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Biomarkers and Thiopurines in IBD Meijer, B.

2017

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Meijer, B. (2017). Biomarkers and Thiopurines in IBD: when two worlds collide.

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CHAPTER

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Berrie Meijer

Margien L Seinen

Tessa Hosman

Ronald K Linskens

Jan-Kees Kneppelhout

Godefridus J Peters

Chris JJ Mulder

Adriaan A van Bodegraven

Nanne KH de Boer

Departments of 1Gastroenterology and Hepatology, 3Medical Oncology VU University Medical Center, Amsterdam

2_{Department of Gastroenterology and Hepatology,}

St. Anna Hospital, Geldrop

4_{Department of Gastroenterology, Geriatrics, Internal and Intensive Care Medicine,}

Zuyderland Medical Center, Sittard-Geleen

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Abstract

Background Thiopurines play an essential role in the management of

inflammatory bowel diseases (IBD, i.e. Crohn’s disease and ulcerative colitis). Over the past decade, several strategies to optimize treatment with thiopurines have been evaluated. One of these therapy optimization strategies is the co-administration of allopurinol, a xanthine-oxidase (XO) inhibitor, to low-dose thiopurine therapy. In this study, we aimed to assess the inter-individual variability of XO-activity between IBD-patients.

Methods We assessed xanthine oxidase activity in serum of IBD-patients of

two medical centers in The Netherlands using the Amplex® Red Xanthine/Xanthine Oxidase Assay Kit.

Results We observed a high inter-individual variability of XO-activity in 119

patients, with a median activity of 16 µU/ml/hour (range 1 - 85 µU/ml/hour). The XO-activity was influenced by gender (male 19.5 vs. female 14.0 µU/ml/hour, P < 0.01), patient’s age (Pearson’s correlation r = 0.21, P = 0.02) and duration of IBD (r = 0.23, P = 0.01). The activity of XO was not affected by the type of IBD, smoking status, body mass index or (type of) thiopurine use (P > 0.05).

Conclusions With this study, we describe the inter-individual variability of

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Introduction

Inflammatory bowel disease (IBD) is the collective term of ulcerative colitis (UC) and Crohn’s disease (CD). Thiopurines, especially the conventional derivatives azathioprine (AZA) and mercaptopurine (MP) have been widely accepted as first-line immunosuppressive therapy and have proven to be effective in maintaining steroid-free remission. [1, 2] Azathioprine is a pro-drug, which needs to be converted into MP and is subsequently activated by an enzymatic pathway, the so-called purine salvage pathway, to the pharmacologically active metabolites 6-thioguaninenucleotides (6-TGN). Xanthine oxidase (XO) degrades MP into 6-thiouric acid (6-TUA) while thiopurine S-methyltransferase (TPMT) can convert MP to 6-methylmercaptopurine (6-MMP). [3] (Fig. 1)

Approximately 40% of the IBD patients are able to maintain remission during therapy with conventional thiopurine derivatives. [4, 5] One mechanism possibly contributing to ineffectiveness of thiopurine therapy might be increased XO activity, thus decreasing the amount of MP available for biotransformation into effective 6-TGN metabolites. [6, 7]

Over recent years, several strategies to optimize individual thiopurine therapy have been suggested. Measurement of thiopurine metabolites and subsequent co-administration of allopurinol to patients with a skewed metabolism has proven its clinical value. [6, 8-10] Allopurinol acts as an XO inhibitor, therefore a possible significant role for XO activity in effectiveness of thiopurine therapy is assumed. [11] Little is known concerning the range of XO variability in IBD patients. [10, 12, 13]

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Methods

In this retrospective cross-sectional study, we identified IBD patients from a database consisting of all IBD-patients from one academic (VU University Medical Center, Amsterdam, The Netherlands) and one district referral hospital (St. Anna Hospital, Geldrop, The Netherlands). Diagnosis of IBD was ascertained by standard clinical, radiological, histological and endoscopic criteria. [14] The patient characteristics extracted from the clinical patient charts were gender, age, type, localization, classification (according to the Montreal guidelines) and duration of IBD, current and historical medication status, surgical history and smoking status. [14] Duration of IBD was calculated from the date of diagnosis to the date of blood withdrawal.

Analysis of samples

Xanthine oxidase activity was determined in serum using the Amplex® Red Xanthine/Xanthine Oxidase Assay Kit (A22182, Invitrogen, Carlsbad, CA, USA). [15] Briefly, xanthine oxidase catalyzes the oxidation of hypoxanthine to uric acid and superoxide. Superoxide subsequently degrades spontaneously to hydrogen peroxide (H2O2) and in the presence of

horseradish peroxide this reacts with the Amplex® Red reagent to produce a red-fluorescent oxidation product, resofurine. [16] The lower limit of detection of this kit was 1 µU/ml/hour, the lower limit of quantification was 2 µU/ml/hour.

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Statistical analysis

Continuous variables were expressed as median with range or mean with standard deviation, according to distribution. Statistical analyses were performed using the Statistical Package for Social Sciences (SPSS, IBM, Armonk, New York, USA). A P-value less than 0.05 was considered significant. To determine differences between 2 groups the Mann-Whitney-U test or the Chi square method was used, according to distribution. Correlations were assessed using the Spearman correlation coefficient. To determine possible confounding variables linear regression was applied.

Results

Patient characteristics

In total, 144 patients were included, of which 91 (63%) patients were treated in an academic hospital and 53 (37%) in a referral hospital. The male to female ratio was 9:10, with a median age of 39 years (range 19 - 86) at the time of blood withdrawal. Crohn’s disease and UC were diagnosed in 77 (54%) and 67 (46%) patients, respectively. Fifty-eight patients (49%) used thiopurine therapy at the time of blood withdrawal, of which the majority used AZA (94%). The median duration of IBD was 7 years (range 0 - 35). Results were obtained in the serum of 119 patients. The remaining 25 patients were excluded due to errors in assay execution. Patient characteristics of the included patients were summarized in Table 1.

Xanthine oxidase activity

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Table 1 Demographic characteristics of included patients.

Total (n=119) (%) Gender Male 60 (50) Female 59 (50) Diagnosis Crohn’s disease (CD) 62 (52)

Ulcerative colitis (UC) 57 (48)

Montreal classification of UC1 E1-E2-E3 11-18-28 (19-32-49) Montreal classification of CD2 A1-A2-A3 9-48-5 (15-77-8) B1-B2-B3 30-20-12 (48-32-20) L1-L2-L3 13-23-26 (21-37-42)

Prior IBD-related surgery3

Yes 36 (30)

No 83 (70)

Thiopurine use at time of blood withdrawal

Yes 58 (49)

No 61 (51)

Median age at blood withdrawal (range) 40 (19-86) years

Median duration of IBD at blood withdrawal (range) 7 (0-35) years

IBD: inflammatory bowel disease

1_{Montreal classification (14):}

Extent: E1: Proctitis, E2: left-sided colitis, E3: pancolitis Age at diagnosis: A1: <17 years; A2: 17-40 years; A3: >40 years

Behavior: B1: non-stricturing, non-penetrating; B2: stricturing; B3: penetrating Localization: L1: ileal; L2: colonic; L3: ileocolonic

3_{IBD-related surgery was defined as ileocecal resection or (hemi)colectomy}

Discussion

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conflicting results have been published regarding the relationship between gender and XO activity. In some studies, no difference in XO activity between men and women

were observed, whereas in other studies a higher XO activity was observed in either women or men. [12, 13, 17, 18, 21] These discrepancies might be explained by the applied method to determine XO activity. In these studies, the urinary caffeine 1U/(1X+1U) metabolic ratio was measured and considered to represent

XO activity, which was calculated from this ratio, without considering other enzymes that might metabolize caffeine. [22] In our study, XO activity was

Figure 1 Simplified overview of thiopurine metabolism.

Azathioprine (AZA) is converted into mercaptopurine (MP), after which MP is subsequently converted into either 6-thiouric acid (6-TUA) by activity of xanthine oxidase (XO), or into 6-methylmercaptopurine (6-MMP) by the activity of thiopurine-S-methyltransferase (TPMT), which reduces the remaining amount of MP available for biotransformation into the pharmacologically active 6-thioguaninenucletoides (6-TGN) by the purine salvage pathway, using hypoxanthine-guanine phosphoribosyl transferase (HGPRT), inosinemonophosphate dehydrogenase (IMPDH) and guanosine -5’-monophosphate synthetase (GMPS).

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Second, we found a correlation between XO activity and age (r = 0.21), which was in line with an earlier observation. [23] This outcome was supported by previous data correlating age with the level of uric acid, which is the end-product of the reaction catalyzed by XO. [24] When evaluating the thiopurine metabolism, a higher XO activity causes shunting towards the XO pathway (hyperoxidators), thereby decreasing the bioavailability of MP for transformation into 6-TGN. [6] Consequently, thiopurines might be less effective at an older age, which was also concluded in a recent systematic review. [25] To our knowledge, no data are currently available monitoring XO activity prospectively with age and correlating this with thiopurine metabolite levels.

Third, duration of IBD correlated positively with XO activity. However, this relationship was not confirmed when stratification for age was applied (P = 0.13). In case IBD turns out to be an independent factor to influence XO activity, the underlying mechanism could be upregulation of xanthine oxidase activity in (active) inflammatory bowel disease, a phenomenon earlier described in patients with celiac disease. [26] Further prospective trials are necessary to support this theory. In this study, unfortunately, no data were available regarding the activity of disease at the moment of blood withdrawal.

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Conclusion

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[2] Dignass A, Lindsay JO, Sturm A, et al. Second European evidence-based consensus on the diagnosis and management of ulcerative colitis part 2: current management. J Crohns Colitis. 2012;6:991-1030.

[3] Van Asseldonk DP, de Boer NK, Peters GJ, et al. On therapeutic drug monitoring of thiopurines in inflammatory bowel disease; pharmacology, pharmacogenomics, drug intolerance and clinical relevance. Curr Drug Metab. 2009;10:981-997.

[4] Teml A, Schaeffeler E, Herrlinger KR, et al. Thiopurine treatment in inflammatory bowel disease: clinical pharmacology and implication of pharmacogenetically guided dosing. Clin Pharmacokinet. 2007;46:187-208.

[5] Timmer A, McDonald JW, Tsoulis DJ, et al. Azathioprine and 6-mercaptopurine for maintenance of remission in ulcerative colitis. Cochrane Database Syst Rev. 2012;9:CD000478. [6] Ansari A, Aslam Z, De Sica A, et al. Influence of xanthine oxidase on thiopurine metabolism in Crohn's disease. Aliment Pharmacol Ther. 2008;28:749-757.

[7] Kudo M, Sasaki T, Ishikawa M, et al. Kinetics of 6-thioxanthine metabolism by allelic variants of xanthine oxidase. Drug Metab Pharmacokinet. 2010;25:361-366.

[8] Ansari A, Patel N, Sanderson J, et al. Low-dose azathioprine or mercaptopurine in combination with allopurinol can bypass many adverse drug reactions in patients with inflammatory bowel disease. Aliment Pharmacol Ther. 2010;31:640-647.

[9] Govani SM, Higgins PD. Combination of thiopurines and allopurinol: adverse events and clinical benefit in IBD. J Crohns Colitis. 2010;4:444-449.

[10] Kiszka-Kanowitz M, Theede K, Mertz-Nielsen A. Randomized clinical trial: a pilot study comparing efficacy of low-dose azathioprine and allopurinol to azathioprine on clinical outcomes in inflammatory bowel disease. Scand J Gastroenterol. 2016;51:1470-1475.

[11] Seinen ML, van Asseldonk DP, de Boer NK, et al. The effect of allopurinol and low-dose thiopurine combination therapy on the activity of three pivotal thiopurine metabolizing enzymes: results from a prospective pharmacological study. J Crohns Colitis. 2013;7:812-819.

[12] Djordjevic N, Carrillo JA, Gervasini G, et al. In vivo evaluation of CYP2A6 and xanthine oxidase enzyme activities in the Serbian population. Eur J Clin Pharmacol. 2010;66:571-578.

[13] Aklillu E, Carrillo JA, Makonnen E, et al. Xanthine oxidase activity is influenced by environmental factors in Ethiopians. Eur J Clin Pharmacol. 2003;59:533-536.

[14] Satsangi J, Silverberg MS, Vermeire S, et al. The Montreal classification of inflammatory bowel disease: controversies, consensus, and implications. Gut. 2006;55:749-753.

[15] van Asseldonk DP, de Boer NK, Smid K, et al. Limited intra-individual variability in hypoxanthine-Guanine phosphoribosyl transferase, thiopurine S-methyl transferase, and xanthine oxidase activity in inflammatory bowel disease patients during 6-thioguanine therapy. Nucleosides Nucleotides Nucleic Acids. 2010;29:284-290.

[16] Johnson I, Spence MTZ. The Molecular Probes Handbook, A Guide to Fluoresecent Probes and Labeling Technologies. 11th edition ed2010.

[17] Relling MV, Lin JS, Ayers GD, et al. Racial and gender differences in N-acetyltransferase, xanthine oxidase, and CYP1A2 activities. Clin Pharmacol Ther. 1992;52:643-658.

[18] Guerciolini R, Szumlanski C, Weinshilboum RM. Human liver xanthine oxidase: nature and extent of individual variation. Clin Pharmacol Ther. 1991;50:663-672.

[19] Kudo M, Moteki T, Sasaki T, et al. Functional characterization of human xanthine oxidase allelic variants. Pharmacogenet Genomics. 2008;18:243-251.

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[21] Begas E, Kouvaras E, Tsakalof A, et al. In vivo evaluation of CYP1A2, CYP2A6, NAT-2 and xanthine oxidase activities in a Greek population sample by the RP-HPLC monitoring of caffeine metabolic ratios. Biomed Chromatogr. 2007;21:190-200.

[22] Madyastha KM, Sridhar GR. A novel pathway for the metabolism of caffeine by a mixed culture consortium. Biochem Biophys Res Commun. 1998;249:178-181.

[23] Aranda R, Domenech E, Rus AD, et al. Age-related increase in xanthine oxidase activity in human plasma and rat tissues. Free Radic Res. 2007;41:1195-1200.

[24] Johnson RJ, Kang DH, Feig D, et al. Is there a pathogenetic role for uric acid in hypertension and cardiovascular and renal disease? Hypertension. 2003;41:1183-1190.

[25] Gisbert JP, Chaparro M. Systematic review with meta-analysis: inflammatory bowel disease in the elderly. Aliment Pharmacol Ther. 2014;39:459-477.