Up to 50% of patients with inflammatory bowel disease

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1 CLINICAL GASTROENTEROLOGY AND HEPATOLOGY 2008;6: Thiopurine Dose in Intermediate and Normal Metabolizers of Thiopurine Methyltransferase May Differ Three-Fold SHARON J. GARDINER,*, RICHARD B. GEARRY,*, EVAN J. BEGG,*, MEI ZHANG,*, and MURRAY L. BARCLAY*,, *Department of Medicine, Department of Clinical Pharmacology, and Department of Gastroenterology, Christchurch Hospital and Christchurch School of Medicine, Christchurch, New Zealand See Van Assche G et al on page 1861 for companion article in the June 2008 issue of Gastroenterology. See CME exam on page 604. Background & Aims: Patients with inflammatory bowel disease (IBD) may have different thiopurine dose requirements in relation to thiopurine methyltransferase (TPMT) genotype and/or phenotype. The purpose of this study was to determine thiopurine dose requirements in intermediate versus normal TPMT metabolism status. Methods: Consecutive patients starting azathioprine or 6-mercaptopurine for IBD were followed up for 9 months. The thiopurine dose was individualized using 6-thioguanine nucleotide (6- TGN) concentrations (range, pmol/ red blood cells [RBCs]) and clinical status. Additional assessments undertaken every three months included measures of disease activity. Results: Eight (10%) of 77 participants were withdrawn because of protocol violation. Fifty-two (75%) of the remaining 69 subjects ( 90% and 10% with the TPMT*1/*1 and *1/*3 genotypes, respectively) completed follow-up on azathioprine (n 46) or 6-mercaptopurine (n 6). The mean initial dose (as azathioprine equivalents) was similar ( 1 mg/kg/d) for the 2 TPMT genotypes, but after 9 months the dose was 50% lower in the TPMT*1/*3 group (0.9 vs 1.8 mg/kg/d, P <.0001). Despite dose adjustment, median 6-TGN concentrations still were 2-fold higher in the TPMT*1/*3 group at the end of the follow-up period (505 vs 273 pmol/ RBCs, P.02). This difference was 3-fold when the concentration was adjusted for dose (578 vs 183 pmol/ per mg/kg/d, P.0007). Results were similar if TPMT phenotype was used instead of genotype. Clinical outcomes did not differ between groups. Conclusions: Initial target doses to attain therapeutic 6-TGN concentrations (>235 pmol/ RBCs) in patients with IBD might be 1 and 3 mg/kg/d in intermediate and normal metabolizers, respectively. Up to 50% of patients with inflammatory bowel disease (IBD) treated with azathioprine and its metabolite, 6-mercaptopurine (6-MP), experience significant toxicity 1,2 or inadequate response. 3 As a result, much research has been directed toward improving outcomes with these drugs. The most successful of these involves testing for thiopurine methyltransferase (TPMT), 4 an enzyme that is expressed polymorphically and integral to the metabolism of both azathioprine and 6-MP (Figure 1). Testing for TPMT by genotype or phenotype (enzyme activity within erythrocytes) is aimed primarily at detecting the 0.3% to 0.6% of individuals with negligible enzyme activity 5,6 who achieve very increased concentrations of the active 6-thioguanine nucleotides (6-TGNs) with standard thiopurine doses and have a high likelihood of profound myelosuppression. 7,8 Although these individuals are best managed with an alternate drug, case reports suggest that a thiopurine dose reduction to approximately 10% of normal may be safe and effective provided that close monitoring may occur. 7,9,10 Because myelosuppression may occur between routine blood counts it seems prudent to restrict the use of thiopurine drugs in cases of TPMT deficiency to those centers where 6-TGN monitoring can be undertaken in a timely manner. There is comparatively less information on how to individualize thiopurine dose in intermediate versus normal metabolizers of TPMT who comprise, when combined, more than 99% of the population ( 10% and 90%, respectively). TPMT activity varies 7-fold across these 2 groups, with 6-TGN concentrations inversely related to TPMT activity. 11,12 When comparing the 2 TPMT groups, intermediate metabolizers have an approximately 5-fold greater risk of mild to moderate leukopenia and an increased likelihood of response compared with normal metabolizers These findings have lead some investigators to suggest that intermediate metabolizers could benefit from a reduction in thiopurine dose to approximately 50% of normal. 13 However, others believe that there is insufficient published evidence to warrant routine implementation of a reduced dose in intermediate metabolizers. 14 One small study showed similar therapeutic outcomes for atopic eczema when intermediate metabolizers received 40% of the azathioprine dose of patients with the normal phenotype (1 and 2.5 mg/kg/d, respectively). 15 Thus, although the clinical use of tests for TPMT (mainly phenotype) seems to be escalating, 4,16,17 more research is required to determine if TPMT-based dose adjustment can improve outcomes of intermediate and normal metabolizers. We aimed to determine the difference in thiopurine dose to attain a similar outcome between individuals with the intermediate Abbreviations used in this paper: CI, confidence interval; CRP, C-reactive protein; ESR, erythrocyte sedimentation rate; IBD, inflammatory bowel disease; IQ, interquartile; 6-MMPN, 6-methylmercaptopurine nucleotides; 6-MP, 6-mercaptopurine; RBC, red blood cell; 6-TGN, 6-thioguanine nucleotides; TPMT, thiopurine methyltransferase by the AGA Institute /08/$34.00 doi: /j.cgh

2 June 2008 THIOPURINE DOSE AND TPMT STATUS 655 Figure 1. Metabolism of azathioprine and 6-MP. GMPS, guanosine monophosphate synthetase; HGPRT, hypoxanthine guanine phosphoribosyltransferase; IMPDH, inosine monophosphate dehydrogenase; TG, thioguanine; TGMP, thioguanine monophosphate; TGN, thioguanine nucleotides; TIMP, thionosine monophosphate; TXMP, thioxanthosine monophosphate. genotype or phenotype of TPMT and those with the normal metabolizer status. Materials and Methods Participants and Study Design A prospective population-based study was undertaken in Canterbury, New Zealand. The characteristics of the individuals with IBD in Canterbury have been described previously. 18 Consecutive patients starting treatment with azathioprine or 6-MP for IBD were identified by their gastroenterologist either via attendance at outpatient clinics or hospitalization. Patients were eligible to participate if they were at least 16 years of age and had normal ( 9.3 IU/mL) or intermediate (5 9.2 IU/mL) TPMT activity. Eligibility criteria were broad to reflect the real-life use of these drugs, with no one excluded on the basis of concurrent disease state or drug therapy unless these constituted a reason to avoid thiopurine treatment (eg, pre-existing neutropenia). Approval was obtained from the Canterbury Ethics Committee (Christchurch, New Zealand). Written informed consent was obtained from all participants. Each subject commenced thiopurine treatment and underwent dose adjustment and monitoring according to the usual practice of their gastroenterologist. This comprised assessment of TPMT activity (phenotype), and dose-individualization based on concentrations of the active 6-TGNs (local target range, pmol/ red blood cells [RBCs]) and assessment of clinical response including hematologic monitoring. Concentrations of the major product of TPMT metabolism, 6-methylmercaptopurine nucleotides (6-MMPN), also were monitored in some patients for clinical purposes, to assess compliance, predict hepatotoxicity, and identify patients who preferentially produce high 6-MMPN and subtherapeutic 6-TGN concentrations. Complete blood counts and liver function tests were monitored as per the usual practice of each gastroenterologist. For the purposes of this noninterventional study, additional assessments were undertaken at monthly intervals during the 9-month follow-up period. Complete blood counts and liver function tests were measured once per month, whereas inflammatory markers (C-reactive protein [CRP] and erythrocyte sedimentation rate [ESR] in blood, and calprotectin in feces) were determined every 3 months. 6-TGN and 6-MMPN concentrations were determined after 1 month of thiopurine treatment and at the same times as the sampling for inflammatory markers (months 3, 6, and 9). Quality of life was assessed every 3 months using the validated short IBD questionnaire. 19 Compliance with these study assessments (blood tests, feces samples, and questionnaire) was encouraged via telephone contact in the week before each scheduled assessment point. At these points of contact, details such as the current thiopurine dose and concomitant drug therapy also were recorded. All decisions regarding patient care including initial thiopurine dose and dose adjustments were made by each patient s treating clinician independently of the research study. The clinicians could access the hematologic results from the study (including TPMT activity) via the hospital intranet but not the calprotectin concentrations or short IBD questionnaire scores. Participants were withdrawn from the study if they repeatedly failed to comply with drug therapy or assessments, or if they had to stop thiopurine treatment for any reason. Subjects with side effects to their initial drug (usually azathioprine in this institution) who then commenced treatment with another thiopurine (6-MP) were re-enrolled in the study where possible. For these subjects, the protocol continued as if it had been uninterrupted. Outcome Definitions The final tolerated thiopurine dose was defined as the dose recorded for the patient at the last assessment point (month 9). The dose of 6-MP was adjusted to azathioprine equivalents by multiplying the 6-MP dose by TGN and 6-MMPN concentrations were both reported as the measured (observed) values (pmol/ RBCs) and as the concentration adjusted for each individual s thiopurine dose and weight (pmol/ RBCs per mg/kg/d). An adverse reaction to thiopurine treatment was defined as one that necessitated cessation of the drug and could be categorized as hepatotoxicity (transaminase concentration greater than 2 times the upper limit of normal), pancreatitis (severe abdominal pain with a serum amylase concentration greater than 3 times the upper limit of normal), myelosuppression (white cell count /L and/or neutrophil count /L), flu-like/hypersensitivity illness (combination of arthralgia, myalgia, fever, and/or rash), nausea/vomiting, or rash. Decisions regarding drug discontinuation because of an adverse reaction were made by the physician responsible on a case-by-case basis. Analytic Procedures The concentrations of 6-TGN and 6-MMPN in RBCs were determined by using a previously described high-performance liquid chromatography method. 21,22 Standard curves were linear over the concentration range of 30 to 2400 pmol/ RBCs for 6-TGN (r ) and 30 to 12,000 for 6-MMPN (r ). Intraday and interday coefficients of variation were less than 10% and the limit of quantification was approximately 30 pmol/ RBCs. TPMT activity was determined using a radiochemical method 23 based on that published by Weinshilboum et al, 24 whereas TPMT genotype was determined using a multiplexed amplification refractory mutation system assay to screen for TPMT*2, *3A, and *3C. 25 Fecal calprotectin concentrations were determined using a commercial single-step enzyme-linked immunosorbent assay (PhiCal Test; Calpro, Oslo,

3 656 GARDINER ET AL CLINICAL GASTROENTEROLOGY AND HEPATOLOGY Vol. 6, No. 6 Norway). Liver function tests, complete blood counts, CRP, and ESR were determined via usual laboratory methods. Statistics Statistical analyses were conducted using GraphPad Prism version 4.0 for Windows (GraphPad Software, San Diego, CA). Comparisons within groups including post hoc analyses were undertaken using the paired t test or the Wilcoxon signed rank test whereas comparisons between groups were made using the unpaired t test, repeated-measures 1-way analysis of variance, the Mann Whitney U test, or the Friedman test as appropriate. Categoric variables were compared using the chisquare test. Relationships were assessed using the Pearson and Spearman correlations for parametric and nonparametric variables, respectively. The percentage variation (r 2 ) in dose owing to TPMT activity was determined from the correlation coefficient (r) identified in the Pearson correlation. For all statistical analyses, 2-sided P values of less than.05 were considered significant. Results Seventy-seven patients consented to participate in this study between September 2003 and May Seven subjects subsequently were withdrawn as a result of failure to commence thiopurine treatment (n 2), poor compliance (n 2), inability to be contacted (n 2), and early drug cessation ( 1 week of treatment) owing to surgery (n 1). Another subject was found to be TPMT deficient and is described separately. 10 The remaining 69 subjects (34 men) had a mean age of 39.2 years (95% confidence interval [CI], y) and weight of 73.5 kilograms (95% CI, kg), respectively. The majority of subjects (76.8%) had Crohn s disease, with the remainder having ulcerative colitis (18.8%) or IBD unspecified (4.3%). Sixty-eight of the 69 subjects were tested for both TPMT genotype and phenotype, with 61 (89.7%) and 7 (10.3%) individuals having the TPMT*1/*1 and *1/*3 genotype, respectively. As expected, the mean TPMT activity was higher in the TPMT*1/*1 genotype group at 13.1 (95% CI, ) IU/mL versus 8.3 (95% CI, ) IU/mL for the TPMT*1/*3 group (P.0001). Genotype did not predict phenotype in 6 of 68 subjects (8.8%). Four of 61 subjects (6.6%) with the TPMT*1/*1 genotype had activity ( IU/mL) in the intermediate range of 5 to 9.2 IU/mL, whereas 2 of 7 (28.6%) subjects with the TPMT*1/*3 genotype had activity (9.8 and 9.9 IU/mL) in the normal range of 9.3 to 17.6 IU/mL. It should be noted that the radiochemical assay used in our study and institution results in lower ranges for TPMT activity than identified via the high-performance liquid chromatography methods used in some laboratories. For example, Prometheus (San Diego, CA) reports a range of 6.7 to 23.6 enzyme units for the intermediate group whereas those with activity greater than 23.6 enzyme units are classified as normal metabolizers. Forty-seven of the 69 subjects (68%) completed the 9-month follow-up on their original thiopurine drug, which was azathioprine in all but 1 case. The remaining 22 (32%) subjects developed toxicity (8 flu-like reactions, 6 hepatotoxicity, 4 nausea/ vomiting, 2 pancreatitis, 1 headache, and 1 abdominal pain) that necessitated discontinuation of azathioprine, which occurred after a median of 30 days (range, 7 99 d) of treatment. After resolution of the adverse reaction, 11 of these 22 subjects attempted therapy with 6-MP. Five had recurrence of the adverse effect (3 flu-like reaction, 1 nausea/vomiting, 1 pancreatitis) whereas 6 subjects (1 flu-like reaction, 4 hepatotoxicity, 1 nausea/vomiting) tolerated the 6-MP and were re-enrolled in the study after a median break from thiopurine treatment of 23 days (interquartile [IQ] range, d). One of these stopped 6-MP after 3 months treatment because of the need for surgery. This left 52 subjects who completed the 9-month evaluation on azathioprine (n 46) or 6-MP (n 6). The majority of these (78%) took mesalazine, which has been suggested to inhibit TPMT, but there was no difference in the median TPMT activity among those who did (12.2; IQ range, ) or did not (13.1; IQ range, ) take mesalazine (P.565). Thiopurine Dose The 52 subjects had a mean initial thiopurine dose of 72 mg/d (95% CI, mg/d) or 1.0 mg/kg/d (95% CI, mg/kg/d) (as azathioprine equivalents). The mean dose increased during the course of the study (P.0001), with post hoc analyses revealing significant differences between month 0 (starting dose) and each of the subsequent monthly assessment points ( 1 vs 1.6 mg/kg/d, P.0001) (Table 1). Most of the dose escalation occurred within the first month of treatment, reflecting the approach of many clinicians to start treatment with a small dose and increase slowly within the first few weeks in an attempt to reduce early side effects. The TPMT*1/*1 and *1/*3 genotypes had comparable mean doses at baseline (month 0) of 1.0 and 1.1 mg/kg/d, respectively (P.780), and after 1 month of treatment (month 1) of 1.5 and 1.2 mg/kg/d, respectively (P.283). However, significant differences were seen from months 2 (1.6 and 1.0 mg/kg/d, P.033) to 9 (1.8 and 0.9 mg/kg/d, P.0006), that is, individuals with the TPMT*1/*1 genotype were titrated to a dose that was 2-fold larger than that of the TPMT*1/*3 group (Figure 2). Similar differences in thiopurine dose were seen between those with the intermediate (5 9.2 IU/mL) versus normal ( 9.3 IU/mL) phenotype (Figure 2). Approximately 30% of the variance in dose was explained by TPMT activity (r , P.0001) (Figure 3). 6-Thioguanine Nucleotide and 6-Methylmercaptopurine Nucleotide Concentrations The mean 6-TGN concentrations for the 52 subjects (including the 5 subjects who had switched to 6-MP within the first 1 2 months of treatment) were stable during evaluation, and were approximately 270 to 280 pmol/ RBCs at months 1, 3, 6, and 9 (Table 1). Mean 6-TGN concentrations at the final assessment point were 2-fold higher in the TPMT*1/*3 genotype than the TPMT*1/*1 group at 505 pmol/ RBCs (95% CI, pmol/ RBCs) versus 273 pmol/ RBCs (95% CI, pmol/ RBCs) despite receiving a 50% lower thiopurine dose (P.016). This difference was 3-fold when the 6-TGN concentration was dose- and weightadjusted, at 578 pmol/ RBCs per mg/kg/d (95% CI, pmol/ RBCs per mg/kg/d) versus 183 pmol/ RBCs per mg/kg/d (95% CI, pmol/ RBCs per mg/kg/d), respectively (P.0007) (Figure 4). Twenty-four of 49 subjects (49%) with evaluable data had 6-TGN concentrations outside the local therapeutic range of 235 to 450 pmol/ RBCs after 9 months of treatment. Those with the TPMT*1/*1 genotype were more likely than those with the

4 June 2008 THIOPURINE DOSE AND TPMT STATUS 657 Table 1. Thiopurine Dose, Metabolite Concentrations, and Markers of Disease Activity Month P value a (between months) Dose, mg 72 b (63 97) 111 (97 125) 110 (97 123) 111 (98 124) 116 ( ) 118 ( ) 117 ( ) 119 ( ) 121 ( ) 122 ( ) <.0001 Dose, mg/kg 1.04 b ( ) 1.56 ( ) 1.56 ( ) 1.57 ( ) 1.64 ( ) 1.65 ( ) 1.64 ( ) 1.66 ( ) 1.64 ( ) 1.66 ( ) < TGN, pmol/8 274 ( ) 266 ( ) 266 ( ) 282 ( ) RBCs 571 ( ) 438 ( ) 511 ( ) 408 ( ) MMPN, pmol/ RBCs Calprotectin, g/g 370 c (64 881) 82 (26 361) 182 (42 403) 158 (50 444).001 CRP, mg/l 9 d (4 29) 5 (4 13) 5 (3 12) 4 (3 8).009 ESR, mm/h 13 (5 27) 10 (5 24) 10 (6 21) 9 (5 19).539 Short IBD 40 e (30 52) 49 f (42 63) 55 (47 62) 56 (47 66) <.0001 questionnaire NOTE. Mean (95% CI) shown for thiopurine dose, and median (IQ range) shown for metabolite concentrations markers of disease activity. a Repeated-measures 1-way analysis of variance (dose) or Friedman test (metabolite concentrations and markers of disease activity). b Month 0 versus months 1 to 9 (P.0001 each). c Month 0 versus month 3 (P.001), vs month 6 (P.01), and vs month 9 (P.02). d Month 0 versus month 3 (P.01), vs month 6 (P.01), and vs month 9 (P.001). e Month 0 versus month 3 (P.0001), vs month 6 (P.0001), and vs month 9 (P.0001). f Month 3 versus month 6 (P.01), and vs month 9 (P.02). Figure 2. (A) Thiopurine dose (as azathioprine equivalents) in TPMT*1/*1 ( ) and *1/*3 (Œ) genotypes and in (B) normal (, 9.3 IU/mL) phenotypes and intermediate (Œ, IU/mL) and presented as mean and 95% CI. Groups became significantly different (P.05). *1/*3 genotype to have concentrations below the range (18 of 44 vs 0 of 5, respectively; P.072) but were less likely to have concentrations above the range (4 of 44 vs 2 of 5, P.046). Within the TPMT*1/*1 group, there was no difference in dose among those who had concentrations above or below 235 pmol/ RBCs (1.7 and 1.8 mg/kg/d, respectively). Measured 6-TGN concentrations did not correlate significantly with either thiopurine dose (mg/kg/d) (r 0.250, P.09) or TPMT activity (r 0.269, P.059). However, when 6-TGN concentrations were adjusted for thiopurine dose (weight-adjusted) there was a significant relationship with TPMT activity (r 0.509, P.0002) (Figure 5). Median 6-MMPN concentrations were lower in individuals with the TPMT*1/*3 versus TPMT*1/*1 genotypes at 154 pmol/ RBCs (IQ range, pmol/ RBCs) and 660 pmol/ RBCs (IQ range, pmol/8 Figure 3. TPMT activity versus weight-adjusted thiopurine dose.

5 658 GARDINER ET AL CLINICAL GASTROENTEROLOGY AND HEPATOLOGY Vol. 6, No. 6 Figure 4. (A) Actual and (B) dose-adjusted 6-TGN concentrations versus TPMT genotype at month 9. Data are presented as mean and 95% CI. Mean actual and dose-adjusted 6-TGN concentrations were 2-fold (P.016) and 3-fold higher (P.0007) in the TPMT*1/*3 versus the *1/*1 genotype, respectively RBCs), respectively (P.001). This difference was less impressive when 6-MMPN concentrations were adjusted for thiopurine dose at 218 pmol/ RBCs per mg/kg/d (IQ range, pmol/ RBCs per mg/kg/d) versus 411 pmol/ RBCs per mg/kg/d (IQ range, pmol/ RBCs per mg/kg/d), respectively (P.058). 6-MMPN concentrations correlated with both thiopurine dose (r 0.610, P.0001) and TPMT activity (r 0.348, P.014) (data not shown). Clinical Outcomes Within subject calprotectin concentrations (P.001), CRP (P.009) and short IBD questionnaire scores (P.0001) improved significantly during the course of the 9-month study (Table 1). The improvements were seen at the first assessment point (3 months) after initiation of thiopurine treatment (P.001, P.01, and P.0001 for comparisons of month 0 vs month 3 for calprotectin, CRP, and short IBD scores, respectively) and were maintained during the 9-month follow-up (Table 1). There was no significant change in ESR during the 9-month follow-up period (P.539). The percentage change in calprotectin (P.352), CRP (P.921), ESR (P.597), or short IBD questionnaire (P.714) did not correlate with thiopurine dose (mg/kg/d), and only the percentage change in short IBD score correlated significantly with 6-TGN concentrations at 9 months (r 0.371, P.01) (data not shown). The patients with TPMT*1/*1 versus *1/*3 genotypes did not experience any difference in the percentage change in calprotectin, CRP, ESR, and short IBD questionnaire from baseline, despite a 2-fold difference in 6-TGN concentrations (data not shown). Consistent with this, no significant differences were observed between the TPMT*1/*3 versus *1/*1 genotypes in the proportion of patients on steroids at completion of the study (2 of 5 and 7 of 46, respectively; P.167) or the median lymphocyte count ( /L each). TPMT genotype did not relate to azathioprine toxicity, with the TPMT*1/*3 genotype comprising approximately 10% of each of the tolerator and nontolerator groups. Further, mean TPMT activity was comparable in the 2 groups at 12.6 (95% CI, ) and 12.8 (95% CI, ), respectively (P.779). There were insufficient data to compare thiopurine dose between the tolerators and nontolerators of azathioprine. However, 14 of the 22 nontolerators underwent blood sampling for metabolite concentrations within 2 days of stopping azathioprine ( 30 days into treatment), enabling crude comparison with the tolerator group at the month 1 (30 day) assessment point. There was a significant difference (P.024) in median 6-TGN concentration between tolerators and nontolerators at 265 pmol/ RBCs (IQ range, pmol/ RBCs) and 165 pmol/ RBCs (IQ range, pmol/ RBCs), respectively. However, this was in the opposite direction to what might be expected if toxicity was concentration related. This may reflect the blood sampling approximately 2 days after drug cessation and the use of lower does in individuals with adverse effects. The median 6-MMPN concentration in the tolerators at month 1 was 583 pmol/ RBCs (IQ range, pmol/ RBCs), which was comparable with the median of 339 pmol/ RBCs (IQ range, pmol/ RBCs) in the nontolerators (P.288). Discussion The principal aim of this prospective study was to determine the difference in thiopurine dose requirements in Figure 5. Dose-adjusted 6-TGN concentrations versus TPMT activity (includes line of best fit plus 95% CI of this line).

6 June 2008 THIOPURINE DOSE AND TPMT STATUS 659 individuals with intermediate versus normal TPMT metabolizer status. The mean initial thiopurine dose (as azathioprine equivalents) was similar ( 1 mg/kg/d) in the 2 groups but with continued treatment individuals with the TPMT*1/*1 genotype were titrated to a dose that was 2-fold higher than that used in the TPMT*1/*3 genotype group (1.8 and 0.9 mg/kg/d, respectively). Despite this difference, individuals with the TPMT*1/*3 genotype attained a 2-fold higher mean concentration of the active 6-TGN metabolites (505 pmol/ RBCs) than the wild-type (273 pmol/ RBCs; P.02). Further, the mean difference in 6-TGN concentrations between groups was 3-fold when each individual s 6-TGN concentrations were adjusted for their thiopurine dose (578 vs 183 pmol/ RBCs per mg/kg/d; P.0007). This suggests that individuals with the TPMT*1/*3 genotype require, on average, one third of the dose of those with the normal genotype to achieve comparable 6-TGN concentrations. These principal findings may not seem surprising in light of the mechanisms for dose adjustments in this study. The clinicians adjusted dose via their usual methods, which included consideration of TPMT activity and 6-TGN concentrations and overall an improvement in IBD disease activity (calprotectin, CRP, and IBD questionnaire score) was seen (Table 1). The final doses achieved, with an approximately 2-fold difference between TPMT*1/*1 and *1/*3 genotypes, are consistent with the suggestion from previous researchers that individuals with the intermediate metabolizer status may require half of the dose of normal metabolizers. 13 However, if 6-TGN concentrations are used to indicate likely efficacy, the true difference in dose could be 3-fold. The use of 6-TGN concentrations as a clinical end point is reasonable because concentrations above 235 to 260 pmol/ RBCs are associated with a 3-fold greater likelihood of remission (odds ratio, 3.27; 95% CI, ). 26 The lack of observation of any other clinical differences (eg, change in inflammatory markers from baseline) between the 2 groups, despite a 2-fold difference in measured 6-TGN concentrations, may reflect some of the difficulties in using clinical end points to assess efficacy (eg, white cell count, steroid use), the small number of intermediate metabolizers studied, and the complexity of thiopurine pharmacokinetics. More detailed examination of the 6-TGN concentrations achieved in the study reinforces the need for differential dose requirements in the 2 TPMT genotype groups. Half of all participants (24 of 49 subjects with evaluable data) achieved 6-TGN concentrations outside the target range of 235 to 450 pmol/ RBCs. Importantly, almost half of those with normal metabolizer status (18 of 44) had 6-TGN concentrations of less than 235 pmol/ RBCs on a mean dose of 1.8 mg/kg/d. This may suggest inadequate drug exposure for many patients, although it also is possible that some patients achieved adequate disease control with values less than 235 pmol/ RBCs, or experienced toxicity with higher concentrations. All 5 individuals with the TPMT*1/*3 genotype and evaluable data had 6-TGN concentrations above 235 pmol/ RBCs (276, 417, 424, 465, and 945 pmol/ RBCs) on a mean dose of approximately 0.9 mg/kg/d. Because the upper limit of the target range (450 pmol/ RBCs) is poorly established 27 and there was no evidence of leukopenia in any of the study participants, it seems reasonable to aim for a mean dose of 1 mg/kg/d in the intermediate metabolizers. However, the dose then should be tailored against clinical outcomes and 6-TGN concentrations, which should be maintained at a reasonable level (perhaps no higher than pmol/ RBCs) because increased concentrations also have been linked with nodular regenerative hyperplasia. 28,29 The reduced dose approach (1 mg/kg/d) in intermediate metabolizers already has been implemented in the practice of some clinicians 30,31 despite the limited supportive evidence. Because there is a 3-fold difference in the dose required to achieve comparable 6-TGN concentrations in the normal metabolizers, it seems reasonable to aim for 3 mg/kg/d in the normal metabolizer group, which is consistent with the upper target dose recommended for patients with IBD, irrespective of TPMT status, in some guidelines. 32 Although either genotyping or phenotyping for TPMT can be used to facilitate individualization of thiopurine dose, phenotyping may have an advantage over genotyping because TPMT activity varies approximately 4-fold across normal and intermediate metabolizers, and varies inversely with 6-TGN concentrations as shown in the current study. However, our study suggests that TPMT activity explains only 30% of the variation in thiopurine dose in the absence of consideration of the 6-TGN concentrations achieved. Examination of further factors influencing thiopurine dose were beyond the scope of this study. Further study is needed to determine whether dosing in direct relationship to measured TPMT activity enables better individualization of thiopurine dose than classification of an individual as 1 of 3 genotypes or phenotypes. Overall, the findings of this study suggest that intermediate metabolizers should receive approximately one third of the dose of normal metabolizers to achieve similar 6-TGN concentrations. Our results support a target dose of 3 mg/kg/d in normal metabolizers and around 1 mg/kg/d for intermediate metabolizers. Lower initial starting doses, for example, 2 and 0.5 mg/ kg/d, initially may help to minimize the occurrence of some of the nonmyelosuppression-related toxicities of this class such as nausea and vomiting or hepatotoxicity, which seem unrelated to either TPMT activity or 6-TGN concentrations. Although these adverse effects usually are regarded as idiosyncratic, there is some evidence of a dose-response relationship. 33 Because the upper limit for 6-TGN concentrations is poorly established and other active metabolites exist, dose adjustments should always occur in conjunction with conventional monitoring including complete blood counts and liver function tests. References 1. Sandborn W, Sutherland L, Pearson D, et al. Azathioprine or 6-mercaptopurine for induction of remission in Crohn s disease. Cochrane Database Syst Rev 2000;2:CD Gearry RB, Barclay ML, Burt MJ, et al. Thiopurine drug adverse effects in a population of New Zealand patients with inflammatory bowel disease. Pharmacoepidemiol Drug Saf 2004;13: Pearson DC, May GR, Fick GH, et al. Azathioprine and 6-mercaptopurine in Crohn disease: a meta-analysis. Ann Intern Med 1995;123: Gardiner SJ, Begg EJ. Pharmacogenetics testing for drug metabolizing enzymes is it happening in practice? 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Am J Gastroenterol 2007;102: Address requests for reprints to: Sharon Gardiner, PhD, Department of Medicine, Christchurch School of Medicine, P.O. Box 4345, Christchurch, New Zealand. sharon.gardiner@cdhb.govt.nz; fax: (64) The authors wish to acknowledge the University of Otago, Tertiary Education Commission, Canterbury Medical Research Foundation, and the Jim and Mary Carney Charitable Trust for financial support of this study.