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The Role of Inosine-5'-Monophosphate Dehydrogenase in Thiopurine Metabolism in Patients With Inflammatory Bowel Disease
Linköping University, Department of Clinical and Experimental Medicine, Gastroenterology and Hepatology. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Medical and Health Sciences, Clinical Pharmacology. Linköping University, Faculty of Health Sciences.
Division of Medical Diagnostics, Laboratory Medicine, Ryhov Hospital, Jönköping;.
Department of Gastroenterology, Lund University Hospital, Lund.
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2011 (English)In: Therapeutic Drug Monitoring, ISSN 0163-4356, E-ISSN 1536-3694, Vol. 33, no 2, 200-208 p.Article in journal (Refereed) Published
Abstract [en]

BACKGROUND:: There is a large interindividual variability in thiopurine metabolism. High concentrations of methylthioinosine-5'-monophosphate (meTIMP) and low concentrations of 6-thioguanine nucleotides (6-TGNs) have been associated with a lower response rate and an increased risk of adverse events. In this study, the role of inosine-5'-monophosphate dehydrogenase (IMPDH) for differences in metabolite patterns of thiopurines was investigated.

METHODS:: IMPDH activity and thiopurine metabolite concentrations were determined in patients with inflammatory bowel disease and a normal thiopurine methyltransferase (TPMT) phenotype and meTIMP/6-TGN concentration ratio > 20 (n = 26), in patients with a metabolite ratio ≤20 (n = 21), in a subgroup with a metabolite ratio <4 (n = 6), and in 10 patients with reduced TPMT activity. In vitro studies were conducted on human embryonic kidney cells (HEK293) with genetically engineered IMPDH and TPMT activities.

RESULTS:: Patients with metabolite ratios >20 had lower IMPDH activity than those with ratios ≤20 (P < 0.001). Metabolic ratios >20 were only observed in patients with normal TPMT activity. Downregulation of IMPDH activity in HEK293 cells was associated with an increase in the concentration of meTIMP (fold change: 17 up to 93, P < 0.001) but, unexpectedly, also of 6-thioguanosine monophosphate (fold change: 2.6 up to 5.0, P < 0.001).

CONCLUSIONS:: These data question the general view of IMPDH as the rate-limiting enzyme in the phosphorylation of thiopurines. Investigations of other mechanisms are needed to more fully explain the various metabolite patterns and outcomes in patients under treatment.

Place, publisher, year, edition, pages
Lippincott Williams & Wilkins, 2011. Vol. 33, no 2, 200-208 p.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-66431DOI: 10.1097/FTD.0b013e31820b42bbISI: 000288498100010PubMedID: 21311411OAI: oai:DiVA.org:liu-66431DiVA: diva2:403835
Available from: 2011-03-15 Created: 2011-03-15 Last updated: 2017-12-11Bibliographically approved
In thesis
1. Interindividual differences in thiopurine metabolism: studies with focus on inflammatory bowel disease
Open this publication in new window or tab >>Interindividual differences in thiopurine metabolism: studies with focus on inflammatory bowel disease
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The thiopurines, 6-mercaptopurine and its prodrug azathioprine, are used in the treatment of inflammatory bowel disease, ulcerative colitis and Crohn´s disease. The main active metabolites are the phosphorylated thioguanine nucleotides (6-TGNs) and methylated thioinosine monophosphate (meTIMP). Both groups contribute to the immunomodulatory effects. About 30-40% of patients fail to benefit from thiopurine treatment. A well-known cause of adverse reactions is decreased or absent thiopurine S-methyltransferase (TPMT) activity. Low TPMT activity is inherited as an autosomal codominant recessive trait and is present in approximately 10% of the population. Although several clinical issues can be solved from determination of TPMT activity, there are cases where it is not possible. In Sweden approximately 25% of IBD-patients display suboptimal 6-TGN concentrations and unexpectedly high concentrations of meTIMP despite a normal TPMT activity. A high meTIMP/6-TGN concentration ratio has been associated with both unresponsiveness to therapy and emergence of adverse reactions. Inosine 5’-monophosphate dehydrogenase (IMPDH) may constitute a candidate gene to explain this metabolite profile, as it is strategically positioned in the metabolic pathway of thiopurines where it competes with TPMT for their common substrate 6-TIMP.

In paper I a pyrosequencing method was developed for genotyping of at that time all known genetic variants of TPMT. The concordance between genotype and phenotype in 30 individuals was 93%. The allele frequencies of TPMT*3A, *3B, *3C and *2 in a Swedish background population (n=800) were in agreement with those in other Caucasian or European populations. In Paper II-IV we explored the molecular basis of different metabolite profiles, i.e. low, normal and high meTIMP/6-TGN concentration ratios. The activity of IMPDH was measured in mononuclear cells (MNC). Patients with high metabolite ratios had lower IMPDH activity than patients with normal or low ratios, explained by an inverse correlation to red blood cells concentration of meTIMP. No correlation to 6-TGN was observed. Downregulation of IMPDH activity in HEK293 cells with genetically engineered TPMT activity was associated with an increase in meTIMP, but unexpectedly also of 6-TGN, irrespective of the TPMT status. These results suggest effects of pharmacogenes other than TPMT and IMPDH. A whole genome expression analysis was performed, (1) to identify new candidate genes that could explain differences in metabolite profiles, and (2) to study genes with known associations to the metabolic pathway of (thio)purines. The whole genome expression analysis did not identify any significant group differences. In analysis of the thiopurine related genes, three clusters of co-regulated genes were defined. A co-operation between expression levels of SLC29A1 and NT5E in explaining the meTIMP/6-TGN concentration ratio was observed, and individually SLC29A1 and NT5E correlated to 6-TGN and meTIMP, respectively.

Pysosequencing is a convenient and flexible method which is now run in parallel to phenotyping in our laboratory. Our results also illustrate the complexity of the thiopurine metabolism and suggest that differences between metabolite profiles are explained either by interactions between several genes, each with a small contribution, or at the post-transcriptional level. Search for more precise tools to explain differences in metabolite profiles is needed. Furthermore, in order to investigate small effects it is necessary to analyse metabolite concentrations and gene expression levels, as well as enzyme activities in the target cells of therapy (MNC).

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2011. 96 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1231
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-66434 (URN)978-91-7393-213-4 (ISBN)
Public defence
2011-04-15, Aulan, Länssjukhuset Ryhov, Jönköping, 13:00 (Swedish)
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Supervisors
Available from: 2011-03-15 Created: 2011-03-15 Last updated: 2012-03-22Bibliographically approved
2. 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)
Opponent
Supervisors
Available from: 2012-10-16 Created: 2012-10-16 Last updated: 2012-11-26Bibliographically approved

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Haglund, SofieVikingsson, SvantePeterson, CurtAlmer, Sven

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