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Interindividual differences in thiopurine metabolism: studies with focus on inflammatory bowel disease
Linköpings universitet, Institutionen för klinisk och experimentell medicin, Gastroenterologi och hepatologi. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Hjärt- och Medicincentrum, Endokrin- och magtarmmedicinska kliniken US.
2011 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
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).

Ort, förlag, år, upplaga, sidor
Linköping: Linköping University Electronic Press , 2011. , s. 96
Serie
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1231
Nationell ämneskategori
Medicin och hälsovetenskap
Identifikatorer
URN: urn:nbn:se:liu:diva-66434ISBN: 978-91-7393-213-4 (tryckt)OAI: oai:DiVA.org:liu-66434DiVA, id: diva2:403844
Disputation
2011-04-15, Aulan, Länssjukhuset Ryhov, Jönköping, 13:00 (Svenska)
Opponent
Handledare
Tillgänglig från: 2011-03-15 Skapad: 2011-03-15 Senast uppdaterad: 2012-03-22Bibliografiskt granskad
Delarbeten
1. Pyrosequencing of TPMT Alleles in a General Swedish Population and in Patients with Inflammatory Bowel Disease
Öppna denna publikation i ny flik eller fönster >>Pyrosequencing of TPMT Alleles in a General Swedish Population and in Patients with Inflammatory Bowel Disease
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2004 (Engelska)Ingår i: Clinical Chemistry, ISSN 0009-9147, E-ISSN 1530-8561, Vol. 50, nr 2, s. 288-295Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

Background: Interindividual differences in therapeutic efficacy in patients treated with thiopurines might be explained by the presence of thiopurine S-methyltransferase (TPMT) alleles that encode for reduced TPMT enzymatic activity. It is therefore of value to know an individual's inherent capacity to express TPMT. Method: We developed a pyrosequencing method to detect 10 single-nucleotide polymorphisms (SNPs) in TPMT. A Swedish population (n = 800) was examined for TPMT*3A, TPMT*3B, TPMT*3C, and TPMT*2. Patients with inflammatory bowel disease (n = 24) and healthy volunteers (n = 6), selected on the basis of TPMT enzymatic activity, were investigated for all 10 SNPs to determine the relationship between TPMT genotype and phenotype. Results: In the general population we identified the following genotypes with nonfunctional alleles: TPMT*1/*3A (*3A allelic frequency, 3.75%), TPMT*1/*3C (*3C allelic frequency, 0.44%), TPMT*1/*3B (*3B allelic frequency, 0.13%), and TPMT*1/*2 (*2 allelic frequency, 0.06%). All nine individuals with normal enzymatic activity were wild-type TPMT*1/*1. Thirteen individuals with intermediate activity were either TPMT*1/*3A (n = 12) or TPMT*1/*2 (n = 1). Eight individuals with low enzymatic activity were TPMT*3A/*3A (n = 4), TPMT*3A/*3C (n = 2), or TPMT*1/*3A (n = 2). Conclusion: Next to wild type, the most frequent alleles in Sweden are TPMT*3A and TPMT*3C. A previously established phenotypic cutoff for distinguishing normal from intermediate metabolizers was confirmed. To identify the majority of cases (90%) with low or intermediate TPMT activity, it was sufficient to analyze individuals for only 3 of the 10 SNPs investigated. Nevertheless, this investigation indicates that other mutations might be of relevance for decreased enzymatic activity. © 2004 American Association for Clinical Chemistry.

Nationell ämneskategori
Medicin och hälsovetenskap
Identifikatorer
urn:nbn:se:liu:diva-45829 (URN)10.1373/clinchem.2003.023846 (DOI)
Tillgänglig från: 2009-10-11 Skapad: 2009-10-11 Senast uppdaterad: 2017-12-13
2. IMPDH activity in thiopurine-treated patients with inflammatory bowel disease - Relation to TPMT activity and metabolite concentrations
Öppna denna publikation i ny flik eller fönster >>IMPDH activity in thiopurine-treated patients with inflammatory bowel disease - Relation to TPMT activity and metabolite concentrations
2008 (Engelska)Ingår i: British Journal of Clinical Pharmacology, ISSN 0306-5251, E-ISSN 1365-2125, Vol. 65, nr 1, s. 69-77Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

AIMS: Azathioprine and 6-mercaptopurine are steroid-sparing drugs used in inflammatory bowel disease (IBD). The polymorphic enzyme thiopurine S-methyltransferase (TPMT) is of importance for thiopurine metabolism and occurrence of adverse events. The role of other thiopurine-metabolizing enzymes is less well known. This study investigated the role of inosine-5′- monophosphate dehydrogenase (IMPDH), which is a key enzyme in the de novo synthesis of guanine nucleotides and also strategically positioned in the metabolic pathway of thiopurines. METHODS: IMPDH was measured in 100 healthy blood donors. IMPDH, TPMT and metabolite concentrations were studied in 50 patients with IBD on stable thiopurine therapy. IMPDH activity was measured in peripheral blood mononuclear cells. TPMT activity, 6-methylthioinosine 5′-monophosphate (meTIMP) and 6-thioguanine nucleotide (6-TGN) concentrations were measured in red blod cells, which is the current practice in clinical monitoring of thiopurines. Enzyme activities were related to metabolite concentrations and clinical characteristics. RESULTS: A wide range of IMPDH activity was observed both in healthy blood donors (median 13.1, range 4.7-24.2 nmol mg-1 protein h-1) and IBD patients (median 14.0, range 7.0-21.7). There was a negative correlation between IMPDH activity and dose-normalized meTIMP concentrations (rs = -0.31, P = 0.03), but no evident correlation to 6-TGN concentration or the meTIMP/6-TGN ratio. There were no significant correlations between TPMT activity and metabolite concentrations. CONCLUSION: Even though the meTIMP concentrations correlated inversely to the IMPDH activity, the role of IMPDH in balancing the formation of methylated and phosphorylated metabolites was not evident. Taken together, the results give cause to question established opinions about thiopurine metabolism. © 2007 The Authors.

Nyckelord
6-Mercaptopurine/*pharmacology Adult Aged Aged, 80 and over Azathioprine/*pharmacology Biological Markers Female Humans IMP Dehydrogenase/genetics/*metabolism Immunosuppressive Agents/*pharmacology Inflammatory Bowel Diseases/*drug therapy Male Methyltran
Nationell ämneskategori
Medicin och hälsovetenskap
Identifikatorer
urn:nbn:se:liu:diva-43268 (URN)10.1111/j.1365-2125.2007.02985.x (DOI)73262 (Lokalt ID)73262 (Arkivnummer)73262 (OAI)
Tillgänglig från: 2009-10-10 Skapad: 2009-10-10 Senast uppdaterad: 2017-12-13Bibliografiskt granskad
3. The Role of Inosine-5'-Monophosphate Dehydrogenase in Thiopurine Metabolism in Patients With Inflammatory Bowel Disease
Öppna denna publikation i ny flik eller fönster >>The Role of Inosine-5'-Monophosphate Dehydrogenase in Thiopurine Metabolism in Patients With Inflammatory Bowel Disease
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2011 (Engelska)Ingår i: Therapeutic Drug Monitoring, ISSN 0163-4356, E-ISSN 1536-3694, Vol. 33, nr 2, s. 200-208Artikel i tidskrift (Refereegranskat) 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.

Ort, förlag, år, upplaga, sidor
Lippincott Williams & Wilkins, 2011
Nationell ämneskategori
Medicin och hälsovetenskap
Identifikatorer
urn:nbn:se:liu:diva-66431 (URN)10.1097/FTD.0b013e31820b42bb (DOI)000288498100010 ()21311411 (PubMedID)
Tillgänglig från: 2011-03-15 Skapad: 2011-03-15 Senast uppdaterad: 2017-12-11Bibliografiskt granskad
4. Pharmacotranscriptomics in thiopurine treated IBD patients with different metabolite profiles
Öppna denna publikation i ny flik eller fönster >>Pharmacotranscriptomics in thiopurine treated IBD patients with different metabolite profiles
2008 (Engelska)Manuskript (preprint) (Övrigt vetenskapligt)
Abstract [en]

Background: Thiopurine drugs are used to induce and maintain remission in inflammatory bowel disease. The methyl thioinosine monophosphate (meTIMP)/6-thioguanine nucleotide (6-TGN) concentration ratio has been related to drug response and adverse reactions. Here we investigated for differences in gene expression levels between patients with different metabolite profiles.

Methods: Transcriptional profiles in blood samples from an exploratory patient cohort (n=21) comprising three groups; patients with normal thiopurine S-methyltransferase phenotype and meTIMP/6-TGN concentration ratio >20, ratio 10.0-14.0 and ratio ≤4, respectively, were assessed by hybridization to microarrays. Results were further evaluated with reverse transcription qPCR [exploratory and a validation cohort of patients (n=33)]. Additionally, known genes of the thiopurine metabolic pathway were analysed separately.

Results: The whole genome expression analysis did not identify any significant differences between metabolite profiles. Analysis of thiopurine related genes revealed a large interindividual variation in gene expression, but only small differences between metabolite profiles. Three clusters of co-regulated genes were defined based on correlations between gene expression levels. The concentration of meTIMP correlated to the expression of NT5E (rs = 0.33, P = 0.02) and TPMT (rs = - 0.37, P = 0.007). The concentration of 6-TGN correlated to the expression of HPRT1 (rs = - 0.31, P = 0.03) and SLC29A1 (rs = 0.33, P = 0.02). With the exception of SLC29A1, these genes belonged to the same cluster of genes.

Conclusions: Our results illustrates 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 in order to explain differences in metabolite profiles is needed.

Nationell ämneskategori
Medicin och hälsovetenskap
Identifikatorer
urn:nbn:se:liu:diva-66432 (URN)
Tillgänglig från: 2011-03-15 Skapad: 2011-03-15 Senast uppdaterad: 2011-03-15Bibliografiskt granskad

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