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Development of new methodology for therapeutic drug monitoringof thiopurine treatment
Linköping University, Department of Medical and Health Sciences, Clinical Pharmacology. Linköping University, Faculty of Health Sciences.
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 [en]
Thiopurines, mercaptopurine, thioguanine, azathioprine, crohn's disease, ulcertive colitis, HPLC, TGN
National Category
Clinical Laboratory Medicine Gastroenterology and Hepatology
Identifiers
URN: urn:nbn:se:liu:diva-84626ISBN: 978-91-7519-808-8 (print)OAI: oai:DiVA.org:liu-84626DiVA: diva2:560819
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
List of papers
1. Monitoring of thiopurine metabolites in patients with inflammatory bowel disease-what is actually measured?
Open this publication in new window or tab >>Monitoring of thiopurine metabolites in patients with inflammatory bowel disease-what is actually measured?
2009 (English)In: Therapeutic Drug Monitoring, ISSN 0163-4356, E-ISSN 1536-3694, Vol. 31, no 3, 345-50 p.Article in journal (Refereed) Published
Abstract [en]

Azathioprine and 6-mercaptopurine are often used in the treatment of patients with inflammatory bowel disease (IBD). They are prodrugs and undergo a complex metabolism to active and inactive metabolites. Thiopurine treatment is monitored in many laboratories by measuring metabolite concentrations in erythrocytes (red blood cells). The metabolites of interest are not measured directly but as hydrolysis products, which can be produced from several metabolites. The aim of this study was to examine which metabolites are actually measured during routine monitoring. Samples from 18 patients treated with a thiopurine were analyzed by a typical routine high-performance liquid chromatography method for therapeutic drug monitoring and by a newly developed specific method measuring thioguanosine monophosphate (TGMP), thioguanosine diphosphate (TGDP), and thioguanosine triphosphate (TGTP), as well as methylthioinosine monophosphate (meTIMP), and the results were compared. 6-Thioguanine nucleotide (TGN) values detected by the routine method were 69% (range 40%-90%) of the sum of TGMP, TGDP, and TGTP measured by the specific method. TGTP and TGDP contributed 85% (range 78%-90%) and 14% (range 10%-21%) of the TGN total, respectively. Thioguanosine was not found in any patient sample. The concentration of meTIMP obtained by the routine method was 548% of the value obtained by the specific method (range 340%-718%). The difference in TGN measurements between the routine and specific methods can be explained by low hydrolysis efficiency in the routine method, although the most likely explanation for the difference in meTIMP values is that not yet identified metabolites are codetermined in the routine high-performance liquid chromatography method. Concentrations reported as TGN during therapeutic drug monitoring of thiopurine metabolites consist of TGDP and TGTP with a minor contribution of the TGMP. Concentrations reported as meTIMP or methyl mercaptopurine consist in part of meTIMP, but other not yet identified metabolites are codetermined.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-18985 (URN)10.1097/FTD.0b013e3181a1ea58 (DOI)19363461 (PubMedID)
Available from: 2009-06-07 Created: 2009-06-07 Last updated: 2017-12-13Bibliographically approved
2. Monitoring of thiopurine metabolites: A high-performance liquid chromatography method for clinical use
Open this publication in new window or tab >>Monitoring of thiopurine metabolites: A high-performance liquid chromatography method for clinical use
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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:nbn:se:liu:diva-84843 (URN)
Available from: 2012-10-24 Created: 2012-10-24 Last updated: 2012-10-24Bibliographically approved
3. Therapeutic drug monitoring of thiopurines in inflammatory bowel disease: Evaluating the benefit of measuring mono-, di-, and triphosphates separately
Open this publication in new window or tab >>Therapeutic drug monitoring of thiopurines in inflammatory bowel disease: Evaluating the benefit of measuring mono-, di-, and triphosphates separately
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

The thiopurines are widely used in the treatment of inflammatory bowel diseases but are limited by poor dose-effect relationship and large interindividual variability in clinical effects. Many attempts have been made to predict response by therapeutic drug monitoring of phosphorylated and methylated metabolites grouped together as thioguanine nucleotides and methylthioinosine monophosphate. We have developed a method to determine the individual metabolites, thioguanosine mono-, di-, and triphosphates, as well as methylthioinosine mono-, di-, and triphosphates, separately in red blood cells.

This aim of this study was to assess the ability of our novel method to predict clinical outcome compared to the routine method in 82 patients with inflammatory bowel diseases.

TPMT wild-type patients with TGN levels below the cut-off level were more likely to have an active disease when TGN was measured by both the routine method (p < 0.05), the novel method (p<0.05), and when TGTP was measured separately (p < 0.01). TGN levels and TGTP were, however, not correlated to disease activity in TPMT defective patients. Patients with meTIN levels above 1500 pmol were more likely to have an active disease (39%, 18/46 vs. 17%, 5/30; p = 0.02). We observed good correlations between the mono-, di-, and triphosphates and their respective sums (R2 > 0.88) and the TGTP ratio (TGTP/(TGDP+TGTP)) was not different in patients with active disease or in clinical remission.

Thiopurine metabolites should still be measured by the routine method, since the novel and technically more challenging method, including determination of TGTP and TGTP ratio, does not offer a clinical advantage compared to the routine method.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-84844 (URN)
Available from: 2012-10-24 Created: 2012-10-24 Last updated: 2012-10-24Bibliographically approved
4. The Role of Inosine-5'-Monophosphate Dehydrogenase in Thiopurine Metabolism in Patients With Inflammatory Bowel Disease
Open this publication in new window or tab >>The Role of Inosine-5'-Monophosphate Dehydrogenase in Thiopurine Metabolism in Patients With Inflammatory Bowel Disease
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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
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-66431 (URN)10.1097/FTD.0b013e31820b42bb (DOI)000288498100010 ()21311411 (PubMedID)
Available from: 2011-03-15 Created: 2011-03-15 Last updated: 2017-12-11Bibliographically approved

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Vikingsson, Svante

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