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Tamoxifen is still an important antihormonal treatment option for patients with breast cancer and estrogen receptor-positive tumors. More than 20% of patients relapse despite treatment. The drug is usually dosed 20 mg/d irrespective of interindividual variation in drug clearance. To study interindividual and intraindividual variation in plasma levels we measured tamoxifen and metabolite levels in plasma on 2 occasions, with at least 4 weeks in between, of 39 women (19 premenopausal and 20 postmenopausal women) on adjuvant treatment (20 mg/d) of early breast cancer.We used an ultra-performance liquid chromatography with a mass spectrometry detection method for identification and quantification of tamoxifen, N-desmethyltamoxifen, 4-OH-tamoxifen, and endoxifen. Follicle-stimulating hormone, luteinizing hormone, and estradiol levels were also measured.The plasma concentrations of tamoxifen and its metabolites showed a pronounced interindividual variation, whereas intraindividual concentrations were rather stable. Despite 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), that is, 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 and individualized dosing could lead to optimal exposure and hopefully better outcome. A randomized outcome study between conventional dosing and therapeutic drug monitoring-guided dosing is needed to show whether this approach works.

The thiopurine antimetabolites, 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) are inactive pro-drugs 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 1μM 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.

Thiopurine efficacy is partly reflected by the genetic polymorphism of the thiopurine methyltransferase (TPMT) enzyme, which is responsible for variation in the metabolism, toxicity and therapeutic efficacy of the thiopurines azathioprine (AZA), 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG). Determination of TPMT activity before administration of thiopurines is thus crucial for individualized dosing in order to prevent toxicity in TPMT deficient individuals. These individuals must be treated with markedly lower (eg, 5-10% of the standard) doses of the prescribed medications. This paper describes a comparison of three different methods for the quantification of TPMT activity in red blood cells (RBC) and cultured human cell lines. We succeeded to perform the measurement of TPMT activity in a minimum amount of 1×10(6) cultured cells with an HPLC-UV system modified and optimized in our laboratory. The TPMT activity was linearly correlated with the cell concentration of the cultured cell line in a range of 1-10×10(6) cells. A significant correlation of determination of TPMT activity in RBC between radiometric detection by HPLC, classic radiochemical detection and UV detection by HPLC, was observed, correlation coefficient (r) were 0.72 and 0.73, respectively. The within-day and day-to-day coefficients of variation of the HPLC-UV-based method were 8% and 16%, respectively. The evaluation of the methods was demonstrated by studying the TPMT activity in RBC isolated from 198 patients, as well as in MOLT4 leukemic cell line and its sub-cell lines with acquired resistance to 6-MP and 6-TG.

Aims: 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. Methods: 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. Results: 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. Conclusion: 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.

Exposure of MOLT4 human T-cell leukemia cells to 6-Mercaptopurine (6-MP) and 6-Thioguanine (6-TG) resulted in acquired resistance associated with attenuated expression of the genes encoding concentrative nucleoside transporter 3 (CNT3) and equilibrative nucleoside transporter 2 (ENT2). To identify other alterations at the RNA and DNA levels associated with 6-MP- and 6-TG resistance, we compared here the patterns of gene expression and DNA copy number profiles of resistant sublines to those of the parental wild-type cells. The mRNA levels for two nucleoside transporters were down-regulated in both of the thiopurine-resistant sublines. Moreover, both of these cell lines expressed genes encoding the enzymes of purine nucleotide composition and synthesis, including adenylate kinase 3-like 1 and guanosine monophosphate synthetase at significantly lower levels than wild-type cells. In addition, expression of the mRNA for a specialized DNA polymerase, human terminal transferase encoded by the terminal deoxynucleotidyl transferase (DNTT) gene, was 122- and 93-fold higher in 6-TG- and 6-MP-resistant cells, respectively. The varying responses to 6-MP- and 6-TG observed here may help identify novel cellular targets and modalities of resistance to thiopurines, as well as indicating new potential approaches to individualization therapy with these drugs.

Aims: The mechanisms behind cellular anthracycline uptake are not completely understood. It is of great interest to gain more insight into cellular uptake mechanisms since this could possibly be used to increase the selectivity of the drugs. We therefore compared the uptake of 5 different anthracyclines, daunorubicin (DNR), doxorubicin (DOX), epirubicin (EPI), idarubicin (IDA), and pirarubicin (PIRA) by leukemic cells and also investigated the possible involvement of specific carriers. Methods: HL-60 cells were incubated with increasing concentrations of anthracyclines for 1 hour and cellular drug uptake was determined. The cells were also preincubated with various protein inhibitors or nucleosides and then with the anthracyclines after which drug uptake was determined with HPLC. Apoptosis was determined with propidium iodine staining and flow cytometry. Results: Of all anthracyclines studied DNR, IDA and PIRA had the highest cellular uptake with a sharp increase in uptake at extracellular concentrations > 1 µM. Uptake of DOX, DNR and IDA was greatly reduced at 0°C. Suramin, a purinergic-2-receptor inhibitor, strongly inhibited the uptake of all anthracyclines except PIRA and dipyridamole, a nucleoside transport inhibitor, only inhibited the uptake of DNR. The addition of nucleosides reduced the uptake of DNR, IDA and PIRA. Conclusion: Our results suggest different uptake mechanisms for various anthracyclines. We found evidence for carrier mediated uptake mechanisms, the data support the involvement of the nucleoside transporter family. Furthermore, our results also indicate an involvement of purinergic-2-receptor signalling in anthracycline uptake. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1705. doi:10.1158/1538-7445.AM2011-1705