Prospective Evaluation of the Pharmacogenetics of Azathioprine in the Treatment of Inflammatory Bowel Disease

A. Ansari; M. Arenas; S. M. Greenfield; D. Morris; J. Lindsay; K. Gilshenan; M. Smith; C. Lewis; A. Marinaki; J. Duley; J. Sanderson

Disclosures

Aliment Pharmacol Ther. 2008;28(8):973-983. 

In This Article

Methods

Patients

Patients between the ages of 18 and 80 years with CD or UC were enrolled into a prospective multi-centre study of AZA undertaken in a number of institutions who were members of the London IBD Forum. The decision to initiate AZA was taken at the discretion of the treating doctor in each centre. Exclusion criteria included age (<18 or >80), previous thiopurine exposure and previous use of biologics. The study protocol was approved by the multi-centre and local research ethics committee at the primary institution and all other institutions recruiting patients (MREC number 00/1/33). Written informed consent was obtained from each patient entering the study. Scientific methodology for TPMT phenotyping, TGN measurements and all genetic analysis have either been developed or are routine practice in the Purine Research Laboratory (PRL). All these tests were performed in the PRL by the authors, M. A., A. M., J. D. and A. A.

Study Protocol

At enrolment, TPMT activity was measured. Treating doctors were blinded to these TPMT results unless a 'very low' result was obtained as it was decided that these patients would be excluded from the study (although none were encountered). Permission was then given to commence treatment and in all cases, AZA was started as near 2 mg/kg daily as possible and without dose alteration.

The study period was 6 months to permit a reasonable assessment of clinical response to AZA. Clinical activity indices (Harvey Bradshaw index for CD and Truelove and Witts score for UC) were recorded at initiation and then at weeks 4, 12 and 24. To correlate these with TPMT and TGN concentrations, blood was also taken at these time intervals. In addition an endoscopic assessment of all patients with UC at the start and end of the study was available to corroborate the Truelove and Witts index (data not shown). Steroids were tapered according to clinical response with the target of achieving complete withdrawal at 12 weeks. Full blood count, erythrocyte sedimentation rate (ESR), standard biochemistry, liver function tests and C-reactive protein were measured at baseline and at each study visit (2, 4, 6, 12, 16 and 24 weeks). Patients were asked to report any adverse events at each visit or, where necessary, between visits by contacting the local treating doctor or study nurse. After the study period all the study centres were asked about any further side effects at 9 months. Definitions for adverse events and criteria for withdrawal or dose reduction are given in Table 1 .

Clinical Response

Indications for starting AZA and the definition of clinical response for each indication are given in Table 2 . Clinical response was assessed at 6 months to allow for the known slow therapeutic onset of thiopurines and to ensure response, especially steroid sparing, was sustained. This was divided into a target of achieving the given definition of response by 12 weeks (e.g. complete steroid withdrawal) followed by documented maintenance of this response for the remaining 3 months. Surgery and initiation of biological or other therapy were also considered as treatment failure.

Pharmacogenetic Profiling

At baseline, blood was taken for measurement of red cell TPMT enzyme activity and for assessment of TPMT and ITPA genotype in each patient. TPMT activity and TGN concentrations were then also measured at 2, 4, 12 and 24 weeks of the study. TPMT activity (phenotype) was assayed by the mass spectroscopy method as previously described.[28] Reference ranges were: 'very low', 0-10 pmol/h/mg Hb, intermediate activity, 11-25 pmol/h/mg Hb and normal, 26-50 pmol/h/mg Hb. Patients were genotyped for the three common TPMT polymorphisms: TPMT*3B (460 G>A, Ala154Thr in exon 7), TPMT*3C (719 A>G, Tyr240Cys in exon 10) and TPMT*3A (presence of both polymorphisms). The two functional ITPA polymorphisms were also genotyped: the exonic ITPA 94C>A [Pro32Thr] mutation and the intron mutation ITPA IVS2 + 21A>C, as previously described.[20] All genotypes were checked for Hardy-Weinberg equilibrium.

Measurement of TGNs

Blood was collected in 4.5 mL EDTA tubes and transported to the laboratory within 5 days of collection. TGNs were extracted and oxidized from 100 μL saline-washed packed RBCs following the method of Rabel et al.[29] An aliquot of 75 μL of the oxidized TGN was injected onto a Waters Alliance HPLC system (Milford, MA, USA) with fluorescent detection. Separation of thioguanine mono-, di- and triphosphate nucleotides (were eluted from a C18 column) were separated by ion-pair reverse phase HPLC (Genesis C18 250 × 4.6, 4 μm; ChromTech, Congleton, UK). Buffer A: 40 mM KH2PO4, 4.7 mM tetrabutylammonium hydrogen sulphate (TBA), pH 3.0. Buffer B: 2 mM KH2PO4, 60% (v/v) methanol, 4.7 mM TBA, pH 3.4. The gradient developed from initial conditions of 95% buffer A and 5% buffer B, going to 75% buffer B over 24 min, then back to initial conditions. The run time was 35 min. The fluorescent detector (Waters 474 scanning fluorescent detector) was set at a λex = 329 nm and λem = 410 nm. A thioguanine mono-, di- and triphosphate standard used for quantitation was a gift from Dr R.A. De Abreu (University Medical Center, Nijmegen, The Netherlands). The method did not allow evaluation of methyl-thiopurine concentrations. Bench side stability and intra-individual reproducibility of TGNs were confirmed by MA and AA before patients were recruited.

Analysis of Results and Statistical Methods

The baseline pretreatment TPMT activity and genotypes of TPMT and ITPA were each compared to withdrawal due to adverse effects, to individual groups of adverse effects and to clinical response (complete response or nil/partial response). Likewise, mean and highest TGN concentrations over the 6 month study period were correlated with clinical response. Analyses of total white cell, neutrophil and lymphocyte counts (mean and lowest), and highest and change in mean cell volume (MCV) were also made against clinical response.

The variables were examined and (apart from TPMT) the distribution was not normally distributed. Fisher's exact test was used to determine whether there was any dependence between TPMT genotype and risk of withdrawal due to adverse effects. An ROC curve was constructed to determine a suitable cut-off point for TPMT activity in an effort to predict clinical response. The cut-off point was visually investigated using overlayed histograms of TPMT activity for the different clinical response categories. The efficacy of this value was examined by cross tabulation. The Student's t-test was used to compare TPMT activity and clinical response; the efficacy of the threshold value for predicting clinical response was tested using Fisher's exact test. A threshold value of TGNs was also determined by the same technique and the Fisher's exact test was also used to compare TGN concentrations and response to AZA. The chi-squared test was used to compare individual types of adverse effects with the ITPA genotype. The TGN concentrations at 4, 12 and 24 weeks were compared to clinical response with the chi-squared test for trends. The effect of the use of 5ASA on TGN concentrations was performed with the Mann-Whitney U-test.

The haematological parameters were compared to clinical response using the t-test. Tests were deemed statistically significant at the 0.05 level. Statistical calculations were performed using the R V2.2.0 program (http://cran.r-project.org/).

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