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Papers by Alan Fotoohi

Research paper thumbnail of Involvement of the concentrative nucleoside transporter 3 and equilibrative nucleoside transporter 2 in the resistance of T-lymphoblastic cell lines to thiopurines

Biochemical and Biophysical Research Communications, Apr 1, 2006

TAS-102, which is effective for refractory metastatic colorectal cancer, is a combination drug of... more TAS-102, which is effective for refractory metastatic colorectal cancer, is a combination drug of anticancer trifluridine (FTD; which is derived from pyrimidine nucleoside) and FTD-metabolizing enzyme inhibitor tipiracil hydrochloride (TPI) at a molecular ratio of 1:0.5. To evaluate the intestinal absorption mechanism of FTD, the uptake and transcellular transport of FTD by human small intestinal epithelial cell (HIEC) monolayer as a model of human intestinal epithelial cells was investigated. The uptake and membrane permeability of FTD by HIEC monolayers were saturable, Na +-dependent, and inhibited by nucleosides. These transport characteristics are mostly comparable with those of concentrative nucleoside transporters (CNTs). Moreover, the uptake of FTD by CNT1-expressing Xenopus oocytes was the highest among human CNT transporters. The obtained K m and V max values of FTD by CNT1 were 69.0 M and 516 pmol/oocyte/30 min, respectively. The transcellular transport of FTD by Caco-2 cells, where CNT1 is heterologously expressed, from apical to basolateral side was greater than that by Mock cells. In conclusion, these results demonstrated that FTD exhibits high oral absorption by the contribution of human CNT1.

Research paper thumbnail of Therapeutic drug monitoring in oncology: International Association of Therapeutic Drug Monitoring and Clinical Toxicology consensus guidelines for imatinib therapy

European Journal of Cancer, Nov 1, 2021

Although therapeutic drug monitoring (TDM) is an important tool in guiding drug dosing for other ... more Although therapeutic drug monitoring (TDM) is an important tool in guiding drug dosing for other areas of medicine including infectious diseases, cardiology, psychiatry and transplant medicine, it has not gained wide acceptance in oncology. For imatinib and other tyrosine kinase inhibitors, a flat dosing approach is utilised for management of oral chemotherapy. There are many published studies examining the correlation of blood concentrations with clinical effects of imatinib. The International Association of Therapeutic Drug Monitoring and Clinical Toxicology (IATDMCT) determined that there was a need to examine the published literature regarding utility of TDM in imatinib therapy and to develop consensus guidelines for TDM based on the available data. This article summarises the scientific evidence regarding TDM of imatinib, as well as the consensus guidelines developed by the IATDMCT.

Research paper thumbnail of Assessment of renal function of cancer patients for dosing carboplatin

Research paper thumbnail of Introducing a simple and cost-effective RT-PCR protocol for detection of DPYD*2A polymorphism: the first study in Kurdish population

Cancer Chemotherapy and Pharmacology

Research paper thumbnail of Comparison of Uptake Mechanisms for Anthracyclines in Human Leukemic Cells

Current Drug Delivery, 2013

The mechanisms behind cellular anthracycline uptake are not completely understood. Knowledge abou... more The mechanisms behind cellular anthracycline uptake are not completely understood. Knowledge about uptake mechanisms could be used to increase the selectivity of the drugs. We compared the uptake patterns of, daunorubicin (DNR), doxorubicin (DOX), epirubicin (EPI), idarubicin (IDA), and pirarubicin (PIRA) by cultured leukemic cells and investigated possible involvement of specific carriers. HL-60 cells were incubated with anthracyclines for 1 hour in the absence or presence of transport inhibitors, suramin, or nucleosides and cellular drug uptake was determined. Cell survival was also determined. MCF-7 breast cancer cells were used as a negative control for concentrative nucleoside transporters (CNTs). Anthracycline concentration was determined with HPLC and fluorometric detection and apoptosis was determined with propidium iodide and flow cytometry. DNR, IDA, and PIRA had higher uptake than DOX and EPI with a prominent increase in uptake at concentrations > 1 µM. Uptake of all anthracyclines was greatly reduced at 0°C. Suramin, a purinergic-2-receptor inhibitor, strongly inhibited the uptake of all anthracyclines except PIRA and increased cell survival. Dipyridamole, an equilibrative NT (ENT) inhibitor, significantly inhibited the uptake of DNR only. The addition of nucleosides significantly inhibited the uptake of DNR, IDA, and PIRA but not in MCF-7 cells lacking functional CNTs. Our results suggest different uptake mechanisms for the anthracyclines studied. We found evidence for carrier mediated uptake mechanisms, supporting involvement of NTs in transmembrane transport of DNR, IDA, and PIRA. The results also showed a strong inhibition of suramin on anthracycline uptake by so far unknown mechanisms.

Research paper thumbnail of Gene expression profiling of leukemia T-cells resistant to methotrexate and 7-hydroxymethotrexate reveals alterations that preserve intracellular levels of folate and nucleotide biosynthesis

Biochemical Pharmacology, 2009

Research paper thumbnail of Molecular mechanisms underlying the enhanced sensitivity of thiopurine-resistant T-lymphoblastic cell lines to methyl mercaptopurineriboside

Biochemical Pharmacology, 2006

Research paper thumbnail of Differential role of thiopurine methyltransferase in the cytotoxic effects of 6-mercaptopurine and 6-thioguanine on human leukemia cells

Biochemical and Biophysical Research Communications, 2013

The thiopurine antimetabolites, 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) are inactive pro... more The thiopurine antimetabolites, 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) are inactive prodrugs that require intracellular metabolism for activation to cytotoxic metabolites. Thiopurine methyltransferase (TPMT) is one of the most important enzymes in this process metabolizing both 6-MP and 6-TG to different methylated metabolites including methylthioinosine monophosphate (meTIMP) and methylthioguanosine monophosphate (meTGMP), respectively, with different suggested pharmacological and cytotoxic properties. While meTIMP is a potent inhibitor of de novo purine synthesis (DNPS) and significantly contributes to the cytotoxic effects of 6-MP, meTGMP, does not add much to the effects of 6-TG, and the cytotoxicity of 6-TG seems to be more dependent on incorporation of thioguanine nucleotides (TGNs) into DNA rather than inhibition of DNPS. In order to investigate the role of TPMT in metabolism and thus, cytotoxic effects of 6-MP and 6-TG, we knocked down the expression of the gene encoding the TPMT enzyme using specifically designed small interference RNA (siRNA) in human MOLT4 leukemia cells. The knock-down was confirmed at RNA, protein, and enzyme function levels. Apoptosis was determined using annexin V and propidium iodide staining and FACS analysis. The results showed a 34% increase in sensitivity of MOLT4 cells to 1 lM 6-TG after treatment with TPMT-targeting siRNA, as compared to cells transfected with non-targeting siRNA, while the sensitivity of the cells toward 6-MP was not affected significantly by down-regulation of the TPMT gene. This differential contribution of the enzyme TPMT to the cytotoxicity of the two thiopurines is probably due to its role in formation of the meTIMP, the cytotoxic methylated metabolite of 6-MP, while in case of 6-TG methylation by TPMT substantially deactivates the drug.

Research paper thumbnail of The pattern of gene expression and gene dose profiles of 6-Mercaptopurine- and 6-Thioguanine-resistant human leukemia cells

Biochemical and Biophysical Research Communications, 2011

Determination of apoptosis by flow cytometry (Study I)……………………………....….30 RNA extraction and cDNA... more Determination of apoptosis by flow cytometry (Study I)……………………………....….30 RNA extraction and cDNA synthesis (Study I & IV)…………………………………………..31 Messenger RNA expression by RT-PCR (Study I & IV)…………………………………...…31 Gene expression by Microarray (Study II)……………………………………………………....32 DNA isolation and array comparative genomic hybridization (Study II)……………32 Preparation of erythrocyte and MOLT4 cell lysates (Study III)………………….………..…33 HPLC instruments and chromatographic conditions (Study III

Research paper thumbnail of Involvement of the concentrative nucleoside transporter 3 and equilibrative nucleoside transporter 2 in the resistance of T-lymphoblastic cell lines to thiopurines

Biochemical and Biophysical Research Communications, 2006

Mechanisms of resistance to thiopurines, 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) were in... more 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.

Research paper thumbnail of Pharmacological and molecular investigations on the mechanisms underlying resistance of human leukaemia cells to the antimetabolites methotrexate, 6-mercaptopurine and 6-thioguanine

The goal of this thesis has been to elucidate the mechanisms underlying resistance to methotrexat... more The goal of this thesis has been to elucidate the mechanisms underlying resistance to methotrexate (MTX), 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG), the antimetabolites widely used in treatment of childhood leukemia, as well as to determine the influence of 7hydroxymethotrexate (7-OHMTX), the major metabolite of MTX, on the therapeutic action of MTX. Resistant sublines of leukemic cell lines were developed by long-term exposure to stepwise increasing concentrations of these different agents. The mechanism underlying resistance to MTX in cells exposed to this drug was a pronounced reduction (> 10-fold) in reduced folate carrier (RFC)–mediated uptake of MTX. In CCRF-CEM cells, this reduction was associated with transcriptional silencing of the RFC gene, due to attenuated or even abolished binding of various transcription factors to their cis-acting elements, including the CRE, E-box, AP1, Mzf-1 and GCbox. In contrast, resistance to 7-OHMTX was due solely to a dramatic decrem...

Research paper thumbnail of Therapeutic drug monitoring in oncology: International Association of Therapeutic Drug Monitoring and Clinical Toxicology consensus guidelines for imatinib therapy

European Journal of Cancer

Research paper thumbnail of Population Pharmacokinetic, Pharmacogenetic, and Pharmacodynamic Analysis of Cyclophosphamide in Ethiopian Breast Cancer Patients

Frontiers in Pharmacology

Cyclophosphamide (CPA) containing chemotherapy regimen is the standard of care for breast cancer ... more Cyclophosphamide (CPA) containing chemotherapy regimen is the standard of care for breast cancer treatment in sub-Saharan Africa. Wide inter-individual variations in pharmacokinetics (PK) of cyclophosphamide (CPA) influence the efficacy and toxicity of CPA containing chemotherapy. Data on the pharmacokinetics (PK) profile of CPA and its covariates among black African patients is lacking. We investigated population pharmacokinetic/pharmacogenetic/pharmacodynamic (PK-PG-PD) of CPA in Ethiopian breast cancer patients. During the first cycle of CPA-based chemotherapy, the population PK parameters for CPA were determined in 267 breast cancer patients. Absolute neutrophil count was recorded at baseline and day 20 post-CPA administration. A population PK and covariate model analysis was performed using non-linear mixed effects modeling. Semi-mechanistic and empiric drug response models were explored to describe the relationship between the area under concentration-time curve (AUC), and neutrophil toxicity. One compartment model better described CPA PK with population clearance and apparent volume of distribution (V D) of 5.41 L/h and 46.5 L, respectively. Inter-patient variability in CPA clearance was 54.5%. Patients carrying CYP3A5*3 or *6 alleles had lower elimination rate constant and longer half-life compared to wild type carriers. CYP2C9 *2 or *3 carriers were associated with increased clearance of CPA. Patients who received 500 mg/m 2 based CPA regimen were associated with a 32.3% lower than average clearance and 37.1% lower than average V D compared to patients who received 600 mg/m 2. A 0.1 m 2 unit increase in body surface area (BSA) was associated with a 5.6% increment in V D. The mean V D (33.5 L) in underweight group (BMI < 18.5 kg/m 2) was significantly lower compared to those of overweight (48.1 L) or

Research paper thumbnail of Vitamin D Status and Association of VDR Genetic Polymorphism to Risk of Breast Cancer in Ethiopia

Nutrients

Emerging evidence associates vitamin D deficiency and vitamin D receptor (VDR) genetic variations... more Emerging evidence associates vitamin D deficiency and vitamin D receptor (VDR) genetic variations with risk for breast cancer. This study investigated the prevalence of vitamin D deficiency and its association with tumor characteristics and the implications of VDR genetic variations for risk of breast cancer in Ethiopia. This unmatched case–control study involved 392 female breast cancer patients and 193 controls. The plasma 25-hydroxyvitamin D (25(OH)D3) level was quantified in chemotherapy-naïve (N = 112) and tamoxifen-treated patients (N = 89). Genotyping for the VDR common variant alleles rs7975232 (ApaI), rs2228570 (FokI), and rs731236 (TaqI) was done. Eighty-six percent of the patients were vitamin D deficient (<50 nmol/L). Chemotherapy-naïve breast cancer patients had a higher prevalence of vitamin D deficiency (91.9% vs. 78.3%) compared to the tamoxifen-treated group (p < 0.001). The prevalence of severe vitamin D deficiency (<25 nmol/L) was significantly higher in ...

Research paper thumbnail of RNA Interference Targeting Thiopurine Methyl Transferase Gene Alters the Cytotoxic Effect of the 6-Mercaptopurine In Human Leukaemia Cells

Blood

2891 Thiopurines; mercaptopurine (6-MP) and 6-thioguanine (6-TG) are important drugs in treatment... more 2891 Thiopurines; mercaptopurine (6-MP) and 6-thioguanine (6-TG) are important drugs in treatment of paediatric cancer patients. The activity of these drugs depends on the activity of several common enzymes in the metabolism pathways such as thiopurine methyl transferase (TPMT) and guanine monphosphate synthetase (GMPS). In the present study the efficacy of thiopurines was investigated upon inhibition of TPMT and GMPS gene expression by RNA interference (siRNA). Treatment of the MOLT4 human T-cell leukemia cells with TPMT and GMPS siRNA resulted in decreased mRNA expression as determined by Real-Time PCR by 60% and 70% respectively. When reducing TPMT mRNA, the MOLT-4 cells were 70% less sensitive to 6-MP while the sensitivity to 6-TG was unchanged. When down-regulating GMPS using siRNA the sensitivity was unchanged upon treatment with both drugs. A microarray experiment was conducted using wild type on MOLT-4, 6-MP and 6-TG resistant MOLT-4 variants. The aim was to identify affecte...

Research paper thumbnail of CYP2D6 Genotype Predicts Plasma Concentrations of Tamoxifen Metabolites in Ethiopian Breast Cancer Patients

Cancers

Tamoxifen displays wide inter-individual variability (IIV) in its pharmacokinetics and treatment ... more Tamoxifen displays wide inter-individual variability (IIV) in its pharmacokinetics and treatment outcome. Data on tamoxifen pharmacokinetics and pharmacogenetics from black African breast cancer patient populations is lacking. We investigated the pharmacokinetic and pharmacogenetic profile of tamoxifen and its major active metabolite, endoxifen, in Ethiopian breast cancer patients. A total of 81 female breast cancer patients on adjuvant tamoxifen therapy were enrolled. Tamoxifen (Tam) and its major metabolites, N-desmethyltamoxifen (NDM), 4-hydroxy-tamoxifen (4-HT), and (Z)-endoxifen (E) were quantified using LC-MS/MS. Genotyping for CYP2D6, CYP2C9, CYP2C19, CYP3A5, POR, and ABCB1 and UGT2B15 and copy number variation for CYP2D6 were done. The proportion of patients with low endoxifen level (<5.9 ng/mL) was 35.8% (median concentration 7.94 ng/mL). The allele frequency of CYP2D6 gene deletion (*5) and duplication (*1×N or *2×N) was 4.3% and 14.8%, respectively. Twenty-six percent ...

Research paper thumbnail of CYP2J2∗7 Genotype Predicts Risk of Chemotherapy-Induced Hematologic Toxicity and Reduced Relative Dose Intensity in Ethiopian Breast Cancer Patients

Frontiers in Pharmacology

Chemotherapy-induced hematologic toxicity is the primary reasons of dose reductions and/or delays... more Chemotherapy-induced hematologic toxicity is the primary reasons of dose reductions and/or delays, low relative dose intensity (RDI), and predicts anticancer response. We investigated the incidence and predictors of chemotherapy-induced hematologic toxicities and reduced RDI in Ethiopian breast cancer patients, and implication of pharmacogenetics variations. Breast cancer patients (n = 249) were enrolled prospectively to receive cyclophosphamide based chemotherapy. Hematological toxicity (neutropenia, anemia, and thrombocytopenia) were monitored throughout chemotherapy cycle. The primary and secondary outcomes were incidence of grade 3 or 4 toxicity and reduced RDI, respectively. CYP2B6 * 6, CYP3A5 * 3, CYP2C9 (* 2, * 3), CYP2C19 (* 2, * 3), CYP2J2 * 7, POR * 28, and ABCB1 (rs3842) genotyping were done. Cox proportional hazard and logistic regression were used to estimate risk predictors of toxicity and reduced RDI, respectively. Majority (73.5%) of the patients were < 45 years of age. The incidence of grade 3 or 4 hematological toxicity was 51.0% (95% CI = 44.54-57.46%). Multivariate Cox proportional hazard regression indicated CYP2J2 * 7 genotype [Hazard ratio (HR) = 1.82; 95% CI = 1.14-2.90], pretreatment grade 1 leukopenia (HR = 2.75; 95% CI = 1.47-5.15) or grade 1 or 2 neutropenia (HR = 2.75; 95% CI = 1.73-4.35) as significant predictors of hematologic toxicities. The odds of having hematologic toxicities was lower in CYP2C9 * 2 or * 3 carriers (p = 0.024). The prevalence of reduced RDI was 56.6% (95% CI = 50.3-62.9%). Higher risk of reduced RDI was associated with CYP2J2 * 7 allele [Adjusted odds ratio (AOR) = 2.79;

Research paper thumbnail of Pharmacological and molecular investigations on the mechanisms underlying resistance of human leukaemia cells to the antimetabolites methotrexate, 6-mercaptopurine and 6-thioguanine

Page 1. From the Department of Oncology-Pathology, Cancer Centrum Karolinska, Karolinska Institut... more Page 1. From the Department of Oncology-Pathology, Cancer Centrum Karolinska, Karolinska Institutet at Karolinska University Hospital, Solna Stockholm, Sweden Pharmacological and molecular investigations on the mechanisms underlying ...

Research paper thumbnail of Mechanisms of antifolate resistance and methotrexate efficacy in leukemia cells

Leukemia Lymphoma, Mar 1, 2008

Antifolates are the first class of antimetabolites introduced to clinic about 6 decades ago. Now,... more Antifolates are the first class of antimetabolites introduced to clinic about 6 decades ago. Now, after several years of administration of antifolates against malignancies and particularly leukemia, we are still trying to achieve a full understanding of the mechanisms of action and resistance to these agents. The present article covers different factors able to influence efficacy of antifolates on leukemic cells, the known mechanisms of resistance to methotrexate (MTX) and strategies to overcome these mechanisms. The dominant factors that are contributed to tolerance to cytocidal effects of MTX including pharmacokinetic factors, impaired transmembrane uptake as the most frequent rote of provoking resistance to MTX, augmented drug efflux, impaired intracellular polyglutamation as a determining process of drug efficacy, alterations in expression or activity of target enzymes and increased intracellular folate pools; and finally role of 7-hydroxymethotrexate on response or resistance to MTX will be discussed in more detail. Finally, strategies to overcome resistance to antifolates are discussed.

Research paper thumbnail of Pronounced inter- but not intraindividual variation in tamoxifen and metabolite levels in plasma during adjuvant treatment of women with early breast cancer

Therapeutic Drug Monitoring, 2015

Tamoxifen is still an important antihormonal treatment option for patients with breast cancer and... more Tamoxifen is still an important antihormonal treatment option for patients with breast cancer and estrogen receptor (ER)-positive tumors. Over 20% of patients relapse in spite of treatment. The drug is usually dosed 20 mg/day irrespective of interindividual variation in drug clearance.In order to study inter- and intraindividual variation in plasma levels we measured tamoxifen and metabolite levels in plasma at two occasions, with at least 4 weeks in between, of 39 women (19 pre- and 20 postmenopausal women) on adjuvant treatment (20 mg/day) of early breast cancer. We used an ultra-performance liquid chromatography with mass spectrometry detection method for identification and quantification of tamoxifen, N-desmethyltamoxifen, 4-OH-tamoxifen, and endoxifen.Follicle-stimulating hormone (FSH), luteinizing hormone (LH), and estradiol levels were also measured. The plasma concentrations of tamoxifen and its metabolites showed a pronounced interindividual variation while intraindividual concentrations were rather stable. In spite of the same dosage, interindividual tamoxifen concentrations varied from 51 to 307 ng/ml (124 ± 57, mean ± SD) and endoxifen values showed a range from 3.2 to 19 ng/ml (10.4 ± 5.2, mean ± SD), i.e. 6-fold variation for both. Large interindividual variation of tamoxifen and endoxifen with stable intraindividual levels, and too low levels of endoxifen in a considerable proportion of patients strongly support that therapeutic drug monitoring (TDM) and individualised dosing could lead to optimal exposure and hopefully better outcome. A randomised outcome study between conventional dosing and TDM-guided dosing is needed to show whether this approach works.

Research paper thumbnail of Involvement of the concentrative nucleoside transporter 3 and equilibrative nucleoside transporter 2 in the resistance of T-lymphoblastic cell lines to thiopurines

Biochemical and Biophysical Research Communications, Apr 1, 2006

TAS-102, which is effective for refractory metastatic colorectal cancer, is a combination drug of... more TAS-102, which is effective for refractory metastatic colorectal cancer, is a combination drug of anticancer trifluridine (FTD; which is derived from pyrimidine nucleoside) and FTD-metabolizing enzyme inhibitor tipiracil hydrochloride (TPI) at a molecular ratio of 1:0.5. To evaluate the intestinal absorption mechanism of FTD, the uptake and transcellular transport of FTD by human small intestinal epithelial cell (HIEC) monolayer as a model of human intestinal epithelial cells was investigated. The uptake and membrane permeability of FTD by HIEC monolayers were saturable, Na +-dependent, and inhibited by nucleosides. These transport characteristics are mostly comparable with those of concentrative nucleoside transporters (CNTs). Moreover, the uptake of FTD by CNT1-expressing Xenopus oocytes was the highest among human CNT transporters. The obtained K m and V max values of FTD by CNT1 were 69.0 M and 516 pmol/oocyte/30 min, respectively. The transcellular transport of FTD by Caco-2 cells, where CNT1 is heterologously expressed, from apical to basolateral side was greater than that by Mock cells. In conclusion, these results demonstrated that FTD exhibits high oral absorption by the contribution of human CNT1.

Research paper thumbnail of Therapeutic drug monitoring in oncology: International Association of Therapeutic Drug Monitoring and Clinical Toxicology consensus guidelines for imatinib therapy

European Journal of Cancer, Nov 1, 2021

Although therapeutic drug monitoring (TDM) is an important tool in guiding drug dosing for other ... more Although therapeutic drug monitoring (TDM) is an important tool in guiding drug dosing for other areas of medicine including infectious diseases, cardiology, psychiatry and transplant medicine, it has not gained wide acceptance in oncology. For imatinib and other tyrosine kinase inhibitors, a flat dosing approach is utilised for management of oral chemotherapy. There are many published studies examining the correlation of blood concentrations with clinical effects of imatinib. The International Association of Therapeutic Drug Monitoring and Clinical Toxicology (IATDMCT) determined that there was a need to examine the published literature regarding utility of TDM in imatinib therapy and to develop consensus guidelines for TDM based on the available data. This article summarises the scientific evidence regarding TDM of imatinib, as well as the consensus guidelines developed by the IATDMCT.

Research paper thumbnail of Assessment of renal function of cancer patients for dosing carboplatin

Research paper thumbnail of Introducing a simple and cost-effective RT-PCR protocol for detection of DPYD*2A polymorphism: the first study in Kurdish population

Cancer Chemotherapy and Pharmacology

Research paper thumbnail of Comparison of Uptake Mechanisms for Anthracyclines in Human Leukemic Cells

Current Drug Delivery, 2013

The mechanisms behind cellular anthracycline uptake are not completely understood. Knowledge abou... more The mechanisms behind cellular anthracycline uptake are not completely understood. Knowledge about uptake mechanisms could be used to increase the selectivity of the drugs. We compared the uptake patterns of, daunorubicin (DNR), doxorubicin (DOX), epirubicin (EPI), idarubicin (IDA), and pirarubicin (PIRA) by cultured leukemic cells and investigated possible involvement of specific carriers. HL-60 cells were incubated with anthracyclines for 1 hour in the absence or presence of transport inhibitors, suramin, or nucleosides and cellular drug uptake was determined. Cell survival was also determined. MCF-7 breast cancer cells were used as a negative control for concentrative nucleoside transporters (CNTs). Anthracycline concentration was determined with HPLC and fluorometric detection and apoptosis was determined with propidium iodide and flow cytometry. DNR, IDA, and PIRA had higher uptake than DOX and EPI with a prominent increase in uptake at concentrations &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt; 1 µM. Uptake of all anthracyclines was greatly reduced at 0°C. Suramin, a purinergic-2-receptor inhibitor, strongly inhibited the uptake of all anthracyclines except PIRA and increased cell survival. Dipyridamole, an equilibrative NT (ENT) inhibitor, significantly inhibited the uptake of DNR only. The addition of nucleosides significantly inhibited the uptake of DNR, IDA, and PIRA but not in MCF-7 cells lacking functional CNTs. Our results suggest different uptake mechanisms for the anthracyclines studied. We found evidence for carrier mediated uptake mechanisms, supporting involvement of NTs in transmembrane transport of DNR, IDA, and PIRA. The results also showed a strong inhibition of suramin on anthracycline uptake by so far unknown mechanisms.

Research paper thumbnail of Gene expression profiling of leukemia T-cells resistant to methotrexate and 7-hydroxymethotrexate reveals alterations that preserve intracellular levels of folate and nucleotide biosynthesis

Biochemical Pharmacology, 2009

Research paper thumbnail of Molecular mechanisms underlying the enhanced sensitivity of thiopurine-resistant T-lymphoblastic cell lines to methyl mercaptopurineriboside

Biochemical Pharmacology, 2006

Research paper thumbnail of Differential role of thiopurine methyltransferase in the cytotoxic effects of 6-mercaptopurine and 6-thioguanine on human leukemia cells

Biochemical and Biophysical Research Communications, 2013

The thiopurine antimetabolites, 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) are inactive pro... more The thiopurine antimetabolites, 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) are inactive prodrugs that require intracellular metabolism for activation to cytotoxic metabolites. Thiopurine methyltransferase (TPMT) is one of the most important enzymes in this process metabolizing both 6-MP and 6-TG to different methylated metabolites including methylthioinosine monophosphate (meTIMP) and methylthioguanosine monophosphate (meTGMP), respectively, with different suggested pharmacological and cytotoxic properties. While meTIMP is a potent inhibitor of de novo purine synthesis (DNPS) and significantly contributes to the cytotoxic effects of 6-MP, meTGMP, does not add much to the effects of 6-TG, and the cytotoxicity of 6-TG seems to be more dependent on incorporation of thioguanine nucleotides (TGNs) into DNA rather than inhibition of DNPS. In order to investigate the role of TPMT in metabolism and thus, cytotoxic effects of 6-MP and 6-TG, we knocked down the expression of the gene encoding the TPMT enzyme using specifically designed small interference RNA (siRNA) in human MOLT4 leukemia cells. The knock-down was confirmed at RNA, protein, and enzyme function levels. Apoptosis was determined using annexin V and propidium iodide staining and FACS analysis. The results showed a 34% increase in sensitivity of MOLT4 cells to 1 lM 6-TG after treatment with TPMT-targeting siRNA, as compared to cells transfected with non-targeting siRNA, while the sensitivity of the cells toward 6-MP was not affected significantly by down-regulation of the TPMT gene. This differential contribution of the enzyme TPMT to the cytotoxicity of the two thiopurines is probably due to its role in formation of the meTIMP, the cytotoxic methylated metabolite of 6-MP, while in case of 6-TG methylation by TPMT substantially deactivates the drug.

Research paper thumbnail of The pattern of gene expression and gene dose profiles of 6-Mercaptopurine- and 6-Thioguanine-resistant human leukemia cells

Biochemical and Biophysical Research Communications, 2011

Determination of apoptosis by flow cytometry (Study I)……………………………....….30 RNA extraction and cDNA... more Determination of apoptosis by flow cytometry (Study I)……………………………....….30 RNA extraction and cDNA synthesis (Study I & IV)…………………………………………..31 Messenger RNA expression by RT-PCR (Study I & IV)…………………………………...…31 Gene expression by Microarray (Study II)……………………………………………………....32 DNA isolation and array comparative genomic hybridization (Study II)……………32 Preparation of erythrocyte and MOLT4 cell lysates (Study III)………………….………..…33 HPLC instruments and chromatographic conditions (Study III

Research paper thumbnail of Involvement of the concentrative nucleoside transporter 3 and equilibrative nucleoside transporter 2 in the resistance of T-lymphoblastic cell lines to thiopurines

Biochemical and Biophysical Research Communications, 2006

Mechanisms of resistance to thiopurines, 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) were in... more 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.

Research paper thumbnail of Pharmacological and molecular investigations on the mechanisms underlying resistance of human leukaemia cells to the antimetabolites methotrexate, 6-mercaptopurine and 6-thioguanine

The goal of this thesis has been to elucidate the mechanisms underlying resistance to methotrexat... more The goal of this thesis has been to elucidate the mechanisms underlying resistance to methotrexate (MTX), 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG), the antimetabolites widely used in treatment of childhood leukemia, as well as to determine the influence of 7hydroxymethotrexate (7-OHMTX), the major metabolite of MTX, on the therapeutic action of MTX. Resistant sublines of leukemic cell lines were developed by long-term exposure to stepwise increasing concentrations of these different agents. The mechanism underlying resistance to MTX in cells exposed to this drug was a pronounced reduction (> 10-fold) in reduced folate carrier (RFC)–mediated uptake of MTX. In CCRF-CEM cells, this reduction was associated with transcriptional silencing of the RFC gene, due to attenuated or even abolished binding of various transcription factors to their cis-acting elements, including the CRE, E-box, AP1, Mzf-1 and GCbox. In contrast, resistance to 7-OHMTX was due solely to a dramatic decrem...

Research paper thumbnail of Therapeutic drug monitoring in oncology: International Association of Therapeutic Drug Monitoring and Clinical Toxicology consensus guidelines for imatinib therapy

European Journal of Cancer

Research paper thumbnail of Population Pharmacokinetic, Pharmacogenetic, and Pharmacodynamic Analysis of Cyclophosphamide in Ethiopian Breast Cancer Patients

Frontiers in Pharmacology

Cyclophosphamide (CPA) containing chemotherapy regimen is the standard of care for breast cancer ... more Cyclophosphamide (CPA) containing chemotherapy regimen is the standard of care for breast cancer treatment in sub-Saharan Africa. Wide inter-individual variations in pharmacokinetics (PK) of cyclophosphamide (CPA) influence the efficacy and toxicity of CPA containing chemotherapy. Data on the pharmacokinetics (PK) profile of CPA and its covariates among black African patients is lacking. We investigated population pharmacokinetic/pharmacogenetic/pharmacodynamic (PK-PG-PD) of CPA in Ethiopian breast cancer patients. During the first cycle of CPA-based chemotherapy, the population PK parameters for CPA were determined in 267 breast cancer patients. Absolute neutrophil count was recorded at baseline and day 20 post-CPA administration. A population PK and covariate model analysis was performed using non-linear mixed effects modeling. Semi-mechanistic and empiric drug response models were explored to describe the relationship between the area under concentration-time curve (AUC), and neutrophil toxicity. One compartment model better described CPA PK with population clearance and apparent volume of distribution (V D) of 5.41 L/h and 46.5 L, respectively. Inter-patient variability in CPA clearance was 54.5%. Patients carrying CYP3A5*3 or *6 alleles had lower elimination rate constant and longer half-life compared to wild type carriers. CYP2C9 *2 or *3 carriers were associated with increased clearance of CPA. Patients who received 500 mg/m 2 based CPA regimen were associated with a 32.3% lower than average clearance and 37.1% lower than average V D compared to patients who received 600 mg/m 2. A 0.1 m 2 unit increase in body surface area (BSA) was associated with a 5.6% increment in V D. The mean V D (33.5 L) in underweight group (BMI < 18.5 kg/m 2) was significantly lower compared to those of overweight (48.1 L) or

Research paper thumbnail of Vitamin D Status and Association of VDR Genetic Polymorphism to Risk of Breast Cancer in Ethiopia

Nutrients

Emerging evidence associates vitamin D deficiency and vitamin D receptor (VDR) genetic variations... more Emerging evidence associates vitamin D deficiency and vitamin D receptor (VDR) genetic variations with risk for breast cancer. This study investigated the prevalence of vitamin D deficiency and its association with tumor characteristics and the implications of VDR genetic variations for risk of breast cancer in Ethiopia. This unmatched case–control study involved 392 female breast cancer patients and 193 controls. The plasma 25-hydroxyvitamin D (25(OH)D3) level was quantified in chemotherapy-naïve (N = 112) and tamoxifen-treated patients (N = 89). Genotyping for the VDR common variant alleles rs7975232 (ApaI), rs2228570 (FokI), and rs731236 (TaqI) was done. Eighty-six percent of the patients were vitamin D deficient (<50 nmol/L). Chemotherapy-naïve breast cancer patients had a higher prevalence of vitamin D deficiency (91.9% vs. 78.3%) compared to the tamoxifen-treated group (p < 0.001). The prevalence of severe vitamin D deficiency (<25 nmol/L) was significantly higher in ...

Research paper thumbnail of RNA Interference Targeting Thiopurine Methyl Transferase Gene Alters the Cytotoxic Effect of the 6-Mercaptopurine In Human Leukaemia Cells

Blood

2891 Thiopurines; mercaptopurine (6-MP) and 6-thioguanine (6-TG) are important drugs in treatment... more 2891 Thiopurines; mercaptopurine (6-MP) and 6-thioguanine (6-TG) are important drugs in treatment of paediatric cancer patients. The activity of these drugs depends on the activity of several common enzymes in the metabolism pathways such as thiopurine methyl transferase (TPMT) and guanine monphosphate synthetase (GMPS). In the present study the efficacy of thiopurines was investigated upon inhibition of TPMT and GMPS gene expression by RNA interference (siRNA). Treatment of the MOLT4 human T-cell leukemia cells with TPMT and GMPS siRNA resulted in decreased mRNA expression as determined by Real-Time PCR by 60% and 70% respectively. When reducing TPMT mRNA, the MOLT-4 cells were 70% less sensitive to 6-MP while the sensitivity to 6-TG was unchanged. When down-regulating GMPS using siRNA the sensitivity was unchanged upon treatment with both drugs. A microarray experiment was conducted using wild type on MOLT-4, 6-MP and 6-TG resistant MOLT-4 variants. The aim was to identify affecte...

Research paper thumbnail of CYP2D6 Genotype Predicts Plasma Concentrations of Tamoxifen Metabolites in Ethiopian Breast Cancer Patients

Cancers

Tamoxifen displays wide inter-individual variability (IIV) in its pharmacokinetics and treatment ... more Tamoxifen displays wide inter-individual variability (IIV) in its pharmacokinetics and treatment outcome. Data on tamoxifen pharmacokinetics and pharmacogenetics from black African breast cancer patient populations is lacking. We investigated the pharmacokinetic and pharmacogenetic profile of tamoxifen and its major active metabolite, endoxifen, in Ethiopian breast cancer patients. A total of 81 female breast cancer patients on adjuvant tamoxifen therapy were enrolled. Tamoxifen (Tam) and its major metabolites, N-desmethyltamoxifen (NDM), 4-hydroxy-tamoxifen (4-HT), and (Z)-endoxifen (E) were quantified using LC-MS/MS. Genotyping for CYP2D6, CYP2C9, CYP2C19, CYP3A5, POR, and ABCB1 and UGT2B15 and copy number variation for CYP2D6 were done. The proportion of patients with low endoxifen level (<5.9 ng/mL) was 35.8% (median concentration 7.94 ng/mL). The allele frequency of CYP2D6 gene deletion (*5) and duplication (*1×N or *2×N) was 4.3% and 14.8%, respectively. Twenty-six percent ...

Research paper thumbnail of CYP2J2∗7 Genotype Predicts Risk of Chemotherapy-Induced Hematologic Toxicity and Reduced Relative Dose Intensity in Ethiopian Breast Cancer Patients

Frontiers in Pharmacology

Chemotherapy-induced hematologic toxicity is the primary reasons of dose reductions and/or delays... more Chemotherapy-induced hematologic toxicity is the primary reasons of dose reductions and/or delays, low relative dose intensity (RDI), and predicts anticancer response. We investigated the incidence and predictors of chemotherapy-induced hematologic toxicities and reduced RDI in Ethiopian breast cancer patients, and implication of pharmacogenetics variations. Breast cancer patients (n = 249) were enrolled prospectively to receive cyclophosphamide based chemotherapy. Hematological toxicity (neutropenia, anemia, and thrombocytopenia) were monitored throughout chemotherapy cycle. The primary and secondary outcomes were incidence of grade 3 or 4 toxicity and reduced RDI, respectively. CYP2B6 * 6, CYP3A5 * 3, CYP2C9 (* 2, * 3), CYP2C19 (* 2, * 3), CYP2J2 * 7, POR * 28, and ABCB1 (rs3842) genotyping were done. Cox proportional hazard and logistic regression were used to estimate risk predictors of toxicity and reduced RDI, respectively. Majority (73.5%) of the patients were < 45 years of age. The incidence of grade 3 or 4 hematological toxicity was 51.0% (95% CI = 44.54-57.46%). Multivariate Cox proportional hazard regression indicated CYP2J2 * 7 genotype [Hazard ratio (HR) = 1.82; 95% CI = 1.14-2.90], pretreatment grade 1 leukopenia (HR = 2.75; 95% CI = 1.47-5.15) or grade 1 or 2 neutropenia (HR = 2.75; 95% CI = 1.73-4.35) as significant predictors of hematologic toxicities. The odds of having hematologic toxicities was lower in CYP2C9 * 2 or * 3 carriers (p = 0.024). The prevalence of reduced RDI was 56.6% (95% CI = 50.3-62.9%). Higher risk of reduced RDI was associated with CYP2J2 * 7 allele [Adjusted odds ratio (AOR) = 2.79;

Research paper thumbnail of Pharmacological and molecular investigations on the mechanisms underlying resistance of human leukaemia cells to the antimetabolites methotrexate, 6-mercaptopurine and 6-thioguanine

Page 1. From the Department of Oncology-Pathology, Cancer Centrum Karolinska, Karolinska Institut... more Page 1. From the Department of Oncology-Pathology, Cancer Centrum Karolinska, Karolinska Institutet at Karolinska University Hospital, Solna Stockholm, Sweden Pharmacological and molecular investigations on the mechanisms underlying ...

Research paper thumbnail of Mechanisms of antifolate resistance and methotrexate efficacy in leukemia cells

Leukemia Lymphoma, Mar 1, 2008

Antifolates are the first class of antimetabolites introduced to clinic about 6 decades ago. Now,... more Antifolates are the first class of antimetabolites introduced to clinic about 6 decades ago. Now, after several years of administration of antifolates against malignancies and particularly leukemia, we are still trying to achieve a full understanding of the mechanisms of action and resistance to these agents. The present article covers different factors able to influence efficacy of antifolates on leukemic cells, the known mechanisms of resistance to methotrexate (MTX) and strategies to overcome these mechanisms. The dominant factors that are contributed to tolerance to cytocidal effects of MTX including pharmacokinetic factors, impaired transmembrane uptake as the most frequent rote of provoking resistance to MTX, augmented drug efflux, impaired intracellular polyglutamation as a determining process of drug efficacy, alterations in expression or activity of target enzymes and increased intracellular folate pools; and finally role of 7-hydroxymethotrexate on response or resistance to MTX will be discussed in more detail. Finally, strategies to overcome resistance to antifolates are discussed.

Research paper thumbnail of Pronounced inter- but not intraindividual variation in tamoxifen and metabolite levels in plasma during adjuvant treatment of women with early breast cancer

Therapeutic Drug Monitoring, 2015

Tamoxifen is still an important antihormonal treatment option for patients with breast cancer and... more Tamoxifen is still an important antihormonal treatment option for patients with breast cancer and estrogen receptor (ER)-positive tumors. Over 20% of patients relapse in spite of treatment. The drug is usually dosed 20 mg/day irrespective of interindividual variation in drug clearance.In order to study inter- and intraindividual variation in plasma levels we measured tamoxifen and metabolite levels in plasma at two occasions, with at least 4 weeks in between, of 39 women (19 pre- and 20 postmenopausal women) on adjuvant treatment (20 mg/day) of early breast cancer. We used an ultra-performance liquid chromatography with mass spectrometry detection method for identification and quantification of tamoxifen, N-desmethyltamoxifen, 4-OH-tamoxifen, and endoxifen.Follicle-stimulating hormone (FSH), luteinizing hormone (LH), and estradiol levels were also measured. The plasma concentrations of tamoxifen and its metabolites showed a pronounced interindividual variation while intraindividual concentrations were rather stable. In spite of the same dosage, interindividual tamoxifen concentrations varied from 51 to 307 ng/ml (124 ± 57, mean ± SD) and endoxifen values showed a range from 3.2 to 19 ng/ml (10.4 ± 5.2, mean ± SD), i.e. 6-fold variation for both. Large interindividual variation of tamoxifen and endoxifen with stable intraindividual levels, and too low levels of endoxifen in a considerable proportion of patients strongly support that therapeutic drug monitoring (TDM) and individualised dosing could lead to optimal exposure and hopefully better outcome. A randomised outcome study between conventional dosing and TDM-guided dosing is needed to show whether this approach works.