Sains Malaysiana 51(6)(2022): 1847-1859

http://doi.org/10.17576/jsm-2022-5106-20

Therapeutic Monitoring of Thiopurine Metabolites: Validation of an HPLC Method and Preliminary Findings from a Small Cohort of Malaysian Patients with Inflammatory Bowel Disease

(Pemantauan Terapeutik MetabolitTiopurina: Pengesahan Kaedah HPLC dan Penemuan Awal daripada Kohort Kecil Pesakit Malaysia dengan Penyakit Radang Usus)

SHENG ZHANG LIM¹, RAJA AFFENDI RAJA ALI², SUZANA MAKPOL³ & ENG WEE CHUA^1,*

¹Centre for Herbal and Drug Development, Faculty of Pharmacy, Universiti Kebangsaan Malaysia, 50300 Kuala Lumpur, Federal Territory, Malaysia

²Department of Medicine, Faculty of Medicine, Universiti Kebangsaan Malaysia, 56000 Cheras, Kuala Lumpur, Federal Territory, Malaysia

³Department of Biochemistry, Faculty of Medicine, Universiti Kebangsaan Malaysia, 56000 Cheras, Kuala Lumpur, Federal Territory, Malaysia

Received: 11 October 2021/Accepted: 15 November 2021

ABSTRACT

Thiopurine therapy of inflammatory bowel disease (IBD) is guided by the relative blood concentrations 6-thioguanine nucleotides (6-TGN) and 6-methylmercaptopurine (6-MMP). However, their action is altered by in vivo phosphorylation, and this is not normally measured in clinical studies. Hence, we trialled a novel method for profiling phosphorylated thiopurine metabolites and revisited the association between thiopurine metabolites and IBD treatment outcomes. We first optimised and validated a published high-performance liquid chromatography (HPLC) method for measuring the blood levels of thioguanosine monophosphate (TGMP), thioguanosine diphosphate (TGDP), thioguanosine triphosphate (TGTP), and methylthioinosine monophosphate (MeTIMP). Then, we assembled a small cohort of IBD patients (n=20), who had been treated with azathioprine for at least three months, and obtained blood samples for analysis of the metabolites. The patients received treatments at the Universiti Kebangsaan Malaysia Specialist Centre between March 2018 and April 2019. They were classified as responders (n = 12) or non-responders (n = 6) to azathioprine based on their disease activity scores (CDAI or Mayo score). The HPLC method was precise with intraday and interday variation < 15% for all the tested metabolites, and the relative accuracy ranged from 40.2 to 114.0%. We noted that the responders had higher median 6-TGN but lower median TGTP levels than the non-responders. However, the differences were not statistically significant (Wilcoxon rank-sum tests; 6-TGN, p = 0.925; TGTP, p = 0.189). The higher median 6-TGN level detected in the responders is in keeping with the findings of prior studies, suggesting that HPLC analysis of phosphorylated thiopurine metabolites is both technically feasible and clinically useful.

Keywords: High-performance liquid chromatography; inflammatory bowel disease; therapeutic drug monitoring; thiopurine metabolites

ABSTRAK

Rawatan tiopurina untuk penyakit radang usus (IBD) adalah berpandukan kepekatan nukleotida 6-tioguanina (6-TGN) dan 6-metilmerkaptopurina (6-MMP) dalam darah. Akan tetapi, tindakan metabolit tiopurina berubah-ubah mengikut tahap fosforilasi in vivo dan fenomena ini sukar dikesan secara tepat dalam kajian klinikal. Oleh itu, kami menguji satu kaedah baharu bagi pengesanan metabolit tiopurina terfosforilasi dan mengkaji semula hubungan antara metabolit tiopurina dan hasil rawatan IBD. Kami mengoptimumkan dan menilai prestasi satu kaedah kromatografi cecair prestasi tinggi (HPLC) yang dilaporkan dalam kajian lepas untuk penentuan paras darah tioguanosina monofosfat (TGMP), tioguanosina difosfat (TGDP), tioguanosina trifosfat (TGTP) dan metiltioinosina monofosfat (MeTIMP). Kemudian, kami merekrut sekumpulan kecil pesakit IBD (n = 20) yang telah dirawat dengan azatioprina untuk sekurang-kurangnya tiga bulan dan memperoleh sampel darah untuk analisis paras metabolit. Pesakit-pesakit tersebut menerima rawatan di Pusat Perubatan Universiti Kebangsaan Malaysia antara Mac 2018 dan April 2019. Mereka dikelaskan kepada kumpulan pesakit yang menunjukkan respons baik terhadap azatioprina, atau responden (n = 12) dan kumpulan yang gagal rawatan, atau bukan responden (n = 6), berdasarkan aktiviti penyakit (CDAI atau skor Mayo). Kaedah HPLC tersebut didapati tepat dengan variasi dalam tempoh sehari dan antara hari < 15% bagi semua metabolit dan ketepatan relatif antara 40.2% dan 114.0%. Kami mendapati bahawa kumpulan pesakit responden mempunyai paras median 6-TGN yang lebih tinggi tetapi paras median TGTP yang lebih rendah daripada pesakit bukan responden. Walau bagaimanapun, perbezaan tersebut tidak signifikan secara statistik (ujian Wilcoxon; 6-TGN, p = 0.925; TGTP, p = 0.189). Selaras dengan kajian terdahulu, kajian ini mendapati bahawa paras median 6-TGN adalah lebih tinggi dalam kalangan pesakit responden. Ini mencadangkan bahawa analisis HPLC metabolit tiopurina terfosforilasi boleh dilaksanakan dan adalah berguna untuk amalan klinikal.

Kata kunci: Kromatografi cecair berprestasi tinggi (HPLC); metabolit tiopurina; pemantauan ubat terapeutik; penyakit radang usus

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^*Corresponding author; email: cew85911@ukm.edu.my