Pharmacology Journal

Treatment of inflammatory bowel disease (IBD) is currently based on aminosalicylates, corticosteroids, immunosuppressives, biologicals, antibiotics, nutritional support and surgery. These therapies are limited by side-effects and may be inefficient in up to 30% of patients. Steroid-refractory and steroid-dependent patients are at great risk of extensive bowel resections, even though surgery cannot cure IBD. These patients are qualified for more extensive immunosuppression and the most successful treatment so far has been thiopurine treatment with azathioprine (AZA) or 6-mercaptopurine (6-MP).

The thiopurines are subject to extensive metabolism with both activating and inactivating pathways. The drugs are activated intracellularly by hypoxanthine guanine phosphoribosyltransferase (HPRT1, EC 2.4.2.8). Phosphorylation to thioguanine nucleotides (6-TGN) and incorporation of 6-TGN in DNA has traditionally been regarded as the most important immunosuppressive mechanism. Competing pathways to 6-TGN formation are methylation regulated by the polymorphic enzyme thiopurine S-methyltransferase (TPMT, EC 2.1.1.67), and oxidation to thiouric acid by xanthine oxidase (EC 1.1.3.22). Both 6-MP and AZA also mediate their effects through inhibition of de novo purine biosynthesis by methylated metabolites (6-methylthioinsoine 5′-monophosphate, meTIMP). Recently, it has also been suggested that thioguanine-triphosphate (thio-GTP) interferes with the Rac1-Vav activation of guanosine 5′-diphosphate (GDP), promoting apoptosis, and that the drugs selectively inhibit the expression of inflammatory genes in activated T lymphocytes. Studies have implicated the inosine triphosphate pyrophosphatase (ITPA, EC 3.6.1.19) 94C→A polymorphism in the development of adverse events of thiopurine drugs, such as rash, flu-like symptoms, pancreatitis and also leukopenia, but with divergent results.

The relevance of thiopurine-metabolizing enzymes other than TPMT to the clinical effects of these drugs has not been extensively evaluated.

Inosine 5′-monophosphate dehydrogenase (IMPDH, EC 1.1.1.205) is a key enzyme in the de novo synthesis of guanine nucleotides and is positioned at the branch point between adenine and guanine biosynthesis. IMPDH has the lowest activity of the purine enzymes. It is also strategically positioned in the metabolic pathway of thiopurines. IMPDH may be a significant rate-limiting enzyme in the metabolism of thiopurine drugs to 6-TGN, and its activity would be expected to correlate positively to 6-TGN concentrations and negatively to meTIMP concentrations.

The IMPDH enzyme is present in two isoforms encoded by two different genes, IMPDH1 and IMPDH2, located on chromosomes 7 and 3, respectively. High expression of IMPDH has been demonstrated in pancreas, kidney, colon and peripheral blood leucocytes compared with other tissues such as liver. Increased enzymatic activity and IMPDH mRNA have been described in rapidly proliferating tumour cells and activated T lymphocytes. In cells that are induced to differentiate, decreased activity and downregulated mRNA expression have been observed.

There are, to our knowledge, no data on IMPDH activity in relation to TPMT activity and the production of 6-TGN or meTIMP in IBD patients. The aims of this study were therefore to investigate (i) IMPDH activity in healthy blood donors and in IBD patients on stable thiopurine therapy, (ii) the relationships between the enzymatic activities of IMPDH and TPMT and metabolite formation, and (iii) whether the ITPA 94C→A polymorphism or concomitant 5-aminosalicylic acid (5-ASA) therapy had any impact on these relationships.

Study subjects

The study was performed in 50 patients with IBD, Crohn’s disease (CD; n = 25) and ulcerative colitis (UC; n = 25), of whom 23 were men aged 20–64 and 27 were women aged 19–80 years. Forty-six were of White and four of non-White ethnicity. All patients had been on stable treatment with thiopurines for at least 3.5 months without dose adjustment. The daily median dose of AZA (n = 39) was 2.06 mg kg^−1 body weight per day (range 0.83–2.94) and that of 6-MP (n = 11) was 0.92 mg kg^−1 body weight per day (range 0.33–1.62). Patients on 6-MP had previously experienced side-effects on AZA but had been successfully changed to 6-MP. Twenty-four patients (CD six, UC 18) were on concomitant treatment with 5-ASA at 2325 mg day^−1 (range 500–6750) and seven (UC four, CD three) were on steroids. Eight women and three men were smokers. None of the patients included in the study had received a blood transfusion within 4 months prior to the study.

Patient and disease characteristics were noted. Disease activity was measured at the time of sampling, using a Walmsley’s index for UC and the Harvey-Bradshaw index for CD. In both of these indices active disease was indicated by a score >5.

The reference group comprised 100 healthy blood donors (28 women aged 23–64 and 72 men aged 24–66 years), who were sampled in order to establish areference interval for IMPDH activity. Their ethnicity was not registered.

Venous blood samples were obtained prior to the morning dose of thiopurine for routine blood chemistry, DNA extraction and IMPDH activity.

The study was reviewed and approved by the Ethics Committee at Linköping University (dnr 03-260). Written informed consent was obtained from all IBD patients and oral informed consent was obtained from the healthy blood donors.

Enzyme and metabolite assays

IMPDH activity was measured in peripheral blood mononuclear cells (MNC) with the ion-pair reversed-phase high-pressure liquid chromatography (HPLC) method previously described by Glander et al. with minor modifications: MNC were isolated using cell prep tubes (CPT tubes 8 ml, 362782) from Becton Dickinson (Franklin Lakes, NJ, USA) with two washing steps instead of Ficoll–Paque. The mobile phase had a higher concentration of tetrabutylammonium bisulphate than in the original method (14 mm instead of 7 mm).

IMPDH activity was expressed as nmol xanthosine 5′-monophosphate (XMP) formed from inosine 5′-monophosphate (IMP) per milligram protein and hour. The assay was run on a Dionex isocratic system (Sunnyvale, CA, USA) with the Chromeleon 6.40 software and an ASI-100 automated sampler, a P680 HPLC pump, and a UV/VIS UVD170U detector. A Prontosil 120-5-C18-AQ 5.0-µm column (Bishoff Chromatography, Leonberg, Germany) with a Brownlee NewGuard MPLC RP18 Aquapor precolumn (Perkin Elmer, Shelton, CT, USA) was utilized.

The standard curve comprised water solutions of XMP at concentrations ranging from 3.7 to 70.0 µmol l^−1.

The sensitivity, specificity, linearity and imprecision were approximately as described by Glander et al. with an imprecision <7% in the incubation conditions.

IMP, nicotinamid adenine dinucleotide (NAD), XMP, KCl and K₂CO₃ were obtained from Sigma Aldrich (St Louis, MO, USA), tetrabutylammonium bisulphate and KH₂PO₄ from Fluka (Buchs, Switzerland) and methanol from Labscan Analytical Sciences (Dublin, Ireland).

TPMT activity was determined in red blood cells (RBC) as previously described. One unit of TPMT enzyme activity represents the formation of 1 nmol 6-methyl-MP from 6-MP per ml packed RBC and hour of incubation (U ml^−1 pRBC). The interassay coefficient of variation at 12 U ml^−1 pRBC was 8.4%. 6-TGN and meTIMP were determined by the method of Lennard and Singleton. The lower limits of quantification of the 6-TGN and meTIMP assays were 20 and 300 pmol per 8 × 10⁸ RBC, respectively. The interassay coefficients of variation at 62 and 692 pmol 6-TGN per 8 × 10⁸ RBC were 21.3% and 18.9%, respectively. The interassay coefficients of variation at 1670 and 17 400 pmol meTIMP per 8 × 10⁸ RBC were 30.3% and 27.4%, respectively.

DNA extraction and genotyping

DNA was isolated using the Biorobot Ez1 and the Ez1 DNA blood kit (Qiagen, Hilden, Germany). The ITPA 94C→A polymorphism was determined using a pyrosequencing method for genotyping.

Statistical analysis

Statistical analysis was performed using the Statistical Package for Social Sciences (SPSS, Inc., Chicago, IL, USA), version 14.0 for Windows.

When evaluating the results, 6-MP doses were converted to AZA doses assuming a conversion factor of 2.08. The dose-normalized metabolite concentrations were expressed as pmol metabolite per mg AZA. The data distribution for each variable was evaluated using the Kolmogorov–Smirnov test. Correlations between variables were evaluated using the Spearman rank order correlation coefficient, r_s. For group comparisons, the Mann–Whitney U-test was used. Median (range) values are given. Two-sided testing was used and considered statistically significant if P < 0.05. from 6-MP to 6-TGN.

Filed under: Pharmacogenetics