| 1. |
- Albertioni, Freidoun
(författare)
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Studies on methotrexate and cladribine : bioanalytical, pharmacokinetic, and clinical aspects
- 1996
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The aim of these studies was to delineate the pharmacokinetics of methotrexate (MTX) and cladribine in order to improve the basis for individualization and optimization of treatment with these two antimetabolites. Methodological aspects of MTX assays in clinical routine usage were investigated as plasma MTX determinations are mandatory in the high-dose treatment of childhood acute Iymphoblastic leukemia (ALL). Four clinically commonly used assays (EIA, EMIT, FPIA1, and FPIA2) were compared with HPLC as the reference method. All non-chromatographic procedures gave very reproducible results but suffered from cross-reactivity with plasma metabolites or endogenous substances. 7-hydroxymethotrexate (7-OHMTX), a major metabolite of MTX interfered with all non- chromatographic assays and this has more significance for plasma samples obtained at later points in time when the metabolite concentration exceeds that of the parent substance. 4-diamino-N10-methylpteroic acid (DAMPA), another plasma MTX metabolite, also interfered in clinical assays using antibodies (EMIT and FPIA). However, the variability of the HPLC method was considerably higher than that of clinical assays. Clinical and pharmacokinetic risk factors regarding toxicity after high dose MTX in children with ALL were studied in 13 patients. Oral mucositis was significantly related to high plasma MTX concentration at 28 h after starting the infusion (p=0.013), to a low ratio of plasma 7-OHMTX/MTX at 66 h after starting the infusion (p=0.049), and to a slow clearance of MTX (p=0.048). The risk of leukopenia increased significantly with the course number (p=0.02). Increasing age (p=0.047) and a long exposure to a high MTX concentration in plasma (p=0.009) correlated with liver toxicity. The occurrence of mucositis was significantly related to the ratio between saliva concentration of 7-OHMTX and MTX at 20 h after starting the infusion (p=0.014), but not at later points in time. Cladribine is a chlorinated analogue of deoxyadenosine which is active against chronic Iymphoproliferative disorders. Plasma pharmacokinetic studies showed that subcutaneous administration gave a drug exposure (AUC) similar to intravenous infusion. The bioavailability of oral cladribine was 50%. Food and omeprazole had practically no influence on the bioavailability of cladribine but food reduced the peak concentration and prolonged the time to reach the maximum plasma concentration. In a perused rat liver experiment, cladribine was highly degraded by cleavage of the N-glycosidic bond by liver enzymes. The fluorinated analogue of cladribine, 2-chloro-2'- arabino-fluoro-2'-deoxyadenosine (CAFdA) was more stable against degradation and showed 2-5 fold lower hepatic extraction ratio as compared to cladribine suggesting that CAFdA is a more suitable drug for oral administration than cladribine. An ion-pair HPLC method was developed for monitoring of the pharmacokinetics of cladribine nucleotides in patients. Pharmacokinetics of cladribine, its deglycosylated product, CAde, and cladribine nucleotides (CdAMP, CdADP and CdATP) were studied in 17 patients with CLL after treatment with cladribine. The concentration of CAde was considerably higher after oral administration. The AUC of CdAMP in leukemic cells was generally higher than that of CdATP (median ratio 1.5). In 4 out of 17 patients the reverse relation was seen. The concentration of the 5'-diphosphate was much lower, in most patients undetectable. The terminal phase had a median t1/2 of 14.6 h for CdAMP and 9.7 h for CdATP. No correlation was observed between the concentration of active metabolite (CdATP) and clinical response.
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| 2. |
- 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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| 3. |
- 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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| 4. |
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| 5. |
- 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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| 6. |
- Fyrberg, Anna, et al.
(författare)
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Cell cycle effect on the activity of deoxynucleoside analogue metabolising enzymes
- 2007
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Ingår i: Biochemical and Biophysical Research Communications - BBRC. - : Elsevier BV. - 0006-291X .- 1090-2104. ; 357:4, s. 847-853
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Tidskriftsartikel (refereegranskat)abstract
- Deoxynucleoside analogues (dNAs) are cytotoxic towards both replicating and indolent malignancies. The impact of fluctuations in the metabolism of dNAs in relation to cell cycle could have strong implications regarding the activity of dNAs. Deoxycytidine kinase (dCK) and deoxyguanosine kinase (dGK) are important enzymes for phosphorylation/activation of dNAs. These drugs can be dephosphorylated/deactivated by 5′-nucleotidases (5′-NTs) and elevated activities of 5′-NTs and decreased dCK and/or dGK activities represent resistance mechanisms towards dNAs. The activities of dCK, dGK, and three 5′-NTs were investigated in four human leukemic cell lines in relationship to cell cycle progression and cytotoxicity of dNAs. Synchronization of cell cultures to arrest in G0/G1 by serum-deprivation was performed followed by serum-supplementation for cell cycle progression. The activities of dCK and dGK increased up to 3-fold in CEM, HL60, and MOLT-4 cells as they started to proliferate, while the activity of cytosolic nucleotidase I was reduced in proliferating cells. CEM, HL60, and MOLT-4 cells were also more sensitive to cladribine, cytarabine, 9-β-d-arabinofuranosylguanine and clofarabine than K562 cells which demonstrated lower levels and less alteration of these enzymes and were least susceptible to the cytotoxic effects of most dNAs. The results suggest that, in the cell lines studied, the proliferation process is associated with a general shift in the direction of activation of dNAs by inducing activities of dCK/dGK and reducing the activity of cN-I which is favourable for the cytotoxic effects of cladribine, cytarabine and, 9-β-d-arabinofuranosylguanine. These results emphasize the importance of cellular proliferation and dNA metabolism by both phosphorylation and dephosphorylation for susceptibility to dNAs. It underscores the need to understand the mechanisms of action and resistance to dNAs in order to increase efficacy of dNAs treatment by new rational. © 2007 Elsevier Inc. All rights reserved.
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| 7. |
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| 8. |
- Fyrberg, Anna, 1981-
(författare)
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Nucleoside analoge cytotoxicity-focus on enzyme regulation, metabolism, and mechanisms of resistance
- 2010
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The aim of this thesis was to determine the role of nucleoside analog activating and deactivating enzymes in nucleoside analog metabolism and resistance development. Nucleoside analogs are anti-cancer drogs and are often used to treat different leukemias, attributably to presence of high levels of nucleoside analog activating enzymes in hematopoietic cells. More recently some of the newer analogs have been used successfully to treat solid tumors as well.We have used human leukemic cell lines, and isolated cells from patients with leukemia, to investigate the nucleoside analog activating enzymes deoxycytidine kinase (dCK) and deoxyguanosine kinase (dGK) and some of the deactivating enzymes called 5'nucleotidases (5'-NTs). We have measured mRNA expressions and enzymatic activities and correlated them with the cytotoxic response to nuc1eoside analogs and changes in cell cycle progression. We optimized and evaluated a siRNA-transfection method and decreased the activities of dCK and dGK in two different cell lines in order to find out more about their respective contribution to activation of these drogs. An expression microarray analysis of a nucleoside analog resistant cell line was also performed in order to clarify which genes are involved in development of resistance.We found that expressions and activities of dCK and dGK were not correlated. The enzyme activities of activating and deactivating enzymes changed during cell cycle progression, giving actively proliferating cells a more favorable enzymatic profile with regard to nucleoside analog cytotoxicity.The activities of dCK and dGK could be reduced transiently in leukemic and solid tumor cell lines, thereby confer either resistance or increased sensitivity to nucleoside analogs to variable degrees. Expression microarray analysis was used to evaluate the effect of the transfection method and the specificity of siRNA. We concluded that cells tolerated the transfection weIl without major effects on gene expression, and considered the siRNA used to be specific to its target.An expression microarray experiment on a nucleoside analog-induced resistant cell line revealed a hypomethylating capacity of the drog and induction of fetal hemoglobin and a multidrog resistance efflux pump as a result of the hypomethylation. This pump should not be affected by nucleoside analoges since they are not a substrate of it, and upregulation of the pump unfortunately renders the cells highly cross-resistant to different types of drogs. Our preliminary data supports our theory that it may be upregulated in order to help excrete hemoglobin that otherwise would be toxic to the cells.
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| 9. |
- Fyrberg, Anna, et al.
(författare)
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RNAi Depletion of Deoxycytidine and Deoxyguanosine Kinase in Human Leukemic CEM Cells
- 2008
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Ingår i: Nucleosides, Nucleotides & Nucleic Acids. - : Informa UK Limited. - 1525-7770 .- 1532-2335. ; 27:6-7, s. 712-719
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Tidskriftsartikel (refereegranskat)abstract
- Resistance toward nucleoside analogues is often due to decreased activities of the activating enzymes deoxycytidine kinase (dCK) and/or deoxyguanosine kinase (dGK). With small interfering RNA (siRNA), dCK and dGK were downregulated by approximately 70% in CEM cells and tested against six nucleoside analogues using the methyl thiazol tetrazolium assay. SiRNA-transfected cells reduced in dCK activity were 3- to 6-fold less sensitive to CdA, AraC, and CAFdA. The sensitivity to AraG and FaraA was unchanged, while the sensitivity toward gemcitabine was significantly increased. dGK depletion in cells resulted in lower sensitivity to FaraA, dFdC, CAFdA, and AraG, but slightly higher sensitivity to CdA and AraC.
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| 10. |
- Fyrberg, Anna, et al.
(författare)
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The role of deoxyguanosine kinase for nucleoside analog activation in leukemic and solid tumor cell lines
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Screening malignant melanoma cell lines against nucleoside analogs revealed as high sensitivity to fludarabine, clofarabine, and gemcitabine as to leukemic cells, and especially in those cells expressing high levels of the mitochondrial enzyme deoxuguanosine kinase (dGK). This enzyme, together with the cytosolic deoxycytidine kinase (dCK), and the mitochondrial thymidine kinase 2 (TK2) contributes to the activation of natural deoxyribonucleosides and nucleoside analogs to phosphorylated compounds. dCK is the most prominent enzyme in hematopoietic cells, while dGK may be high in cells harbouring many mitochondria, such as neurons and melanocytes. We found that dGK mRNA and protein expression was considerably higher in melanoma cells than in a leukemic cell line, while the difference at the activity level was less profound. Downregulation of dGK in the melanoma cell line RaH5 using siRNA led to a compensatory increase in TK2 activity, which led to significantly increased sensitivity of the cells to gemcitabine. In contrast, downregulation of dGK in the human leukemic CEM cell line decreased TK2 activity, and rendered the cells more resistant to the drugs. The compensatory regulation of deoxynucleoside kinases with over-lapping substrate specificity differed in leukemic and melanoma cell lines probably because they preferably rely on different deoxynucleoside kinases for nucleoside and nucleoside analog activation. dGK and TK2 that are both located in the mitochondria, seems to be able to compensate for each other to a higher extent in the dGK-dependent melanoma cells compared to CEM cells that possess high dCK activity. Solid tumors, such as melanoma, expressing high levels of dGK should be considered for nucleoside analog therapy preferably in combination with their standard treatment.
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