-1464; E-mail: [email protected].
methyl transferase (TPMT) activity, the key catabolic enzyme in 6-MP
(AZA) have proven efficacy in the maintenance of disease remission in patients with inflammatory bowel disease. Inherent differences in drug metabolism based on genetic polymorphism in thiopurine metabolism, have been shown to influence patient responsiveness to therapy. Indeed, tailoring the dose of AZA based on TPMT activity has been proposed as a useful clinical tool to improve overall clinical response and to avoid untoward side effects. Recent studies have shown that patients with intermediate TPMT activity should be initiated on a low (1 mg/kg) dosage of AZA and carefully monitored in order to minimize the risk of bone marrow suppression. Patients with very high TPMT enzyme activity levels (>15 U/mL) may very well respond to a higher (>2 mg/kg) dosage of AZA. However, these patients may remain refractory to conventional-dose therapy by shunting 6-MP metabolism away from the production of its active 6-thioguanine (6-TGn) nucleotide metabolite. Individualized AZA dosing based on TPMT enzyme activity is based on the notion of achieving therapeutic erythrocyte 6-TGn metabolite levels between 235–260 pmol/8×108 red blood cells. Future prospective controlled trials are still needed to validate the universal application of drug monitoring strategies in clinical practice.
C Keywords Azathioprine, 6-mercaptopurine, metabolites, IBD, therapy.
58
ritical-dose drugs are defined as medications that are used to treat critical disease states. Furthermore, these therapeutic agents must also demonstrate a wide interpatient variability in drug metabolism that may influence patient responsiveness to therapy and patient susceptibility to drug-induced cytotoxicity. Based on this definition, these inherent differences in drug metabolism would also necessitate establishing a therapeutic window of drug efficacy and toxicity through drug monitoring.1 Two critical-dose drugs that are well known for their immunosuppressive effects in the management of patients with Crohn’s disease (CD) and ulcerative colitis (UC) are 6-mercaptopurine (6-MP) and its prodrug azathioprine (AZA).1,2 The metabolism of 6-MP and AZA as it applies to the management of patients with inflammatory bowel disease (IBD) are herein reviewed. The role of metabolite testing will also be discussed based on a review of the
Gastroenterology & Hepatology Volume 2, Issue 1 January 2006
A Z AT H I O P R I N E M E TA B O L I T E T E S T I N G I N I B D
TPMT HPRT AZA
6-MP
thioinosine 5’ monophosphate
6-TG
XO
6-thiouric acid
Figure 1. The metabolism of azathioprine. AZA = azathioprine; HPRT = hypoxanthine phosphoribosyl transferase; 6-MP = 6-mercaptopurine; 6-TG = 6-thioguanine; TPMT = thiopurine S-methyltransferase; XO = xanthine oxidase.
literature, and several recommendations will be made on applying this technology in clinical practice. 6-MP Metabolism The immunosuppressive properties of 6-MP and AZA are most likely mediated through their interference with protein synthesis and nucleic acid metabolism in the sequence that follows antigen stimulation, as well as by their direct cytotoxic effects on lymphoid cells.3,4 Since 6-MP and AZA are by themselves inactive, they must be transformed into their active ribonucleotides, which function as purine antagonists. These purine antagonists are then incorporated into DNA, thereby interfering with DNA and protein interactions involved in ribonucleotide replication.4 The metabolism of 6-MP and AZA occurs intracellularly along the competing routes catalyzed by hypoxanthine phosphoribosyl transferase and thiopurine S-methyltransferase (TPMT), giving rise to 6-thioguanine nucleotides (6-TGn) and 6-methyl mercaptopurine (6-MMP), respectively (Figure 1).2 6-TGn is the active ribonucleotide of 6-MP that functions as a purine antagonist inducing lymphocytotoxicity and immunosuppression.5 An apparent genetic polymorphism has been observed in TPMT activity among white and black populations, with low TPMT levels noted in 0.3% of individuals and intermediate levels in 11% of individuals.2 TPMT enzyme deficiency is inherited as an autosomal recessive trait and, to date, 10 mutant alleles and several silent and intronic mutations have been described.6 In patients with the homozygous recessive and heterozygous TPMT genotype, 6-MP metabolism is shunted preferentially into the production of 6-TGn. Although 6-TGn is thought to be lymphocytoxic and therefore beneficial in the treatment of
patients with leukemia and lymphoma, patients with low or intermediate TPMT activity are at risk for bone marrow suppression by achieving potentially toxic erythrocyte 6-TGn levels on standard doses of 6-MP.7-10 Among these patients with leukemia with low TPMT enzyme activity levels, therapeutic erythrocyte 6-TGn metabolite levels can still be achieved without untoward cytotoxicity by lowering the dose of 6-MP 10- to 15-fold.11 This inherent variability in 6-MP metabolism has led many pediatric oncologists to implement pharmacogenomic screening prior to instituting maintenance 6-MP therapy in patients with lymphoma and leukemia.11 Pharmacogenetic differences in 6-MP and AZA metabolism are also known to influence clinical responsiveness to therapy in patients with leukemia. Patients with high TPMT activity shunt 6-MP metabolism away from the production of 6-TGn and into the preferential formation of 6-MMP metabolites. Despite the use of conventional therapeutic 6-MP dosing practices, these patients with leukemia remain either refractory to maintenance 6-MP therapy or at an increased risk for disease relapse. Azathioprine in IBD Ulcerative colitis and CD are critical disease states that are often difficult to manage clinically. Also, although 6-MP and AZA have proven clinical efficacy in the induction and maintenance of remission in patients with steroiddependent disease, therapeutic responsiveness to therapy ranges from 20% to 70% and is independent of drug dose.12-14 Indeed, the wide therapeutic dosing range used in clinical practice today, as well as the variation in clinical response time, seem to suggest that pharmacokinetic differences in drug metabolism may also influence clinical responsiveness to therapy. Clinical Parameters A meta-analysis by Pearson and colleagues13 of several well-controlled clinical trials in patients with CD affirmed that clinical response to AZA therapy is largely dependent on duration of therapy. Indeed, patients that are weaned from prednisone before AZA can achieve its effect do not have a favorable clinical response to therapy. Interestingly, a therapeutic response would also seem to require a delay in clinical response time of at least 4 months in most patients.15 As a consequence, many clinicians are either reluctant to prescribe these slow-acting agents on account of the delayed clinical response times or will prematurely discontinue drug therapy in patients with a less than favorable clinical response. Most often, physicians will measure drug efficacy based on either an improvement in their patients’ clinical symptoms and quality of life or their ability to maintain
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C U F FA R I
Table 1. Clinical Responsiveness to 6-MP and AZA Therapy Based on Threshold* Erythrocyte 6-TGn Metabolite Levels 6-TGn Response Threshold Study
N (Response)
Above
Below
Odds Ratio
Dubinsky et al
92 (30)
.78
.40
5.0
Gupta et al
101 (47)
.56
.43
1.7
Belaiche et al
28 (19)
.75
.65
1.6
Cuffari et al25
82 (47)
.86
.35
11.6
Achar et al26
60 (24)
.51
.22
3.8
170 (114)
.64
.68
0.9
74 (14)
.24
.18
1.5
22
23 24
Lowry et al
27
Goldenberg et al
28
* 235–250 pmol/8 × 108 red blood cells. AZA = azathioprine; 6-MP = 6-mercaptopurine; 6-TGn = 6-thioguanine nucleotide.
60
remission while weaning off of corticosteroid therapy. In general, physicians will tend to rely on their clinical judgment and experience in determining the dose of AZA to be used in treating patients with IBD.16,17 Colonna and Korelitz18 have proposed that clinical responsiveness to therapy can best be achieved by tailoring the dose of 6-MP to induce leukopenia in patients with CD. In 51 patients with moderate to severe CD on long-term 6-MP therapy, clinical responsiveness correlated well with drug-induced leukopenia. None of their patients developed clinical signs of bone marrow suppression, required hospitalization for concurrent infection, or required transfusions despite total leukocyte counts of less than 5 × 103/mm3. Indeed, a true separation between immunosuppression19 and cytotoxicity has yet to be defined since the dosing of 6-MP and AZA has been based largely on clinical outcome. The wide range in AZA dosing used in clinical practice would suggest that a safe and established therapeutic dose has yet to be determined. As a consequence, the clinician must always remain aware of potential adverse effects, including allergic reactions, hepatitis, pancreatitis, bone marrow suppression, and lymphoma, while attempting to achieve an optimal therapeutic response.20 In pediatric oncology, the dosage of either 6-MP or AZA will often be tailored based on the measure of erythrocyte 6-TGn metabolite levels. The same notion of adhering to a therapeutic window of clinical efficacy and toxicity based on 6-TGn metabolite monitoring has yet to be universally accepted in the management of patients with IBD.
erythrocyte 6-TGn metabolite levels showed a strong inverse correlation with disease activity in adolescent patients with CD.21 Although a wide range of erythrocyte 6-TGn levels was associated with clinical responsiveness to therapy, patients with 6-TGn levels greater than 250 pmol/8 × 108 red blood cells (RBCs) were uniformly asymptomatic. Moreover, the lack of clinical response was clearly associated with low erythrocyte 6-TGn metabolite levels. In one patient, noncompliance was easily suspected in view of very low (15 U/mL blood) TPMT enzyme activity levels were also less likely to respond to therapy.35 High hepatic TPMT activity may draw most of the 6-MP from the plasma, thereby limiting the amount of substrate available for the bone marrow and peripheral leukocytes.37 This concept of rapid AZA metabolism interfering with therapeutic response could explain the low response rate in a recently published controlled trial in CD that compared high-dose oral (2 mg/kg per day) AZA therapy with and without initiating a short course of high-dose intravenous (40 mg/kg) AZA therapy. That study was confined to individuals with high median (>15 U/mL blood) TPMT enzyme activity levels so that the intravenous AZA treatment group could be studied safely. Even at 2 mg/kg per day of oral AZA therapy, only 20% of these rapid metabolizers in both groups achieved clinical remission,38 a clinical response that is lower than that reported in most consecutive patient publications.13-18 Normal Activity AZA metabolism is clearly influenced by inherent differences in TPMT activity present within the population.
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C U F FA R I
Figure 2. Azathioprine dosage recommendations based on TPMT phenotype. TPMT = thiopurine S-methyltransferase.
Instead of using a fixed dosage of either AZA or 6-MP for induction therapy, the knowledge of TPMT activity levels may allow physicians to predict clinical response and dose AZA in order to maximize efficacy while minimizing the risk of toxicity. A recent study has also shown that patients with above average (>12 U/mL blood) TPMT activity levels were more likely to require higher doses (2 mg/kg per day) of AZA from the outset in order to optimize erythrocyte 6-TGn metabolite levels. Patients with above average TPMT activity had a mean erythrocyte 6-TGn level that reached a plateau below a presumed therapeutic (250 pmol/8 × 108 RBCs) mean erythrocyte 6-TGn levels after 16 weeks of induction AZA therapy. This occurred even though both groups received a similar dosage of AZA. Clinically, 69% of patients with TPMT activity levels of less than or equal to 12 U/mL blood achieved a clinical response with therapeutic erythrocyte 6-TGn metabolite levels after 4 months of continuous therapy. In comparison, less than 30% of patients with above average (>12 U/mL blood) TPMT activity achieved a clinical response. These patients ultimately required the dosage of AZA to be further optimized to achieve therapeutic 6-TGn metabolite levels, thereby delaying their clinical response time.35 Concluding Remarks The careful monitoring of complete blood count and erythrocyte 6-TGn metabolite levels are indicated in
62
patients with either low or intermediate (15 U/mL blood) may very well respond to a high (>2 mg/kg per day) dose of AZA; however, the physician must realize that these patients may remain refractory to conventional-dose therapy due to shunting of 6-MP metabolism away from 6-TGn production.3 Moreover, further dose escalation in these patients may also render them at risk for AZA-induced hepatotoxicity (Figure 2). Phenotype testing will allow physicians to clearly identify this important subgroup of patients among those with a presumed normal genotype. It remains the author’s opinion that relying on either the white blood cell count or mean corpuscular volume as the only measure of dosing adequacy should be done with caution.39 The putative cytotoxicity of methylated metabolites in developing 6-MP–induced leukopenia suggest that leukopenia may not be an appropriate endpoint for all patients on antimetabolite therapy, especially those patients with high (>15 U/mL blood) TPMT activity. Although several studies have entertained a therapeutic index of clinical responsiveness to AZA therapy based on achieving therapeutic erythrocyte 6-TGn levels of 235–260 pmol/8 × 108 RBCs,22-28 prospective controlled trials are still needed to determine whether individualized AZA dosing strategies should be adopted based on inherent differences in TPMT activity in order to improve therapeutic effectiveness and clinical response time and lessen adverse side effects to AZA therapy. References 1. Kelly P, Kahan BD. Review: metabolism of immunosuppressant drugs. Curr Drug Metab. 2002;3:275-287. 2. Weinshilboum RN, Sladek Sl. Mercaptopurine pharmacogenetics: monogenic inheritance of erythrocyte thiopurine methyl transferase activity. Am J Hum Genet. 1980;32:651-662. 3. Christie NT, Drake S, Meyn RE. 6-thioguanine induced DNA damage as a determinant of cytotoxicity in cultured hamster ovary cells. Cancer Res. 1986;44:3665-3671. 4. Fairchild CR, Maybaum J, Kennedy KA. Concurrent unilateral chromatid damage and DNA strand breaks in response to 6-thioguanine treatment. Biochem Pharmacol. 1986;35:3533-3541. 5. Lennard L. The clinical pharmacology of 6-mercaptopurine in acute lymphoblastic leukemia. Eur J Clin Pharmacol. 1992;43:329-339. 6. Alves S, Prata MJ, Ferreira F, Amorim A. Screening of thiopurine methyl Stransferase mutations by horizontal conformation-sensitive gel electrophoresis. Human Mutat. 2000;15:246-253. 7. Evans WE, Horner M, Chu YQ, et al. Altered mercaptopurine metabolism,
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toxic effects, and dosage requirements in a thiopurine methyltransferase deficient child with acute lymphoblastic leukemia. J Pediatr. 1991;119:985-989. 8. Bostrom B, Erdman G. Cellular pharmacology of 6-mercaptopurine in acute lymphoblastic leukemia. Am J Pediatr Hematol Oncol. 1993;15:80-86. 9. Lennard L, Rees CA, Lilleyman JS, et al. Childhood leukemia: a relationship between intracellular 6-mercaptopurine metabolites and neutropenia. Br J Clin Pharmacol. 1993;16:359-363. 10. Zimm S, Collins JM, Riccardi R, et al. Variable bioavailability of oral mercaptopurine: is maintenance chemotherapy in acute lymphoblastic leukemia being optimally delivered. N Engl J Med. 1983;308:1005-1009. 11. McLeod HL, Relling MV, Liu Q, Pui CH, Evans WE. Polymorphic thiopurine methyl transferase in erythrocytes is indicative of activity in leukemic blasts from children with acute lymphoblastic leukemia. Blood. 1995;85:1897-7-1902. 12. Kombluth A, Sachar DB, Salomon P. Crohn’s disease. In: Sleisenger MH, Fordtran JS, eds. Gastrointestinal Diseases. Philadelphia, Pa: WB Saunders;1998:1708-1734. 13. Pearson DC, May GR, Fick GH, et al. Azathioprine and 6-mercaptopurine in Crohn’s disease: a meta-analysis. Ann Intern Med. 1995;122:132-142. 14. Ewe K, Press AG, Singe CC, et al. Azathioprine combined with prednisolone or monotherapy with prednisolone in active Crohn’s disease. Gastroenterology. 1993;105:367-372. 15. O’Brien JJ, Bayless TM, Bayless JA. Use of azathioprine or 6-mercaptopurine in the treatment of Crohn’s disease. Gastroenterology. 1991;101:39-46. 16. Korelitz BI, Adler DJ, Mendelsohn RA, et al. Long-term experience with 6-mercaptopurine in the treatment of Crohn’s disease. Am J Gastroenterol. 1993;88:1198-1205. 17. Present DH, Korelitz BI, Wisch N, et al. Treatment of Crohn’s disease with 6-mercaptopurine: a long-term, randomized, double-blind study. N Engl J Med. 1980;302:981-987. 18. Colonna T, Korelitz BI. The role of leukopenia in 6-mercaptopurine-induced remission of refractory Crohn’s disease. Am J Gastroenterol. 1993;89:362-366. 19. Brogan M, Hiserot J, Olicer M. The effects of 6-mercaptopurine on natural killer cell activities in Crohn’s disease. J Clin Immunol. 1985;5:204-211. 20. Present DH, Meltzer SJ, Krumholz MP, et al. 6-mercaptopurine in the management of inflammatory bowel disease: short and long-term toxicity. Ann Intern Med. 1995;111:641-649. 21. Cuffari C, Theoret Y, Latour S, et al. 6-mercaptopurine metabolism in Crohn’s disease: correlation with efficacy and toxicity. Gut. 1996;39:401-406. 22. Dubinsky MC, Lamothe S, Yang HY, et al. Pharmacogenomics and metabolite measurement for 6-mercaptopurine therapy in inflammatory bowel disease. Gastroenterology. 2000;118;705-713. 23. Gupta P, Gokhlae R, Kirschner BS. 6-mercaptopurine metabolite levels in children with inflammatory bowel disease. J Pediatr Gastroenterol Nutr. 2001;33:450-454. 24. Belaiche J, Desager JP, Horsman Y, Louis E. Therapeutic drug monitoring of
azathioprine and 6-mercaptopurine metabolites in Crohn’s disease. Scand J Gastroenterol. 2001;36:71-76. 25. Cuffari C, Hunt S, Bayless TM. Utilization of erythrocyte 6-thioguanine metabolite levels to optimize therapy in IBD. Gut. 2001;48:642-646. 26. Achar JP, Stevens T, Brzezinski A, Seidner D, Lashner B. 6-Thioguanine levels versus white blood cell counts in guiding 6-mercaptopruine and azathioprine therapy. Am J Gastroenterol. 2000;95:A272. 27. Lowry PW, Franklin CL, Weaver AL, et al. Leukopenia resulting from a drug interaction between azathioprine or 6-mercaptopurine and mesalamine, sulphasalazine or balsalazide. Gut. 2001;49:656-664. 28. Goldenberg BA, Rawsthorne P, Berstein CN. The utility of 6-thioguanine metabolite levels in managing patients with inflammatory bowel disease. Am J Gastroenterol. 2004;99:1744-1748. 29. 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;122:904-915. 30. Bo J, Schroder H, Kristinsoon J, et al. Possible carcinogenic effect of 6-mercaptopurine on bone marrow stem cells: relation to thiopurine metabolism. Cancer. 1999;86:1080-1086. 31. McLeod HL, Krynetski EY, Relling MV, Evans WE. Genetic polymorphism of thiopurine methyltransferase and its clinical relevance for childhood acute lymphoblastic leukemia. Leukemia. 2000;14:567-572. 32. Black AJ, McLeod HL, Capell HA. Thiopurine methyl transferase predicts therapy-limiting severe toxicity from azathioprine. Ann Intern Med. 1998;129:716-718. 33. Szumlanski C, Weinshilboum RN. Sulphasalazine inhibition of thio methyl transferase: possible mechanism of interaction with 6-mercaptopurine. Br J Clin Pharmacol. 1995;39:456-459. 34. Proujansky R, Maxwell M, Johnson J, et al. Molecular genotyping predicts complications of 6-mercaptopurine therapy in childhood IBD. Gastroenterology. 1999;116:A800. 35. Cuffari C, Dassoupolus T. Bayless TM. Thiopurine methyl-transferase activity influences clinical response to azathioprine therapy in patients with IBD. Clin Gastroenterol Hepatol. 2004;2:410-417. 36. Kaskas BA, Louis E, Hinderof U, et al. Safe treatment of thiopurine S-transferase deficient Crohn’s disease patients with azathioprine. Gut. 2003;52:140-142. 37. Lennard L, Lilleyman JS. Variable mercaptopurine metabolism and treatment outcome in childhood lymphoblastic leukemia. J Clin Oncol. 1989;7:1816-1823. 38. Sandborn WJ, Tremaine WJ, Wolfe DC, et al. Lack of effect of intravenous administration on time to respond to azathioprine for steroid-treated Crohn’s disease. Gastroenterology. 1999; 117:527-535. 39. Garza A, Sninsky CA. Changes in red cell mean corpuscular volume (MCV) during azathioprine or 6-mercaptopurine therapy for Crohn’s disease may indicate optimal dose titration. Gastroenterology. 2001;120;A3166.
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Dr. Cuffari is Associate Professor of Pediatrics at The Johns Hopkins School of Medicine, Division of Pediatric Gastroenterology, in Baltimore, Md.
Abstract: 6-Mercaptopurine (6-MP) and its prodrug azathioprine
Address correspondence to: C. Cuffari, MD, FRCP, The Johns Hopkins Hospital, Department of Pediatrics, Division of Pediatric Gastroenterology and Nutrition, 600 N. Wolfe Street, Brady 320, Baltimore, MD 21287-2631; Tel: 410-955-8769; Fax: 410-955-1464; E-mail: [email protected].
methyl transferase (TPMT) activity, the key catabolic enzyme in 6-MP
(AZA) have proven efficacy in the maintenance of disease remission in patients with inflammatory bowel disease. Inherent differences in drug metabolism based on genetic polymorphism in thiopurine metabolism, have been shown to influence patient responsiveness to therapy. Indeed, tailoring the dose of AZA based on TPMT activity has been proposed as a useful clinical tool to improve overall clinical response and to avoid untoward side effects. Recent studies have shown that patients with intermediate TPMT activity should be initiated on a low (1 mg/kg) dosage of AZA and carefully monitored in order to minimize the risk of bone marrow suppression. Patients with very high TPMT enzyme activity levels (>15 U/mL) may very well respond to a higher (>2 mg/kg) dosage of AZA. However, these patients may remain refractory to conventional-dose therapy by shunting 6-MP metabolism away from the production of its active 6-thioguanine (6-TGn) nucleotide metabolite. Individualized AZA dosing based on TPMT enzyme activity is based on the notion of achieving therapeutic erythrocyte 6-TGn metabolite levels between 235–260 pmol/8×108 red blood cells. Future prospective controlled trials are still needed to validate the universal application of drug monitoring strategies in clinical practice.
C Keywords Azathioprine, 6-mercaptopurine, metabolites, IBD, therapy.
58
ritical-dose drugs are defined as medications that are used to treat critical disease states. Furthermore, these therapeutic agents must also demonstrate a wide interpatient variability in drug metabolism that may influence patient responsiveness to therapy and patient susceptibility to drug-induced cytotoxicity. Based on this definition, these inherent differences in drug metabolism would also necessitate establishing a therapeutic window of drug efficacy and toxicity through drug monitoring.1 Two critical-dose drugs that are well known for their immunosuppressive effects in the management of patients with Crohn’s disease (CD) and ulcerative colitis (UC) are 6-mercaptopurine (6-MP) and its prodrug azathioprine (AZA).1,2 The metabolism of 6-MP and AZA as it applies to the management of patients with inflammatory bowel disease (IBD) are herein reviewed. The role of metabolite testing will also be discussed based on a review of the
Gastroenterology & Hepatology Volume 2, Issue 1 January 2006
A Z AT H I O P R I N E M E TA B O L I T E T E S T I N G I N I B D
TPMT HPRT AZA
6-MP
thioinosine 5’ monophosphate
6-TG
XO
6-thiouric acid
Figure 1. The metabolism of azathioprine. AZA = azathioprine; HPRT = hypoxanthine phosphoribosyl transferase; 6-MP = 6-mercaptopurine; 6-TG = 6-thioguanine; TPMT = thiopurine S-methyltransferase; XO = xanthine oxidase.
literature, and several recommendations will be made on applying this technology in clinical practice. 6-MP Metabolism The immunosuppressive properties of 6-MP and AZA are most likely mediated through their interference with protein synthesis and nucleic acid metabolism in the sequence that follows antigen stimulation, as well as by their direct cytotoxic effects on lymphoid cells.3,4 Since 6-MP and AZA are by themselves inactive, they must be transformed into their active ribonucleotides, which function as purine antagonists. These purine antagonists are then incorporated into DNA, thereby interfering with DNA and protein interactions involved in ribonucleotide replication.4 The metabolism of 6-MP and AZA occurs intracellularly along the competing routes catalyzed by hypoxanthine phosphoribosyl transferase and thiopurine S-methyltransferase (TPMT), giving rise to 6-thioguanine nucleotides (6-TGn) and 6-methyl mercaptopurine (6-MMP), respectively (Figure 1).2 6-TGn is the active ribonucleotide of 6-MP that functions as a purine antagonist inducing lymphocytotoxicity and immunosuppression.5 An apparent genetic polymorphism has been observed in TPMT activity among white and black populations, with low TPMT levels noted in 0.3% of individuals and intermediate levels in 11% of individuals.2 TPMT enzyme deficiency is inherited as an autosomal recessive trait and, to date, 10 mutant alleles and several silent and intronic mutations have been described.6 In patients with the homozygous recessive and heterozygous TPMT genotype, 6-MP metabolism is shunted preferentially into the production of 6-TGn. Although 6-TGn is thought to be lymphocytoxic and therefore beneficial in the treatment of
patients with leukemia and lymphoma, patients with low or intermediate TPMT activity are at risk for bone marrow suppression by achieving potentially toxic erythrocyte 6-TGn levels on standard doses of 6-MP.7-10 Among these patients with leukemia with low TPMT enzyme activity levels, therapeutic erythrocyte 6-TGn metabolite levels can still be achieved without untoward cytotoxicity by lowering the dose of 6-MP 10- to 15-fold.11 This inherent variability in 6-MP metabolism has led many pediatric oncologists to implement pharmacogenomic screening prior to instituting maintenance 6-MP therapy in patients with lymphoma and leukemia.11 Pharmacogenetic differences in 6-MP and AZA metabolism are also known to influence clinical responsiveness to therapy in patients with leukemia. Patients with high TPMT activity shunt 6-MP metabolism away from the production of 6-TGn and into the preferential formation of 6-MMP metabolites. Despite the use of conventional therapeutic 6-MP dosing practices, these patients with leukemia remain either refractory to maintenance 6-MP therapy or at an increased risk for disease relapse. Azathioprine in IBD Ulcerative colitis and CD are critical disease states that are often difficult to manage clinically. Also, although 6-MP and AZA have proven clinical efficacy in the induction and maintenance of remission in patients with steroiddependent disease, therapeutic responsiveness to therapy ranges from 20% to 70% and is independent of drug dose.12-14 Indeed, the wide therapeutic dosing range used in clinical practice today, as well as the variation in clinical response time, seem to suggest that pharmacokinetic differences in drug metabolism may also influence clinical responsiveness to therapy. Clinical Parameters A meta-analysis by Pearson and colleagues13 of several well-controlled clinical trials in patients with CD affirmed that clinical response to AZA therapy is largely dependent on duration of therapy. Indeed, patients that are weaned from prednisone before AZA can achieve its effect do not have a favorable clinical response to therapy. Interestingly, a therapeutic response would also seem to require a delay in clinical response time of at least 4 months in most patients.15 As a consequence, many clinicians are either reluctant to prescribe these slow-acting agents on account of the delayed clinical response times or will prematurely discontinue drug therapy in patients with a less than favorable clinical response. Most often, physicians will measure drug efficacy based on either an improvement in their patients’ clinical symptoms and quality of life or their ability to maintain
Gastroenterology & Hepatology Volume 2, Issue 1 January 2006
59
C U F FA R I
Table 1. Clinical Responsiveness to 6-MP and AZA Therapy Based on Threshold* Erythrocyte 6-TGn Metabolite Levels 6-TGn Response Threshold Study
N (Response)
Above
Below
Odds Ratio
Dubinsky et al
92 (30)
.78
.40
5.0
Gupta et al
101 (47)
.56
.43
1.7
Belaiche et al
28 (19)
.75
.65
1.6
Cuffari et al25
82 (47)
.86
.35
11.6
Achar et al26
60 (24)
.51
.22
3.8
170 (114)
.64
.68
0.9
74 (14)
.24
.18
1.5
22
23 24
Lowry et al
27
Goldenberg et al
28
* 235–250 pmol/8 × 108 red blood cells. AZA = azathioprine; 6-MP = 6-mercaptopurine; 6-TGn = 6-thioguanine nucleotide.
60
remission while weaning off of corticosteroid therapy. In general, physicians will tend to rely on their clinical judgment and experience in determining the dose of AZA to be used in treating patients with IBD.16,17 Colonna and Korelitz18 have proposed that clinical responsiveness to therapy can best be achieved by tailoring the dose of 6-MP to induce leukopenia in patients with CD. In 51 patients with moderate to severe CD on long-term 6-MP therapy, clinical responsiveness correlated well with drug-induced leukopenia. None of their patients developed clinical signs of bone marrow suppression, required hospitalization for concurrent infection, or required transfusions despite total leukocyte counts of less than 5 × 103/mm3. Indeed, a true separation between immunosuppression19 and cytotoxicity has yet to be defined since the dosing of 6-MP and AZA has been based largely on clinical outcome. The wide range in AZA dosing used in clinical practice would suggest that a safe and established therapeutic dose has yet to be determined. As a consequence, the clinician must always remain aware of potential adverse effects, including allergic reactions, hepatitis, pancreatitis, bone marrow suppression, and lymphoma, while attempting to achieve an optimal therapeutic response.20 In pediatric oncology, the dosage of either 6-MP or AZA will often be tailored based on the measure of erythrocyte 6-TGn metabolite levels. The same notion of adhering to a therapeutic window of clinical efficacy and toxicity based on 6-TGn metabolite monitoring has yet to be universally accepted in the management of patients with IBD.
erythrocyte 6-TGn metabolite levels showed a strong inverse correlation with disease activity in adolescent patients with CD.21 Although a wide range of erythrocyte 6-TGn levels was associated with clinical responsiveness to therapy, patients with 6-TGn levels greater than 250 pmol/8 × 108 red blood cells (RBCs) were uniformly asymptomatic. Moreover, the lack of clinical response was clearly associated with low erythrocyte 6-TGn metabolite levels. In one patient, noncompliance was easily suspected in view of very low (15 U/mL blood) TPMT enzyme activity levels were also less likely to respond to therapy.35 High hepatic TPMT activity may draw most of the 6-MP from the plasma, thereby limiting the amount of substrate available for the bone marrow and peripheral leukocytes.37 This concept of rapid AZA metabolism interfering with therapeutic response could explain the low response rate in a recently published controlled trial in CD that compared high-dose oral (2 mg/kg per day) AZA therapy with and without initiating a short course of high-dose intravenous (40 mg/kg) AZA therapy. That study was confined to individuals with high median (>15 U/mL blood) TPMT enzyme activity levels so that the intravenous AZA treatment group could be studied safely. Even at 2 mg/kg per day of oral AZA therapy, only 20% of these rapid metabolizers in both groups achieved clinical remission,38 a clinical response that is lower than that reported in most consecutive patient publications.13-18 Normal Activity AZA metabolism is clearly influenced by inherent differences in TPMT activity present within the population.
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C U F FA R I
Figure 2. Azathioprine dosage recommendations based on TPMT phenotype. TPMT = thiopurine S-methyltransferase.
Instead of using a fixed dosage of either AZA or 6-MP for induction therapy, the knowledge of TPMT activity levels may allow physicians to predict clinical response and dose AZA in order to maximize efficacy while minimizing the risk of toxicity. A recent study has also shown that patients with above average (>12 U/mL blood) TPMT activity levels were more likely to require higher doses (2 mg/kg per day) of AZA from the outset in order to optimize erythrocyte 6-TGn metabolite levels. Patients with above average TPMT activity had a mean erythrocyte 6-TGn level that reached a plateau below a presumed therapeutic (250 pmol/8 × 108 RBCs) mean erythrocyte 6-TGn levels after 16 weeks of induction AZA therapy. This occurred even though both groups received a similar dosage of AZA. Clinically, 69% of patients with TPMT activity levels of less than or equal to 12 U/mL blood achieved a clinical response with therapeutic erythrocyte 6-TGn metabolite levels after 4 months of continuous therapy. In comparison, less than 30% of patients with above average (>12 U/mL blood) TPMT activity achieved a clinical response. These patients ultimately required the dosage of AZA to be further optimized to achieve therapeutic 6-TGn metabolite levels, thereby delaying their clinical response time.35 Concluding Remarks The careful monitoring of complete blood count and erythrocyte 6-TGn metabolite levels are indicated in
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patients with either low or intermediate (15 U/mL blood) may very well respond to a high (>2 mg/kg per day) dose of AZA; however, the physician must realize that these patients may remain refractory to conventional-dose therapy due to shunting of 6-MP metabolism away from 6-TGn production.3 Moreover, further dose escalation in these patients may also render them at risk for AZA-induced hepatotoxicity (Figure 2). Phenotype testing will allow physicians to clearly identify this important subgroup of patients among those with a presumed normal genotype. It remains the author’s opinion that relying on either the white blood cell count or mean corpuscular volume as the only measure of dosing adequacy should be done with caution.39 The putative cytotoxicity of methylated metabolites in developing 6-MP–induced leukopenia suggest that leukopenia may not be an appropriate endpoint for all patients on antimetabolite therapy, especially those patients with high (>15 U/mL blood) TPMT activity. Although several studies have entertained a therapeutic index of clinical responsiveness to AZA therapy based on achieving therapeutic erythrocyte 6-TGn levels of 235–260 pmol/8 × 108 RBCs,22-28 prospective controlled trials are still needed to determine whether individualized AZA dosing strategies should be adopted based on inherent differences in TPMT activity in order to improve therapeutic effectiveness and clinical response time and lessen adverse side effects to AZA therapy. References 1. Kelly P, Kahan BD. Review: metabolism of immunosuppressant drugs. Curr Drug Metab. 2002;3:275-287. 2. Weinshilboum RN, Sladek Sl. Mercaptopurine pharmacogenetics: monogenic inheritance of erythrocyte thiopurine methyl transferase activity. Am J Hum Genet. 1980;32:651-662. 3. Christie NT, Drake S, Meyn RE. 6-thioguanine induced DNA damage as a determinant of cytotoxicity in cultured hamster ovary cells. Cancer Res. 1986;44:3665-3671. 4. Fairchild CR, Maybaum J, Kennedy KA. Concurrent unilateral chromatid damage and DNA strand breaks in response to 6-thioguanine treatment. Biochem Pharmacol. 1986;35:3533-3541. 5. Lennard L. The clinical pharmacology of 6-mercaptopurine in acute lymphoblastic leukemia. Eur J Clin Pharmacol. 1992;43:329-339. 6. Alves S, Prata MJ, Ferreira F, Amorim A. Screening of thiopurine methyl Stransferase mutations by horizontal conformation-sensitive gel electrophoresis. Human Mutat. 2000;15:246-253. 7. Evans WE, Horner M, Chu YQ, et al. Altered mercaptopurine metabolism,
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toxic effects, and dosage requirements in a thiopurine methyltransferase deficient child with acute lymphoblastic leukemia. J Pediatr. 1991;119:985-989. 8. Bostrom B, Erdman G. Cellular pharmacology of 6-mercaptopurine in acute lymphoblastic leukemia. Am J Pediatr Hematol Oncol. 1993;15:80-86. 9. Lennard L, Rees CA, Lilleyman JS, et al. Childhood leukemia: a relationship between intracellular 6-mercaptopurine metabolites and neutropenia. Br J Clin Pharmacol. 1993;16:359-363. 10. Zimm S, Collins JM, Riccardi R, et al. Variable bioavailability of oral mercaptopurine: is maintenance chemotherapy in acute lymphoblastic leukemia being optimally delivered. N Engl J Med. 1983;308:1005-1009. 11. McLeod HL, Relling MV, Liu Q, Pui CH, Evans WE. Polymorphic thiopurine methyl transferase in erythrocytes is indicative of activity in leukemic blasts from children with acute lymphoblastic leukemia. Blood. 1995;85:1897-7-1902. 12. Kombluth A, Sachar DB, Salomon P. Crohn’s disease. In: Sleisenger MH, Fordtran JS, eds. Gastrointestinal Diseases. Philadelphia, Pa: WB Saunders;1998:1708-1734. 13. Pearson DC, May GR, Fick GH, et al. Azathioprine and 6-mercaptopurine in Crohn’s disease: a meta-analysis. Ann Intern Med. 1995;122:132-142. 14. Ewe K, Press AG, Singe CC, et al. Azathioprine combined with prednisolone or monotherapy with prednisolone in active Crohn’s disease. Gastroenterology. 1993;105:367-372. 15. O’Brien JJ, Bayless TM, Bayless JA. Use of azathioprine or 6-mercaptopurine in the treatment of Crohn’s disease. Gastroenterology. 1991;101:39-46. 16. Korelitz BI, Adler DJ, Mendelsohn RA, et al. Long-term experience with 6-mercaptopurine in the treatment of Crohn’s disease. Am J Gastroenterol. 1993;88:1198-1205. 17. Present DH, Korelitz BI, Wisch N, et al. Treatment of Crohn’s disease with 6-mercaptopurine: a long-term, randomized, double-blind study. N Engl J Med. 1980;302:981-987. 18. Colonna T, Korelitz BI. The role of leukopenia in 6-mercaptopurine-induced remission of refractory Crohn’s disease. Am J Gastroenterol. 1993;89:362-366. 19. Brogan M, Hiserot J, Olicer M. The effects of 6-mercaptopurine on natural killer cell activities in Crohn’s disease. J Clin Immunol. 1985;5:204-211. 20. Present DH, Meltzer SJ, Krumholz MP, et al. 6-mercaptopurine in the management of inflammatory bowel disease: short and long-term toxicity. Ann Intern Med. 1995;111:641-649. 21. Cuffari C, Theoret Y, Latour S, et al. 6-mercaptopurine metabolism in Crohn’s disease: correlation with efficacy and toxicity. Gut. 1996;39:401-406. 22. Dubinsky MC, Lamothe S, Yang HY, et al. Pharmacogenomics and metabolite measurement for 6-mercaptopurine therapy in inflammatory bowel disease. Gastroenterology. 2000;118;705-713. 23. Gupta P, Gokhlae R, Kirschner BS. 6-mercaptopurine metabolite levels in children with inflammatory bowel disease. J Pediatr Gastroenterol Nutr. 2001;33:450-454. 24. Belaiche J, Desager JP, Horsman Y, Louis E. Therapeutic drug monitoring of
azathioprine and 6-mercaptopurine metabolites in Crohn’s disease. Scand J Gastroenterol. 2001;36:71-76. 25. Cuffari C, Hunt S, Bayless TM. Utilization of erythrocyte 6-thioguanine metabolite levels to optimize therapy in IBD. Gut. 2001;48:642-646. 26. Achar JP, Stevens T, Brzezinski A, Seidner D, Lashner B. 6-Thioguanine levels versus white blood cell counts in guiding 6-mercaptopruine and azathioprine therapy. Am J Gastroenterol. 2000;95:A272. 27. Lowry PW, Franklin CL, Weaver AL, et al. Leukopenia resulting from a drug interaction between azathioprine or 6-mercaptopurine and mesalamine, sulphasalazine or balsalazide. Gut. 2001;49:656-664. 28. Goldenberg BA, Rawsthorne P, Berstein CN. The utility of 6-thioguanine metabolite levels in managing patients with inflammatory bowel disease. Am J Gastroenterol. 2004;99:1744-1748. 29. 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;122:904-915. 30. Bo J, Schroder H, Kristinsoon J, et al. Possible carcinogenic effect of 6-mercaptopurine on bone marrow stem cells: relation to thiopurine metabolism. Cancer. 1999;86:1080-1086. 31. McLeod HL, Krynetski EY, Relling MV, Evans WE. Genetic polymorphism of thiopurine methyltransferase and its clinical relevance for childhood acute lymphoblastic leukemia. Leukemia. 2000;14:567-572. 32. Black AJ, McLeod HL, Capell HA. Thiopurine methyl transferase predicts therapy-limiting severe toxicity from azathioprine. Ann Intern Med. 1998;129:716-718. 33. Szumlanski C, Weinshilboum RN. Sulphasalazine inhibition of thio methyl transferase: possible mechanism of interaction with 6-mercaptopurine. Br J Clin Pharmacol. 1995;39:456-459. 34. Proujansky R, Maxwell M, Johnson J, et al. Molecular genotyping predicts complications of 6-mercaptopurine therapy in childhood IBD. Gastroenterology. 1999;116:A800. 35. Cuffari C, Dassoupolus T. Bayless TM. Thiopurine methyl-transferase activity influences clinical response to azathioprine therapy in patients with IBD. Clin Gastroenterol Hepatol. 2004;2:410-417. 36. Kaskas BA, Louis E, Hinderof U, et al. Safe treatment of thiopurine S-transferase deficient Crohn’s disease patients with azathioprine. Gut. 2003;52:140-142. 37. Lennard L, Lilleyman JS. Variable mercaptopurine metabolism and treatment outcome in childhood lymphoblastic leukemia. J Clin Oncol. 1989;7:1816-1823. 38. Sandborn WJ, Tremaine WJ, Wolfe DC, et al. Lack of effect of intravenous administration on time to respond to azathioprine for steroid-treated Crohn’s disease. Gastroenterology. 1999; 117:527-535. 39. Garza A, Sninsky CA. Changes in red cell mean corpuscular volume (MCV) during azathioprine or 6-mercaptopurine therapy for Crohn’s disease may indicate optimal dose titration. Gastroenterology. 2001;120;A3166.
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