| 1. |
- Knaust, Eva, 1944-, et al.
(författare)
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Different effects of metabolic inhibitors and cyclosporin A on daunorubicin transport in leukemia cells from patients with AML
- 2003
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Ingår i: Leukemia Research. - 0145-2126 .- 1873-5835. ; 27:2, s. 183-191
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Tidskriftsartikel (refereegranskat)abstract
- The objective of this study was to determine the role of transport proteins in daunorubicin (Dnr) accumulation and efflux in leukemia cells from 36 patients with acute myeloid leukemia (AML). Mononuclear cells were isolated and incubated with 1 μM Dnr with/without addition of 3 μM cyclosporin A (CyA) or metabolic inhibitors (MI). Cellular Dnr concentration in leukemia blast cells was measured with flow cytometry. After washing and reincubation of the cells in drug-free medium, Dnr efflux was followed with/without addition of CyA or MI. Levels of mRNA expression for mdr1, multidrug resistance associated protein (mrp) and lung resistance protein (lrp) were determined with reverse transcriptase-polymerase chain reaction (RT-PCR). MI enhanced cellular Dnr accumulation to a higher extent than CyA whereas CyA reduced Dnr efflux more efficiently than MI (P<0.001). There was a significant difference in Dnr accumulation between samples with low and high mdr1 mRNA levels but only in the presence of MI or CyA. Our results imply that other factors than P-glycoprotein (Pgp) are of major importance for in vitro Dnr accumulation in AML blasts and that the role of Pgp as a drug efflux pump is not conclusive.
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| 2. |
- Knaust, Eva, 1944-, et al.
(författare)
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Heterogeneity of isolated mononuclear cells from patients with acute myeloid leukemia affects cellular accumulation and efflux of daunorubicin
- 2000
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Ingår i: Haematologica. - 0390-6078 .- 1592-8721. ; 85:2, s. 124-132
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND AND OBJECTIVE: Pharmacologic studies on blasts from patients with leukemia are generally performed on density gradient isolated blood or bone marrow cells. Thereby, cellular drug accumulation and efflux are determined as mean values of the entire cell population. The objective of the present study was to characterize the heterogeneity in the accumulation and efflux of daunorubicin in various subpopulations of mononuclear cells isolated from patients with acute myeloid leukemia (AML).DESIGN AND METHODS: Mononuclear cells from 33 patients with AML were isolated from peripheral blood by density gradient centrifugation on Lymphoprep (1. 077 g/mL). Cellular accumulation of fluorescent daunorubicin was determined by flow cytometry after incubation of the cells at +37C for 1 hour. Thereafter, the cells were washed and reincubated in drug-free medium. Kinetics of drug efflux were determined by frequent determination of cellular fluorescence during 30 min. Daunorubicin accumulation and efflux were compared in the total isolated mononuclear cell population and in the various blast cell populations gated on FSC/SSC according to the results of immunophenotyping.RESULTS: In 8 of these 33 (24%) patient samples, two distinct blast cell populations could be identified. In 7 out of 8 these cases the more immature blasts had a lower drug accumulation and in 6 out of the 8 cases also a higher efflux rate than the differentiating cell population. Cyclosporin A increased daunorubicin accumulation and reduced efflux in the immature blast population. In the differentiating cell population cyclosporin A increased both the accumulation and the efflux. In patients with a single blast cell population, the gated blast cells had a significantly lower drug accumulation but also a lower drug efflux rate than the total cell population.INTERPRETATION AND CONCLUSIONS: The results imply that drug transport studies on cells isolated from patients with AML give somewhat different results depending on the cell population studied. Some, but not all, of these differences in daunorubicin accumulation and efflux as well as in the effect of cyclo-sporin A can be explained by a heterogenous expression of the mdr1-gene. The observed heterogeneity may be of special relevance with regard to drug resistance. The presence of even a small resistant cell clone may jeopardize the effect of the chemotherapy due to expansion resulting in relapse of disease.
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| 3. |
- Palucka, Karolina A., et al.
(författare)
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Intraclonal heterogeneity in the in vitro daunorubicin-induced apoptosis in acute myeloid leukemia
- 1999
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Ingår i: Leukemia and Lymphoma. - : Informa UK Limited. - 1042-8194 .- 1029-2403. ; 32:3-4, s. 309-316
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Tidskriftsartikel (refereegranskat)abstract
- Leukemic cells from ten patients with acute myeloid leukemia (AML) were sorted on the basis of in vitro daunorubicin (DNR) uptake. The obtained subpopulations with high and low DNR accumulation were compared with regard to induction of apoptosis, expression of bcl-2 and p53. Heterogeneous induction of apoptosis, confined to subpopulations with high DNR uptake, was observed. The size of the DNR-induced apoptotic fraction (4% to 16%) within a given AML blast population was determined by intracellular drug accumulation and was not related to the level of bcl-2 expression. All tested leukemic samples displayed expression of p53 in a growth promoter orientation, i.e. PAb1620-/PAb240+. In two samples, however, sub-populations expressing a growth suppressor orientation of p53, i.e. PAb1620+/PAb240-, were also present. These subpopulations were confined to high-DNR-uptake fractions and associated with the induction of apoptosis. We conclude that intraclonal heterogeneity in the intracellular drug accumulation and subsequently in DNR-induced apoptosis might allow the selection of inherently drug-resistant AML clones thus contributing to relapse of leukemia.
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| 4. |
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| 5. |
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| 6. |
- Fotoohi, Alan Kambiz, et al.
(författare)
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Impaired transport as a mechanism of resistance to thiopurines in human T-lymphoblastic leukemia cells
- 2006
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Ingår i: Nucleosides, Nucleotides & Nucleic Acids. - : Informa UK Limited. - 1525-7770 .- 1532-2335. ; 25:9-11, s. 1039-1044
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Tidskriftsartikel (refereegranskat)abstract
- In order to better understand the mechanisms of resistance to thiopurines, we studied two sublines of the MOLT4 T-lymphoblastic leukemia cell line, resistant to 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG). We found that the underlying mechanism of resistance in both resistant cell lines was a markedly reduction in initial transport of 6-MP (3- and 5-fold, respectively, in 6-MP- and 6-TG-resistant cells). No significant alteration of activities of hypoxanthine-guanine phosphoribosyl transferase, thiopurine methyltransferase or inosine monophosphate dehydrogenase, the key enzymes involved in the metabolism of thiopurines was detected. We conclude that defected initial transport of thiopurines by cells may very well explain their resistance to these drugs. Copyright © Taylor & Francis Group, LLC.
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| 7. |
- Fotoohi, Alan Kambiz, et al.
(författare)
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Involvement of the concentrative nucleoside transporter 3 and equilibrative nucleoside transporter 2 in the resistance of T-lymphoblastic cell lines to thiopurines
- 2006
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Ingår i: Biochemical and Biophysical Research Communications - BBRC. - : Elsevier BV. - 0006-291X .- 1090-2104. ; 343:1, s. 208-215
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Tidskriftsartikel (refereegranskat)abstract
- Mechanisms of resistance to thiopurines, 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) were investigated in human leukemia cell lines. We developed two 6-MP- and 6-TG-resistant cell lines from the human T-lymphoblastic cell line (MOLT-4) by prolonged exposure to these drugs. The resistant cells were highly cross resistant to 6-MP and 6-TG, and exhibited marked reduction in cellular uptake of 6-MP (70% and 80%, respectively). No significant modification of the activities of hypoxanthine-guanine phosphoribosyl transferase, thiopurine methyltransferase or inosine monophosphate dehydrogenase was observed. Real-time PCR of concentrative nucleoside transporter 3 (CNT3) and equilibrative nucleoside transporter 2 (ENT2) of resistant cells showed substantial reductions in expression of messenger RNAs. Small interfering RNA designed to silence the CNT3 and ENT2 genes down-regulated the expression of these genes in leukemia cells. These decreases were accompanied by reduction of transport of 6-MP (47% and 21%, respectively) as well as its cytocidal effect (30% and 21%, respectively). Taken together these results show that CNT3 and ENT2 play a key role in the transport of 6-MP and 6-TG by leukemia cells. From a clinical point of view determination of CNT3 and ENT2 levels in leukemia cells may be useful in predicting the efficacy of thiopurine treatment. © 2006 Elsevier Inc. All rights reserved.
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| 8. |
- Fotoohi, A K, et al.
(författare)
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Molecular mechanisms underlying the enhanced sensitivity of thiopurine-resistant T-lymphoblastic cell lines to methyl mercaptopurineriboside
- 2006
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Ingår i: Biochemical Pharmacology. - : Elsevier BV. - 0006-2952 .- 1356-1839. ; 72:7, s. 816-823
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Tidskriftsartikel (refereegranskat)abstract
- Methylmercaptopurine riboside (meMPR), a cellular metabolite of 6-mercaptopurine (6-MP), is a potent inhibitor of de novo purine synthesis (DNPS). Human MOLT4 T-lymphoblastic leukaemia cells that have acquired resistance to 6-MP or 6-thioguanine (6-TG) as a consequence of defective transport exhibit enhanced sensitivity to meMPR. HPLC-based analysis of the transport of meMPR revealed normal uptake of this compound by our thiopurine-resistant cell sublines, suggesting a route of transport distinct from that for 6-MP and 6-TG. Studies on the wild-type parental leukemic cells showed that adenosine, dipyridamole and nitrobenzylthioinosine inhibit uptake of meMPR to a significant extent, whereas Na+ ions have no influence on this process. Transfection of these leukemic cells with small interference RNA molecules targeting the gene encoding the first member of the family of equiliberative nucleoside transporters (ENT1) strongly reduced the initial rate of meMPR transport. Our resistant cell lines exhibited 30-52% reductions (p < 0.005) in their levels of mRNA encoding several proteins involved in de novo purine synthesis, i.e., aminoimidazole carboxamide ribonucleotide formyltransferase, glycinamide ribonucleotide transformylase and guanine monophosphate synthetase. Consequently, the rate of de novo purine synthesis in these resistant sublines was decreased by 50%. Furthermore, the levels of ribonucleoside triphosphates in these cells were significantly lower than in the non-resistant parental cells. In combination, a reduced rate of de novo purine synthesis together with low levels of ribonucleoside triphosphates can explain the enhanced sensitivity of our thiopurine-resistant cell lines to meMPR. In this manner, meMPR bypasses the mechanisms of resistance to thiopurines and is even more cytotoxic towards resistant than towards wild-type cells. © 2006.
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| 9. |
- Fotoohi, K, et al.
(författare)
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Interference of 7-hydroxymethotrexate with the determination of methotrexate in plasma samples from children with acute lymphoblastic leukemia employing routine clinical assays
- 2005
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Ingår i: Journal of Chromatography. B. - : Elsevier BV. - 1873-376X .- 1570-0232. ; 817:2, s. 139-144
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Tidskriftsartikel (refereegranskat)abstract
- The accuracy of two clinical assays, the enzyme-multiplied immunoassay (EMIT) and fluorescence polarization immunoassay (FPIA2), universally employed for measurement of plasma levels of methotrexate (MTX) in children administered a high dose of this drug for treatment of acute lymphoblastic leukemia was evaluated here. Because of its superior specificity, sensitivity, and precision, high performance liquid chromatography (HPLC) was selected as the reference method with which the other two procedures were compared using approximately 420 different plasma samples for method comparison. 7-Hydroxymethotrexate (7-OHMTX), the major plasma metabolite of MTX, that can be detected in plasma at relatively high concentrations for long periods following infusion of a high dose of MTX, was also quantitated by HPLC. Forty-two and 66 h after infusion, the plasma level of MTX was overestimated in 2% and 3% of the samples by the FPIA2 procedure in 5% and 31% by the EMIT assay. The overall correlation coefficients (r(2)) for the values obtained by FPIA2 or EMIT versus those based on HPLC were 0.989 and 0.663, respectively. The presence of 7-OHMTX exerted a highly significant influence (p = 0.0007 as determined by the unpaired t-test) on MTX measurement by the EMIT assay. We conclude that the rapid automated procedures routinely used at present and in particular EMIT, suffer from cross-reactivity with metabolites of MTX. Thus, the relatively high percentage of samples in which the level of MTX is overestimated at check-points by EMIT may result in longer periods of hospitalization, higher costs and prolonged administration of elevated doses of "rescue" leucovorin with an increased risk for relapse.
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| 10. |
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| 11. |
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| 12. |
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| 13. |
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| 14. |
- Haglund, Sofie, 1975-, et al.
(författare)
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IMPDH activity in thiopurine-treated patients with inflammatory bowel disease - Relation to TPMT activity and metabolite concentrations
- 2008
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Ingår i: British Journal of Clinical Pharmacology. - : Wiley. - 0306-5251 .- 1365-2125. ; 65:1, s. 69-77
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 15. |
- Haglund, Sofie, 1975-, et al.
(författare)
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Pharmacotranscriptomics in thiopurine treated IBD patients with different metabolite profiles
- 2008
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 16. |
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| 17. |
- Hindorf, Ulf, et al.
(författare)
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Pharmacogenetics during standardised initiation of thiopurine treatment in inflammatory bowel disease
- 2006
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Ingår i: Gut. - : BMJ. - 0017-5749 .- 1468-3288. ; 55:10, s. 1423-1431
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Tidskriftsartikel (refereegranskat)abstract
- Background: Firm recommendations about the way thiopurine drugs are introduced and the use of thiopurine methyltransferase (TPMT) and metabolite measurements during treatment in inflammatory bowel disease (IBD) are lacking. Aim: To evaluate pharmacokinetics and tolerance after initiation of thiopurine treatment with a fixed dosing schedule in patients with IBD. Patients: 60 consecutive patients with Crohn's disease (n = 33) or ulcerative colitis (n = 27) were included in a 20 week open, prospective study. Methods: Thiopurine treatment was introduced using a predefined dose escalation schedule, reaching a daily target dose at week 3 of 2.5 mg azathioprine or 1.25 mg 6-mercaptopurine per kg body weight. TPMT and ITPA genotypes, TPMT activity, TPMT gene expression, and thiopurine metabolites were determined. Clinical outcome and occurrence of adverse events were monitored. Results: 27 patients completed the study per protocol, while 33 were withdrawn (early protocol violation (n = 5), TPMT deficiency (n = 1), thiopurine related adverse events (n = 27)), 67% of patients with adverse events tolerated long term treatment on a lower dose (median 1.32 mg azathioprine/kg body weight). TPMT activity did not change during the 20 week course of the study but a significant decrease in TPMT gene expression was found (TPMT/huCYC ratio, p = 0.02). Patients with meTIMP concentrations > 11 450 pmol/8 × 108 red blood cells during steady state at week 5 had an increased risk of developing myelotoxicity (odds ratio = 45.0, p = 0.015). Conclusions: After initiation of thiopurine treatment using a fixed dosing schedule, no general induction of TPMT enzyme activity occurred, though TPMT gene expression decreased. The development of different types of toxicity was unpredictable, but we found that measurement of meTIMP early in the steady state phase helped to identify patients at risk of developing myelotoxicity.
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| 18. |
- Hursti, T. J., et al.
(författare)
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Effect of chemotherapy on circulating gastrointestinal hormone levels in ovarian cancer patients: relationship to nausea and vomiting
- 2005
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Ingår i: Scand J Gastroenterol. - : Taylor & Francis. - 0036-5521 .- 1502-7708. ; 40:6, s. 654-61
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Tidskriftsartikel (refereegranskat)abstract
- OBJECTIVE: The introduction of 5-HT3 receptor antagonists greatly reduced the problems associated with nausea and vomiting immediately after cancer chemotherapy. However, delayed nausea and vomiting is still a major problem and the underlying mechanism is obscure. MATERIAL AND METHODS: We studied the effect of cisplatin-containing combination chemotherapy in 14 ovarian cancer patients on the levels of gastrin and a panel of other hormones as well as glucose and prostaglandin F2a. Blood samples were obtained once daily in the morning before chemotherapy and for 4 days after chemotherapy. RESULTS: Concentrations of many hormones including gastrin were generally high. A pronounced increase in plasma insulin levels occurred on the day after chemotherapy accompanied by a modest increase in plasma glucose concentrations. Minor increases were observed for gastrin, oxytocin and prostaglandin F2a. In contrast, a transient decrease after chemotherapy was observed for motilin. Plasma cortisol decreased markedly after chemotherapy as expected since betamethasone was given as an antiemetic prophylaxis. Certain trends concerning the relationship between some hormones and nausea and vomiting were noted. A high plasma gastrin concentration before chemotherapy was related to delayed vomiting. Relative day-to-day variability of cholecystokinin tended to correlate positively with delayed nausea, whereas an inverse relationship was observed for gastrin variability. CONCLUSIONS: Changes in hormone plasma levels were found but only few could be distinguished as possible mediators of delayed nausea and vomiting.
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| 19. |
- Knaust, Eva, 1944-
(författare)
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Experimental studies on multidrug resistance in human leukaemia : role of cellular heterogeneity for daunorubicin kinetics
- 2005
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Cellular resistance to chemotherapy is a major cause of treatment failure in acute myeloid leukaemia (AML) and still the majority of the patients die from their disease. Drug resistance 1s multifactorial, the most studied mechanism being multidrug resistance (MDR), mediated by the P-glycoprotein (Pgp). Pgp is an energy-dependent transport protein, encoded by the mdr1 gene, with the power to extrude the cytotoxic drugs out of the cells; thus causing reduced effect of the drug on the leukaemic cells. MDR is characterised by cross-resistance to a wide range of chemotherapeutics of natural origin. Other transport proteins, involved in drug resistance, are the multidrug resistance associated protein (MRP) and the lung resistance protein (Lrp).The aims of this thesis were to elucidate transport kinetics of the anthracycline, daunorubicin, (Dnr) and to investigate the effects of reversing agents on heterogeneity of drug accumulation in cells from patients with AML. The ultimate goal is to improve treatment based on each patient's individual resistance patterns.Density gradient isolated mononuclear cells from patients with AML were incubated with Dnr. Incubated cells were sorted with flow cytometry (FC) on the basis of accumulation levels of the autofluorescent Dnr. Gene expression of the Pgp and the MRP in sorted subpopulations were analysed with polymerase chain reaction (PCR). Apoptosis, expression of p53 and bcl-2 in the sorted subpopulations were determined with monoclonal antibodies and FC. Drug accumulation and efflux, with/without the resistance modifier Cyclosporin A (CyA) and energy-depleting metabolic inhibitors (MJ), were also determined in the leukaemic cell populations with FC.Gene expressions of mdr1 and mrp1 were shown to be heterogeneous in the leukaemic samples and drug accumulation correlated inversely to the gene expression. Cell populations with the higher drug accumulation entailed more apoptosis. The leukaemic cell lopulation, defined by immunopenotyping, differed in drug accumulation an efflux compared to the total mononuclear cell population that also contains normal lymphocytes and monocytes. In leukaemic samples with two blast cell populations, the more immature blast ceUs accumulated drug to a lesser extent and bad a higher efflux rate than the differentiating blast cells. CyA reduced Dnr efflux more efficiently than MI, but MJ increased drug accumulation in leukaemic cells more than CyA.In conclusion: analysis of the total mononuclear population does not give an accurate picture of the leukaemic cell population as concerns resistance mechanisms. Heterogeneity in the leukaemic cell population ought to be taken into account since two or more leukaemic cell populations often exist. The most immature blast cell population should be analysed as relapse usually derives from this population. Furthermore the role of Pgp in MDR is not conclusive as results with reversing agents differed from what was expected.
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| 20. |
- Lindqvist Appell, Malin, 1976-, et al.
(författare)
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Identification of two novel sequence variants affecting thiopurine methyltransferase enzyme activity
- 2004
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Ingår i: Pharmacogenetics. - : Ovid Technologies (Wolters Kluwer Health). - 0960-314X .- 1473-561X. ; 14:4, s. 261-265
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Tidskriftsartikel (refereegranskat)abstract
- The polymorphic enzyme thiopurine methyltransferase (TPMT) is involved in the methylation of thiopurines. On comparing the phenotype with the genotype in Swedish patients with inflammatory bowel disease and healthy individuals, we found two discordant cases with low TPMT enzyme activity (0.3 and 0.4 U/ml packed red blood cells (pRBC). Genotyping by pyrosequencing revealed that they carried the nucleotide substitutions 460G>A and 719A>G, giving two possible genotypes (TPMT*1/*3A or TPMT*3B/ *3C). DNA sequencing of exon III to X was performed in the patients and their parents. We identified an A>G transition in the start codon (exon III, 1A>G, Met>Val, TPMT*14) in one of the patients and her father (6.3 U/ml pRBC). The mother in this family carried the 460G>A and 719A>G nucleotide substitutions (TPMT*3A, 5.0 U/ml pRBC). In the second family, sequencing revealed a G>A transition in the acceptor splice site in intron VII/exon VIII (IVS7 - 1G>A, TPMT*15) in the patient and his mother (6.9 U/ml pRBC). His father was genotyped as TPMT*1/*3A (6.0 U/ml pRBC). Hence, we report the identification of two novel sequence variants, present in highly conserved nucleotide positions of the human TPMT gene, resulting in a loss of enzyme activity.
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| 21. |
- Lindqvist Appell, Malin, 1976-, et al.
(författare)
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No induction of thiopurine methyltransferase during thiopurine treatment in inflammatory bowel disease
- 2006
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Ingår i: Nucleosides, Nucleotides & Nucleic Acids. - : Informa UK Limited. - 1525-7770 .- 1532-2335. ; 25:9-11, s. 1033-1037
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Tidskriftsartikel (refereegranskat)abstract
- The aim of this study was to follow, during standardized initiation of thiopurine treatment, thiopurine methyltransferase (TPMT) gene expression and enzyme activity and thiopurine metabolite concentrations, and to study the role of TPMT and ITPA 94C > A polymorphisms for the development of adverse drug reactions. Sixty patients with ulcerative colitis or Crohn's disease were included in this open and prospective multi-center study. Thiopurine naive patients were prescribed azathioprine (AZA), patients previously intolerant to AZA received 6-mercaptopurine (6-MP). The patients followed a predetermined dose escalation schedule, reaching target dose at Week 3; 2.5 and 1.25 mg/kg body weight for AZA and 6-MP, respectively. The patients were followed every week during Weeks 1-8 from baseline and then every 4 weeks until 20 weeks. TPMT activity and thiopurine metabolites were determined in erythrocytes, TPMT and ITPA genotypes, and TPMT gene expression were determined in whole blood. One homozygous TPMT-deficient patient was excluded. Five non compliant patients were withdrawn during the first weeks. Twenty-seven patients completed the study per protocol; 27 patients were withdrawn because of adverse events. Sixty-seven percent of the withdrawn patients tolerated thiopurines at a lower dose at Week 20. There was no difference in baseline TPMT enzyme activity between individuals completing the study and those withdrawn for adverse events (p = 0.45). A significant decrease in TPMT gene expression (TPMT/huCYC ratio, p = 0.02) was found, however TPMT enzyme activity did not change. TPMT heterozygous individuals had a lower probability of remaining in the study on the predetermined dose (p = 0.039). The ITPA 94C > A polymorphism was not predictive of adverse events (p = 0.35).
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| 22. |
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| 23. |
- Lindqvist Appell, Malin, 1976-, et al.
(författare)
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Real-time RT-PCR methodology for quantification of thiopurine methyltransferase gene expression
- 2003
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Ingår i: European Journal of Clinical Pharmacology. - : Springer Science and Business Media LLC. - 0031-6970 .- 1432-1041. ; 59:3, s. 207-211
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Tidskriftsartikel (refereegranskat)abstract
- Objective: The aim of the present study was to develop a real-time reverse-transcription polymerase chain reaction (RT-PCR) methodology for the quantification of thiopurine methyltransferase (TPMT) gene expression in whole blood and compare it with the TPMT enzyme activity measured in red blood cells. Methods: TPMT gene expression was quantified relative to the housekeeping gene cyclophilin (huCYC) and expressed as a TPMT/huCYC ratio. TPMT activity in red blood cells was determined by measuring the formation rate of 6- 14C-methylmercaptopurine from 6-MP using S-adenosyl-L-( 14C-methyl)-methlonine as methyl donor. Thirty-nine individuals were included in the study. A cut-off value of 9 U/ml pRBC was used to distinguish intermediate TPMT enzyme activity from high TPMT enzyme activity. Results: Sequencing of the real-time RT-PCR amplicon proved that the method was specific for the TPMT cDNA, without co-amplification of the highly similar TPMT processed pseudogene. The intra-assay coefficients of variation (CVs), as determined by the threshold cycle, were 0.7% for TPMT and 0.5% for huCYC. The interassay CVs were 1.5% for TPMT and 4.0% for huCYC. The intra- and interassay CVs, as determined by the TPMT/huCYC ratio, were 8.6% and 25%, respectively. There was a statistically significant correlation between TPMT enzyme activity and mRNA level in blood cells from individuals with an enzyme activity above 9 U/ml pRBC (rs = 0.66, P = 0.0001). However, we did not find any statistically significant correlation in individuals with lower enzyme activity or when analysing the whole population. Conclusion: We present a specific and robust real-time RT-PCR method for quantifying TPMT gene expression. The method may be used for studies on TPMT gene regulation.
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| 24. |
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| 25. |
- Lindqvist Appell, Malin, 1976-, et al.
(författare)
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Thiopurines in inflammatory bowel disease - The role of pharmacogenetics and therapeutic drug monitoring
- 2006
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Ingår i: Current Pharmacogenomics. - : Bentham Science Publishers Ltd.. - 1570-1603. ; 4:4, s. 285-300
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Tidskriftsartikel (refereegranskat)abstract
- Pharmacogenetics represents the study of variability in drug response due to genetic variations. Inflammatory bowel disease (IBD, i.e. primarily Crohn's disease and ulcerative colitis) is characterized by a chronic or relapsing inflammation of the digestive tract. The thiopurines 6-mercaptopurine (6-MP) and azathioprine (AZA), an imidazol derivative and pro-drug of 6-MP, are widely used in IBD, particularly in Crohn's disease. The metabolism of thiopurines is complex and individually variable. Thiopurine methyltransferase (TPMT) is a key enzyme in this metabolism and exhibits a genetic variability due to a number of variant alleles coding for a defective enzyme. The formation of biologically active thioguanine nucleotides (TGN) and methylated metabolites may vary considerably due to the TPMT activity. Patients with decreased TPMT activity are at increased risk of developing severe side effects if treated with conventional thiopurine doses, due to the accumulation of toxic metabolites. Determination of the TPMT phenotype or genotype is often used to identify individuals with increased risk for adverse events. Twenty-one variant TPMT alleles have been described, of which three are more common than the others. An association between inosine triphosphate pyrophosphatase polymorphisms and adverse events during thiopurine treatment has also been proposed. In this review, the clinical value of TPMT status determination and pharmacological monitoring of thiopurine metabolites are discussed as well as the increased interest in the use of 6-thioguanine, a thiopurine with a less complex metabolism, as an alternative for patients who do not tolerate AZA or 6-MP. It can be concluded that TPMT determination before start of thiopurine therapy is of value to identify individuals with increased risk for adverse reactions due to genetic enzyme deficiency. However, large prospective studies are still needed to evaluate the true benefit of monitoring thiopurine metabolites during thiopurine treatment. © 2006 Bentham Science Publishers Ltd.
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