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Monitoring of thiopurine metabolites: A high-performance liquid chromatography method for clinical use
Linköping University, Department of Medical and Health Sciences, Clinical Pharmacology. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Clinical and Experimental Medicine, Gastroenterology and Hepatology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Heart and Medicine Centre, Department of Endocrinology and Gastroenterology UHL.
Linköping University, Department of Medical and Health Sciences, Clinical Pharmacology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre for Surgery, Orthopaedics and Cancer Treatment, Department of Oncology UHL.
Linköping University, Department of Medical and Health Sciences, Clinical Pharmacology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre for Diagnostics, Department of Clinical Pharmacology.
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

high-performance liquid chromatography method capable of measuring thiopurine mono-, di-, and triphosphates separately in red blood cells (RBCs) was developed. RBC:s were isolated from whole blood using centrifugation. Proteins were precipitated using dichloromethane and methanol. The thioguanine nucleotides (TGNs) were derivatised using potassium permanganate before analysis. Analytes were separated by ion-pairing liquid chromatography using tetrabutylammonium ions and detected using UV absorption and fluorescence. The method was designed for use in clinical trials in thiopurine therapy and proven valid by analysis of authentic patient samples.

The method measured thioguanosine mono- (TGMP), di- (TGDP), and triphosphate (TGTP), as well as methylthioinosine mono- (meTIMP), di- (meTIDP) and triphosphate (meTITP) in RBCs collected from patients treated with thiopurine drugs (azathioprine, 6-mercaptopurine, and 6-thioguanine).

LOQ was 0.3, 3, 2, 30, 30 and 40 pmol/8x10^8 RBC, for TGMP, TGDP, TGTP, meTIMP, meTIDP and meTITP, respectively. Between-day precision were below 14% for all analytes at all concentrations and samples were stable at 5 °C for 8 hours after sampling.

National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-84843OAI: oai:DiVA.org:liu-84843DiVA: diva2:562443
Available from: 2012-10-24 Created: 2012-10-24 Last updated: 2012-10-24Bibliographically approved
In thesis
1. Development of new methodology for therapeutic drug monitoringof thiopurine treatment
Open this publication in new window or tab >>Development of new methodology for therapeutic drug monitoringof thiopurine treatment
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The three thiopurine drugs azathioprine (AZA), 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) are used to treat several diseases, including inflammatory bowel disease (IBD). They are pro-drugs and are believed to act through the formation of thioguanine nucleotides (TGNs). Other important metabolites are the methylthioinosine nucleotides (meTINs). These metabolites are active in the white blood cells (WBCs).Most patients respond well to the thiopurine drugs but up to a third have to modify or discontinue their treatment due to adverse events or a lack of therapeutic effects. This could be caused by inter-patient variability in the metabolism of the drugs. Therapeutic drug monitoring (TDM) of thiopurine nucleotides in red blood cells (RBCs) is used to guide treatment. Current routine assays measure the nucleotides after hydrolysation to nucleic bases and are therefore unable to distinguish between mono-, di-, and triphosphates. Recently it was shown that these assays failed to predict the clinical outcome in about 40% of the patients. It has been suggested that measuring thioguanosine triphosphate (TGTP) (believed to be the most active of the TGNs) separately might increase the clinical value.An assay suitable for measuring thioguanosine mono- (TGMP) and diphosphate (TGDP) and TGTP, as well as methylthioinosine mono- (meTIMP), di- (meTIDP) and triphosphate (meTITP) separately in RBCs in clinical samples has been developed. In clinical studies of 82 IBD patients, we found no correlation between the thiopurine dose and metabolite levels in RBCs, thus illustrating the importance of metabolite measurements in the TDM of thiopurines.The TGN peak measured by the routine assay during TDM of patients treated with thiopurines consisted of TGTP and TGDP with a small contribution from TGMP. The meTIN also consisted of mono-, di- and triphosphates, but in different proportions, indicating differences in the formation. The inter-individual differences in nucleotide distribution were very small and a strong correlation between the different nucleotides and their respective sums was observed. As a consequence, measuring the mono-, di- and triphosphates separately was not beneficial in predicting remission, which was confirmed by the results from the clinical study.Further research into the metabolism and mode of action of thiopurine drugs is needed to understand the inter-patient variability in response and metabolite formation. An assay suitable for such studies, measuring TGNs and meTINs in cultured cells, has also been developed.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2012. 57 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1323
Keyword
Thiopurines, mercaptopurine, thioguanine, azathioprine, crohn's disease, ulcertive colitis, HPLC, TGN
National Category
Clinical Laboratory Medicine Gastroenterology and Hepatology
Identifiers
urn:nbn:se:liu:diva-84626 (URN)978-91-7519-808-8 (ISBN)
Public defence
2012-11-23, Linden, Campus US, Linköpings Universitet, Linköping, 10:00 (English)
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Supervisors
Available from: 2012-10-16 Created: 2012-10-16 Last updated: 2012-11-26Bibliographically approved

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Vikingsson, SvanteAlmer, SvenPeterson, CurtCarlsson, BjörnJosefsson, Martin

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Clinical PharmacologyFaculty of Health SciencesGastroenterology and HepatologyDepartment of Endocrinology and Gastroenterology UHLDepartment of Oncology UHLDepartment of Clinical PharmacologyDepartment of Physics, Chemistry and BiologyThe Institute of Technology
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