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Zimdahl, Anna
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Publications (4 of 4) Show all publications
Zimdahl, A. (2020). Pharmacogenetic studies of thiopurine methyltransferase genotype-phenotype concordance and effect of methotrexate on thiopurine metabolism. (Doctoral dissertation). Linköping: Linköping University Electronic Press
Open this publication in new window or tab >>Pharmacogenetic studies of thiopurine methyltransferase genotype-phenotype concordance and effect of methotrexate on thiopurine metabolism
2020 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The thiopurines (6-mercaptopurine [6-MP], 6-thioguanine and azathioprine) are cytotoxic drugs used in the treatment of acute lymphoblastic leukemia (ALL), inflammatory bowel diseases, certain autoimmune diseases and after transplantation. The metabolism of thiopurines is complex with several enzymes involved in the conversion into active drug metabolites. One of the enzymes, thiopurine methyltransferase (TPMT), is one of the best examples of implemented pharmacogenetics so far. Due to lowered TPMT enzyme activity caused by genetic polymorphism, carriers of heterozygous or homozygous defective TPMT alleles need dose reduction to avoid cytotoxic adverse reactions like myelosuppression or hepatotoxicity if treated with thiopurines.

To determine TPMT status before the start of treatment, genotyping (for the three most occurring TPMT alleles) and/or phenotyping (TPMT enzyme activity measurements) are used in the clinical setting. In the focus of this thesis, concordance of these methods was investigated in a large cohort of unique samples (n=12,663) collected in the routine analysis service of TPMT status determinations in Linköping. By sequencing all exons in samples where the results of the two methods differed, rare or novel TPMT alleles were discovered. Four TPMT alleles (TPMT*41, *42, *43, *44), not previously described, were characterized in terms of clinical in vivo data as well as protein structure and stability data obtained from recombinant human TPMT (rTPMT) produced by E. Coli and biophysical methods.

The clinical cohort was also used in the search for other factors (except genetic factors) that influence TPMT enzyme activity, and both age and gender turned out to affect TPMT enzyme activity level. In addition, TPMT enzyme activity in the early treatment of ALL was investigated and shown to be significantly lower at time of ALL diagnosis.

In the treatment protocol of ALL, the combined treatment using 6-MP and methotrexate (MTX) has increased the positive outcomes since the start in the 1950s. Despite this, the synergistic effect of these drugs is not yet fully understood. To evaluate the effect of MTX on thiopurine metabolism specifically, TPMT enzyme activity, TPMT gene expression, and thiopurine metabolite levels were determined before and after MTX infusions in vivo and after cotreatment in lymphoblasts in vitro. In the presence of MTX, TPMT enzyme activity and metabolite levels decreased, both in vivo and in vitro, although dose- and time-dependent. In addition, MTX bound to rTPMT and caused inhibition of rTPMT enzyme activity.

The results found in the scope of this thesis may be used for further individualization of thiopurine treatment.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2020. p. 111
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1723
National Category
Pharmacology and Toxicology
Identifiers
urn:nbn:se:liu:diva-163614 (URN)10.3384/diss.diva-163614 (DOI)9789179299408 (ISBN)
Public defence
2020-03-06, Granitsalen, Building 448, Entrance 7, Campus US, Linköping, 09:00 (Swedish)
Opponent
Supervisors
Available from: 2020-02-17 Created: 2020-02-17 Last updated: 2020-03-13Bibliographically approved
Niklasson, M., Andrésen, C., Helander, S., Roth, M., Zimdahl Kahlin, A., Lindqvist Appell, M., . . . Lundström, P. (2015). Robust and convenient analysis of protein thermal and chemical stability. Protein Science, 24(12), 2055-2062
Open this publication in new window or tab >>Robust and convenient analysis of protein thermal and chemical stability
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2015 (English)In: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 24, no 12, p. 2055-2062Article in journal (Refereed) Published
Abstract [en]

We present the software CDpal that is used to analyze thermal and chemical denaturation data to obtain information on protein stability. The software uses standard assumptions and equations applied to two-state and various types of three-state denaturation models in order to determine thermodynamic parameters. It can analyze denaturation monitored by both circular dichroism and fluorescence spectroscopy and is extremely flexible in terms of input format. Furthermore, it is intuitive and easy to use because of the graphical user interface and extensive documentation. As illustrated by the examples herein, CDpal should be a valuable tool for analysis of protein stability.

Place, publisher, year, edition, pages
WILEY-BLACKWELL, 2015
Keywords
protein stability; thermal denaturation; chemical denaturation; circular dichroism; fluorescence; curve fitting; protein stability software; protein denaturation software
National Category
Chemical Sciences Clinical Medicine
Identifiers
urn:nbn:se:liu:diva-124648 (URN)10.1002/pro.2809 (DOI)000368292000014 ()26402034 (PubMedID)
Note

Funding Agencies|Swedish Research Council [2012-5136]; LiU Cancer

Available from: 2016-02-08 Created: 2016-02-08 Last updated: 2017-11-30
Haglund, S., Zimdahl Kahlin, A., Vikingsson, S., Almér, S. & Söderman, J. (2014). P658 Effects of allopurinol on thiopurine metabolism and gene expression levels in HepG2 cells. In: : . Paper presented at 9th Congress of ECCO - the European Crohn's and Colitis Organisation (pp. 1:S345). , 8
Open this publication in new window or tab >>P658 Effects of allopurinol on thiopurine metabolism and gene expression levels in HepG2 cells
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2014 (English)Conference paper, Published paper (Other academic)
National Category
Clinical Medicine
Identifiers
urn:nbn:se:liu:diva-117883 (URN)10.1016/S1873-9946(14)60777-7 (DOI)
Conference
9th Congress of ECCO - the European Crohn's and Colitis Organisation
Available from: 2015-05-12 Created: 2015-05-12 Last updated: 2015-05-26
Wennerstrand, P., Mårtensson, L.-G., Söderhäll, S., Lindqvist Appell, M. & Zimdahl, A. (2013). Methotrexate binds to recombinant thiopurine S-methyltransferase and inhibits enzyme activity after high-dose infusions in childhood leukaemia. European Journal of Clinical Pharmacology, 69(9), 1641-1649
Open this publication in new window or tab >>Methotrexate binds to recombinant thiopurine S-methyltransferase and inhibits enzyme activity after high-dose infusions in childhood leukaemia
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2013 (English)In: European Journal of Clinical Pharmacology, ISSN 0031-6970, E-ISSN 1432-1041, Vol. 69, no 9, p. 1641-1649Article in journal (Refereed) Published
Abstract [en]

Purpose

Important drugs in the treatment of childhood acute lymphoblastic leukaemia (ALL) are 6-mercaptopurine (6-MP) and methotrexate (MTX). Thiopurine methyltransferase (TPMT) is a polymorphic enzyme causing variability in 6-MP response and toxicity. The aim of this study was to investigate the fluctuation in TPMT enzyme activity over time and the effect of high-dose MTX infusions on TPMT enzyme activity and 6-MP metabolites in paediatric ALL patients.

Methods

Fifty-three children with ALL treated according to the NOPHO-ALL 2000 protocol were included in the study. TPMT enzyme activity was measured at six different times starting from diagnosis until after the end of maintenance treatment. TPMT and 6-MP metabolites were measured before the initiation of high-dose MTX (HD-MTX) infusions and at 66 h post-infusion. The interaction between MTX and TPMT was investigated in vitro using recombinant TPMT protein and a leukaemic cell line.

Results

Forty percent of TPMT wild-type individuals had deceptively low TPMT enzyme activity according to genotype at the time of diagnosis. TPMT activity had decreased significantly 66 h after the start of HD-MTX infusions (−9.2 %; p = 0.013). MTX bound to recombinant TPMT protein severely inhibiting TPMT enzyme activity (remaining activity 16 %).

Conclusions

Our results show that TPMT genotyping should be performed in children with ALL, since 40 % of the children in our study who carried the wild-type TPMT gene were at risk of initial underdosing of 6-MP in cases where only TPMT enzyme activity was determined. MTX inhibits the TPMT enzyme activity after HD-MTX infusions due to protein binding.

Place, publisher, year, edition, pages
Springer Berlin/Heidelberg, 2013
Keywords
Leukaemia, 6-mercaptopurine, methotrexate, pharmacogenetics, thiopurine s-methyltransferase
National Category
Natural Sciences
Identifiers
urn:nbn:se:liu:diva-80190 (URN)10.1007/s00228-013-1521-9 (DOI)000323429900003 ()
Available from: 2012-08-22 Created: 2012-08-22 Last updated: 2020-02-17Bibliographically approved
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