CLINICAL GASTROENTEROLOGY AND HEPATOLOGY 2008;6:654 – 660
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 require-
ments 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: Con-
secutive 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, 235– 450 pmol/8 ⴛ 108 red
blood cells [RBCs]) and clinical status. Additional assess-
ments 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were withdrawn because of protocol violation.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 ⴝ 46) or 6-mercaptopurine (n ⴝ
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/8 ⴛ 108 RBCs, P ⴝ .02). This difference was 3-fold
when the concentration was adjusted for dose (578 vs 183
pmol/8 ⴛ 108 per mg/kg/d, P ⴝ .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 concentra-
tions (>235 pmol/8 ⴛ 108 RBCs) in patients with IBD
might be 1 and 3 mg/kg/d in intermediate and normal
metabolizers, respectively.
U p to 50% of patients with inflammatory bowel disease
(IBD) treated with azathioprine and its metabolite,
6-mercaptopurine (6-MP), experience significant toxicity1,2 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 meth-
yltransferase (TPMT),4 an enzyme that is expressed polymor-
phically and integral to the metabolism of both azathioprine
and 6-MP (Figure 1).
Testing for TPMT by genotype or phenotype (enzyme activ-
ity within erythrocytes) is aimed primarily at detecting the 0.3%
to 0.6% of individuals with negligible enzyme activity5,6 who
achieve very increased concentrations of the active 6-thiogua-
nine nucleotides (6-TGNs) with standard thiopurine doses and
have a high likelihood of profound myelosuppression.7,8 Al-
though 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 pro-
vided that close monitoring may occur.7,9,10 Because myelosup-
pression 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 individ-
ualize thiopurine dose in intermediate versus normal metabo-
lizers of TPMT who comprise, when combined, more than 99%
of the population (⬃10% and ⬃90%, respectively). TPMT activ-
ity varies 7-fold across these 2 groups, with 6-TGN concentra-
tions inversely related to TPMT activity.11,12 When comparing
the 2 TPMT groups, intermediate metabolizers have an approx-
imately 5-fold greater risk of mild to moderate leukopenia and
an increased likelihood of response compared with normal
metabolizers.11–13 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 re-
quired to determine if TPMT-based dose adjustment can im-
prove 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, inflamma-
tory bowel disease; IQ, interquartile; 6-MMPN, 6-methylmercaptopurine
nucleotides; 6-MP, 6-mercaptopurine; RBC, red blood cell; 6-TGN, 6-thio-
guanine nucleotides; TPMT, thiopurine methyltransferase.
© 2008 by the AGA Institute
1542-3565/08/$34.00
doi:10.1016/j.cgh.2008.02.032
June 2008
Figure 1. Metabolism of azathioprine and 6-MP. GMPS, guanosine
monophosphate synthetase; HGPRT, hypoxanthine guanine phospho-
ribosyltransferase; IMPDH, inosine monophosphate dehydrogenase;
TG, thioguanine; TGMP, thioguanine monophosphate; TGN, thiogua-
nine nucleotides; TIMP, thionosine monophosphate; TXMP, thioxan-
thosine 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 individ-
uals 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 (ⱖ9.3 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 consti-
tuted a reason to avoid thiopurine treatment (eg, pre-existing
neutropenia). Approval was obtained from the Canterbury Eth-
ics Committee (Christchurch, New Zealand). Written informed
consent was obtained from all participants.
Each subject commenced thiopurine treatment and under-
went dose adjustment and monitoring according to the usual
practice of their gastroenterologist. This comprised assess-
ment of TPMT activity (phenotype), and dose-individualiza-
tion based on concentrations of the active 6-TGNs (local
target range, 235– 450 pmol/8 ⫻ 108 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 func-
tion tests were monitored as per the usual practice of each
gastroenterologist. For the purposes of this noninterventional
study, additional assessments were undertaken at monthly in-
tervals 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
THIOPURINE DOSE AND TPMT STATUS 655
treatment and at the same times as the sampling for inflam-
matory markers (months 3, 6, and 9). Quality of life was
assessed every 3 months using the validated short IBD ques-
tionnaire.19 Compliance with these study assessments (blood
tests, feces samples, and questionnaire) was encouraged via
telephone contact in the week before each scheduled assess-
ment 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 repeat-
edly 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 2.08.20 6-TGN and
6-MMPN concentrations were both reported as the measured
(observed) values (pmol/8 ⫻ 108 RBCs) and as the concentra-
tion adjusted for each individual’s thiopurine dose and weight
(pmol/8 ⫻ 108 RBCs per mg/kg/d). An adverse reaction to
thiopurine treatment was defined as one that necessitated ces-
sation 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 ⬍3.0 ⫻
109/L and/or neutrophil count ⬍2.0 ⫻ 109/L), flu-like/hyper-
sensitivity 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-perfor-
mance liquid chromatography method.21,22 Standard curves
were linear over the concentration range of 30 to 2400 pmol/8 ⫻
108 RBCs for 6-TGN (r2 ⬎ 0.99) and 30 to 12,000 for 6-MMPN
(r2 ⬎ 0.99). Intraday and interday coefficients of variation were
less than 10% and the limit of quantification was approximately
30 pmol/8 ⫻ 108 RBCs. TPMT activity was determined using a
radiochemical method23 based on that published by Wein-
shilboum 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 concen-
trations were determined using a commercial single-step en-
zyme-linked immunosorbent assay (PhiCal Test; Calpro, Oslo,
656 GARDINER ET AL
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 chi-
square test. Relationships were assessed using the Pearson and
Spearman correlations for parametric and nonparametric vari-
ables, respectively. The percentage variation (r2) in dose owing
to TPMT activity was determined from the correlation coeffi-
cient (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 2005. Seven subjects
subsequently were withdrawn as a result of failure to commence
thiopurine treatment (n ⫽ 2), poor compliance (n ⫽ 2), inabil-
ity 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], 35.4 – 42.9 y) and weight of 73.5
kilograms (95% CI, 69.7–77.3 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 pheno-
type, 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, 12.5–13.7) IU/mL versus 8.3 (95% CI,
6.8 –9.8) IU/mL for the TPMT*1/*3 group (P ⬍ .0001). Geno-
type 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
(7.9 –9.2 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 exam-
ple, 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 azathio-
prine in all but 1 case. The remaining 22 (32%) subjects devel-
oped toxicity (8 flu-like reactions, 6 hepatotoxicity, 4 nausea/
vomiting, 2 pancreatitis, 1 headache, and 1 abdominal pain)
that necessitated discontinuation of azathioprine, which oc-
curred after a median of 30 days (range, 7–99 d) of treatment.
After resolution of the adverse reaction, 11 of these 22 subjects
CLINICAL GASTROENTEROLOGY AND HEPATOLOGY Vol. 6, No. 6
attempted therapy with 6-MP. Five had recurrence of the ad-
verse effect (3 flu-like reaction, 1 nausea/vomiting, 1 pancreati-
tis) 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, 14 –33 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, 10.3–14.3) or did not
(13.1; IQ range, 10.4 –14.5) take mesalazine (P ⫽ .565).
Thiopurine Dose
The 52 subjects had a mean initial thiopurine dose of
72 mg/d (95% CI, 63–97 mg/d) or 1.0 mg/kg/d (95% CI, 0.9 –1.2
mg/kg/d) (as azathioprine equivalents). The mean dose in-
creased 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 (ⱖ9.3 IU/mL) pheno-
type (Figure 2). Approximately 30% of the variance in dose was
explained by TPMT activity (r2 ⫽ 0.295, 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/8 ⫻ 108 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/8 ⫻ 108 RBCs
(95% CI, 188 – 823 pmol/8 ⫻ 108 RBCs) versus 273 pmol/8 ⫻ 108
RBCs (95% CI, 234 –312 pmol/8 ⫻ 108 RBCs) despite receiving
a 50% lower thiopurine dose (P ⫽ .016). This difference was
3-fold when the 6-TGN concentration was dose- and weight-
adjusted, at 578 pmol/8 ⫻ 108 RBCs per mg/kg/d (95% CI,
407–749 pmol/8 ⫻ 108 RBCs per mg/kg/d) versus 183 pmol/8
⫻ 108 RBCs per mg/kg/d (95% CI, 142–224 pmol/8 ⫻ 108 RBCs
per mg/kg/d), respectively (P ⫽ .0007) (Figure 4). Twenty-four
of 49 subjects (49%) with evaluable data had 6-TGN concentra-
tions outside the local therapeutic range of 235 to 450 pmol/8
⫻ 108 RBCs after 9 months of treatment. Those with the
TPMT*1/*1 genotype were more likely than those with the
 Table 1. Thiopurine Dose, Metabolite Concentrations, and Markers of Disease Activity

Month P
June 2008

valuea
(between
0 1 2 3 4 5 6 7 8 9 months)

Dose, mg 72b (63–97) 111 (97–125) 110 (97–123) 111 (98–124) 116 (103–129) 118 (105–130) 117 (104–130) 119 (106–131) 121 (108–134) 122 (109–135) <.0001
Dose, mg/kg 1.04b (0.90–1.18) 1.56 (1.37–1.74) 1.56 (1.37–1.75) 1.57 (1.39–1.74) 1.64 (1.46–1.81) 1.65 (1.48–1.82) 1.64 (1.46–1.81) 1.66 (1.49–1.82) 1.64 (1.47–1.82) 1.66 (1.48–1.84) <.0001
6-TGN, pmol/8 ⫻ 274 (183–372) 266 (182–374) 266 (214–407) 282 (195–401) .797
108 RBCs
6-MMPN, pmol/8 ⫻ 571 (364–1407) 438 (240–1278) 511 (216–1716) 408 (275–1117) .829
108 RBCs
Calprotectin, ␮g/g 370c (64–881) 82 (26–361) 182 (42–403) 158 (50–444) .001
CRP, mg/L 9d (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 40e (30–52) 49f (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.
aRepeated-measures 1-way analysis of variance (dose) or Friedman test (metabolite concentrations and markers of disease activity).
bMonth 0 versus months 1 to 9 (P ⬍ .0001 each).
cMonth 0 versus month 3 (P ⬍ .001), vs month 6 (P ⬍ .01), and vs month 9 (P ⫽ .02).
dMonth 0 versus month 3 (P ⬍ .01), vs month 6 (P ⬍ .01), and vs month 9 (P ⬍ .001).
eMonth 0 versus month 3 (P ⬍ .0001), vs month 6 (P ⬍ .0001), and vs month 9 (P ⬍ .0001).
fMonth 3 versus month 6 (P ⬍ .01), and vs month 9 (P ⫽ .02).

TPMT activity (r ⫽ ⫺0.509, P ⫽ .0002) (Figure 5).

pmol/8 ⫻ 108 RBCs (1.7 and 1.8 mg/kg/d, respectively).
THIOPURINE DOSE AND TPMT STATUS

Figure 3. TPMT activity versus weight-adjusted thiopurine dose.

and 660 pmol/8 ⫻ 108 RBCs (IQ range, 334 –1470 pmol/8 ⫻
pmol/8 ⫻ 108 RBCs (IQ range, 86 –185 pmol/8 ⫻ 108 RBCs)
among those who had concentrations above or below 235
concentrations above the range (4 of 44 vs 2 of 5, P ⫽ .046).

with the TPMT*1/*3 versus TPMT*1/*1 genotypes at 154

Median 6-MMPN concentrations were lower in individuals
.09) or TPMT activity (r ⫽ ⫺0.269, P ⫽ .059). However, when
cantly with either thiopurine dose (mg/kg/d) (r ⫽ ⫺0.250, P ⫽
TPMT*1/*1 (⌬) and *1/*3 (Œ) genotypes and in (B) normal (⌬, ⱖ9.3

(weight-adjusted) there was a significant relationship with

6-TGN concentrations were adjusted for thiopurine dose
Measured 6-TGN concentrations did not correlate signifi-
Within the TPMT*1/*1 group, there was no difference in dose
44 vs 0 of 5, respectively; P ⫽ .072) but were less likely to have
*1/*3 genotype to have concentrations below the range (18 of
657

IU/mL) phenotypes and intermediate (Œ, 5–9.2 IU/mL) and presented

Figure 2. (A) Thiopurine dose (as azathioprine equivalents) in

as mean and 95% CI. †Groups became significantly different (P ⬍ .05).

658 GARDINER ET AL
Figure 4. (A) Actual and (B) dose-adjusted 6-TGN concentrations ver-
sus 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.
108 RBCs), respectively (P ⫽ .001). This difference was less
impressive when 6-MMPN concentrations were adjusted for
thiopurine dose at 218 pmol/8 ⫻ 108 RBCs per mg/kg/d (IQ
range, 126 –260 pmol/8 ⫻ 108 RBCs per mg/kg/d) versus 411
pmol/8 ⫻ 108 RBCs per mg/kg/d (IQ range, 231– 804 pmol/8 ⫻
108 RBCs per mg/kg/d), respectively (P ⫽ .058). 6-MMPN con-
centrations 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
CLINICAL GASTROENTEROLOGY AND HEPATOLOGY Vol. 6, No. 6
month 3 for calprotectin, CRP, and short IBD scores, respec-
tively) 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 concentra-
tions 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 cal-
protectin, CRP, ESR, and short IBD questionnaire from base-
line, 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 (1.5 ⫻ 109/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,
11.8 –13.4) and 12.8 (95% CI, 11.4 –14.1), respectively (P ⫽ .779).
There were insufficient data to compare thiopurine dose be-
tween the tolerators and nontolerators of azathioprine. How-
ever, 14 of the 22 nontolerators underwent blood sampling for
metabolite concentrations within 2 days of stopping azathio-
prine (⬃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/8 ⫻ 108 RBCs (IQ range, 180 –370 pmol/8 ⫻ 108 RBCs)
and 165 pmol/8 ⫻ 108 RBCs (IQ range, 130 –259 pmol/8 ⫻ 108
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/8 ⫻ 108 RBCs (IQ range, 291–1422
pmol/8 ⫻ 108 RBCs), which was comparable with the median of
339 pmol/8 ⫻ 108 RBCs (IQ range, 245–1220 pmol/8 ⫻ 108
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).
June 2008
individuals with intermediate versus normal TPMT metabolizer
status. The mean initial thiopurine dose (as azathioprine equiv-
alents) 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, respec-
tively). 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/8 ⫻ 108 RBCs) than the
wild-type (273 pmol/8 ⫻ 108 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/8 ⫻ 108 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 clini-
cians 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 be-
tween 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/8 ⫻ 108 RBCs are associated with a 3-fold greater likeli-
hood of remission (odds ratio, 3.27; 95% CI, 1.71– 6.27).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 complex-
ity 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 par-
ticipants (24 of 49 subjects with evaluable data) achieved
6-TGN concentrations outside the target range of 235 to 450
pmol/8 ⫻ 108 RBCs. Importantly, almost half of those with
normal metabolizer status (18 of 44) had 6-TGN concentra-
tions of less than 235 pmol/8 ⫻ 108 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/8 ⫻ 108 RBCs, or experienced toxicity with higher con-
centrations. All 5 individuals with the TPMT*1/*3 genotype and
evaluable data had 6-TGN concentrations above 235 pmol/8 ⫻
108 RBCs (276, 417, 424, 465, and 945 pmol/8 ⫻ 108 RBCs) on
a mean dose of approximately 0.9 mg/kg/d. Because the upper
limit of the target range (450 pmol/8 ⫻ 108 RBCs) is poorly
established27 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
THIOPURINE DOSE AND TPMT STATUS 659
6-TGN concentrations, which should be maintained at a rea-
sonable level (perhaps no higher than 400 –500 pmol/8 ⫻ 108
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 clinicians30,31 despite
the limited supportive evidence. Because there is a 3-fold dif-
ference 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 guide-
lines.32
Although either genotyping or phenotyping for TPMT can
be used to facilitate individualization of thiopurine dose, phe-
notyping 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 in-
dividualization of thiopurine dose than classification of an indi-
vidual 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 concentra-
tions. Our results support a target dose of 3 mg/kg/d in normal
metabolizers and around 1 mg/kg/d for intermediate metabo-
lizers. 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.
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Address requests for reprints to: Sharon Gardiner, PhD, Department
of Medicine, Christchurch School of Medicine, P.O. Box 4345,
Christchurch, New Zealand. e-mail: sharon.gardiner@cdhb.govt.nz; fax:
(64) 3-364-1003.
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.