Intracellular cytarabine triphosphate in circulating blasts post-treatment predicts remission status in patients with acute myeloid leukemia.

Cytarabine remains the backbone of therapy in acute myeloid leukemia (AML). The ability to assess intracellular cytarabine triphosphate (ara-CTP) levels in patients receiving cytarabine represents a major goal in the prediction of treatment response. This study, conducted within a clinical setting, aimed to assess ara-CTP levels in circulating peripheral blasts from non-M3 AML patients receiving cytarabine at one of three dosing levels, using a novel biosensor assay. Results from the initial 72 hours post-commencement were correlated with day 28 remission status, with feasibility parameters concurrently assessed. Intracellular ara-CTP was detectable in ex vivo blasts post-treatment for standard-dose (SD) and high-dose (HD) patients (p < 0.05), and quantification revealed a 27-fold increase in intracellular steady-state concentration between the two dosing levels. For low-dose cytarabine, high rates of patient discharge and low intracellular concentrations limited analysis; however, assessment of intracellular ara-CTP concentration was achievable in a dwindling population of blasts for SD and HD treatment cohorts, with 4 hours post-treatment commencement potentially being most predictive of clinical response (r = -0.912, p = 0.0113). Concurrent assessment of peripheral leukemia-associated immunophenotype (LAIP)-positive cells revealed a decline in burden (0-72 hours), which correlated with remission status (p < 0.05). Unexpectedly high rates of night sampling led to challenges associated with sampling rates, but did not have an impact on patient compliance. Additional training of night staff improved feasibility substantially. Multiple peripheral sampling during the initial 72 hours of treatment is feasible in newly diagnosed patients, and ara-CTP is detectable over the initial 24 hours, facilitating prediction of chemosensitivity of leukemic blasts to cytarabine.

Simultaneous determination of 1-ß-d-Arabinofuranosylcytosine and two metabolites, 1-ß-d-Arabinofuranosyluracil and 1-ß-d-Arabinofuranosylcytosine triphosphate in leukemic cell by HPLC-MS/MS and the application to cell pharmacokinetics.

A specific and reliable HPLC-MS/MS method was developed and validated for the simultaneous determination of 1-ß-d-Arabinofuranosylcytosine (ara-C), 1-ß-d-Arabinofuranosyluracil (ara-U) and 1-ß-d-Arabinofuranosylcytosine triphosphate (ara-CTP) in the leukemic cells for the first time. The analytes were separated on a C18 column (100mm×2.1mm, 1.8µm) and a triple-quadrupole mass spectrometry equipped with an electrospray ionization (ESI) source was used for detection. The ion-pairing reagent, NFPA, was added to the mobile phase to retain the analytes in the column. The cell homogenates sample was prepared by the simple protein precipitation. The calibration curves were linear over a concentration range of 3.45-3450.0ng/mL for ara-C, 1.12-1120.0ng/mL for ara-U and 4.13-4130.0ng/mL for ara-CTP. The intra-day and inter-day precision was less than 15% and the relative error (RE) were all within ±15%. The validated method was successfully applied to assess the disposition characteristics of ara-C and support cell pharmacokinetics after the patients with leukemia were intravenously infused with SDAC and HiDAC. The result of the present study would provide the valuable information for the ara-C therapy.

Pharmacogenetics of deoxycytidine kinase: identification and characterization of novel genetic variants.

Deoxycytidine kinase (DCK) is a rate-limiting enzyme in the activation of nucleoside analogs such as cytarabine (ara-C), gemcitabine, clofarabine, and others. The present study was undertaken to identify and to determine the functional consequences of genetic variants in DCK. We sequenced 1.5 kilobases of the DCK proximal promoter and all seven coding exons in International HapMap Project panels (n = 90 each) with European (Centre d' Etude du Polymorphisme Humain; CEPH) or African (Yoruba people in Ibadan, Nigeria; YRI) ancestry. Sixty-four genetic polymorphisms, including three nonsynonymous coding changes (I24V, A119G, and P122S) were identified. Compared with DCK-wild-type (WT) protein, the activity of the recombinant DCK24Val, DCK119Gly, and DCK122Ser proteins was 85 +/- 5, 66 +/- 3, and 43 +/- 4%, respectively. DCK119Gly and DCK122Ser mutants had lower Km (p < 0.01) and Vmax (p < 0.001) compared with DCK-WT protein. Lymphoblast cell lines from subjects heterozygous for the coding changes had significantly lower DCK activity compared with homozygous WT subjects. Ethnic differences were observed, with African ancestry subjects demonstrating significantly higher DCK mRNA expression compared with subjects with European ancestry. In both CEPH and YRI subjects, the C allele of a 3'-untranslated region single-nucleotide polymorphism (SNP) (35708 C>T) was significantly associated with lower DCK mRNA expression. This SNP was strongly linked with other intronic SNPs, forming a major haplotype block in both ethnic groups. In an exploratory analysis, the 35708C allele was also associated with lower blast ara-C-5'-triphosphate (ara-CTP) levels in acute myeloid leukemia patients receiving ara-C as continuous infusion. These results suggest that genetic variation in DCK influences its activity and expression and may predict the variability observed in intracellular levels of the ara-C active metabolite ara-CTP.

Rapid quantitative determination of L-FMAU-TP from human peripheral-blood mononuclear cells of hepatitis B virus-infected patients treated with L-FMAU by ion-pairing, reverse-phase, liquid chromatography/electrospray tandem mass spectrometry.

The purpose of this study was to develop an analytical method for the determination of 2'-fluoro-5-methyl-beta-l-arabinofuranosyl uracil triphosphate (L-FMAU-TP) in human peripheral blood mononuclear cells (PBMCs), and its application in the determination of cellular levels of L-FMAU-TP in PBMCs isolated from patients treated with 2'-fluoro-5-methyl-beta-l-arabinofuranosyl uracil (L-FMAU). An ion-pairing liquid chromatography (IPC) method, coupled with negative ion electrospray ionization tandem mass spectrometry (ESI-MS/MS), was developed for the accurate and repeatable detection of L-FMAU-TP, with a limit of detection of 1.6 pmol/10 cells. The calibration curve for L-FMAU-TP was linear over the concentration range 1.6 to 80 pmol/10(6) cells. The intra- and inter-day precision was lower than 11.2%, and the accuracy was between 97.1 and 106.9%. When applied to the determination of L-FMAU-TP in PBMCs isolated from HBV-infected patients undergoing L-FMAU treatment, the levels reached a steady state concentration 4 weeks after daily single oral administration of 20 mg L-FMAU, and these levels were maintained for up to 12 weeks, but then decreased 12 weeks after drug cessation. The terminal half-life of L-FMAU-TP in PBMCs after drug cessation was estimated to be 15.6 days.

Close correlation of 1-beta-D-arabinofuranosylcytosine 5'-triphosphate, an intracellular active metabolite, to the therapeutic efficacy of N(4)-behenoyl-1-beta-D-arabinofuranosylcytosine therapy for acute myelogenous leukemia.

N(4)-Behenoyl-1-beta-D-arabinofuranosylcytosine (BHAC), a prodrug of 1-beta-D-arabinofuranosylcytosine, is used effectively for the treatment of leukemia in Japan. BHAC therapy may be more effective if it is delivered in conjunction with monitoring of 1-beta-D-arabinofuranosylcytosine 5'-triphosphate (ara-CTP), the intracellular active metabolite of ara-C derived from BHAC. However, previous monitoring methods for ara-CTP were insufficiently sensitive. Here, using our new sensitive method, we evaluated the ara-CTP pharmacokinetics in relation to the therapeutic response in 11 acute myelogenous leukemia patients who received a 2-h infusion of BHAC (70 mg / m(2)) in combination remission induction therapy. ara-CTP could be monitored at levels under 1 mM. BHAC maintained effective levels of plasma ara-C and intracellular ara-CTP for a longer time, even compared with historical values of high-dose ara-C. The area under the concentration-time curve of ara-CTP was significantly greater in the patients with complete remission than in the patients without response. This greater amount of ara-CTP was attributed to the higher ara-CTP concentrations achieved in the responding patients. There was no apparent difference of plasma ara-C pharmacokinetics between the two groups. Thus, for the first time, the ara-CTP pharmacokinetics was evaluated in relation to the therapeutic effect of BHAC, and the importance of ara-CTP was proven. Administration of optimal BHAC therapy may require monitoring of the ara-CTP pharmacokinetics in each individual patient.

Pharmacokinetic and pharmacodynamic studies of fludarabine and cytosine arabinoside administered as loading boluses followed by continuous infusions after a phase I/II study in pediatric patients with relapsed leukemias. The Children's Cancer Group.

The sequential administration of fludarabine followed by cytosine arabinoside (ara-C) has demonstrated significant synergistic effects against the CEM human leukemic cell line. This in vitro synergism was investigated in a Phase I trial in pediatric patients with relapsed acute leukemia. The optimum concentrations of 9-beta-D-arabinofuranosyl 2-fluoroadenine and ara-C necessary to achieve significant drug synergism from in vitro studies were between 10 and 20 microM. Fludarabine was infused at a dose to attain a target plasma concentration of 10 microM for 48 h, followed by a continuous infusion of escalated ara-C doses to maintain plasma ara-C concentrations of 10, 12.5, 15, or 17.5 microM for 72 h. Thirteen patients with acute lymphocytic leukemia and 18 with acute myelocytic leukemia were entered into the study, 30 of whom were clinically evaluable for toxicity. Pharmacokinetic and pharmacodynamic studies were performed on specimens from 20 patients. The optimal 9-beta-D-arabinofuranosyl 2-fluoroadenine and ara-C concentrations in plasma were easily achieved after continuous infusion regimens of both drugs. Cellular ara-CTP is augmented 5-8-fold in leukemic cells from patients receiving fludarabine phosphate treatment followed by ara-C. The maximum tolerated plasma concentrations for this combination regimen was 10 microM fludarabine for 48 h followed by 72 h of 15 microM ara-C, which were achieved at dose level 3. A significant number of responses were also seen. Nine of 18 evaluable patients (50%) with acute myelocytic leukemia achieved complete or partial responses, and 3 of 9 evaluable patients with acute lymphocytic leukemia achieved complete or partial responses. Fludarabine and ara-C successfully eradicated bone marrow disease in 16 of 27 patients (59%), 23 patients of which had been treated previously with high-dose ara-C. These results verified the synergistic effect fludarabine exhibited in augmenting ara-CTP concentrations in patients' leukemic blasts, thus improving the clinical response in relapsed pediatric leukemias.

Clinical and laboratory studies of 2-chlorodeoxyadenosine +/- cytosine arabinoside for relapsed or refractory acute myelogenous leukemia in adults.

Previous studies in pediatric patients with acute myelogenous leukemia (AML) have suggested that 2-chlorodeoxyadenosine (2CdA) is an effective therapeutic agent. Santana et al (J Clin Oncol 1992; 10: 364-370) reported a CR rate of 8/17 (95% Cl 23-72%) in children with relapsed AML and a median first CR of 21 months. The activity of 2CdA in adults with relapsed or refractory leukemia was therefore investigated in a phase I study. In the phase II study, based on biochemical modulation rationale, 2CdA was combined with Ara-C for adults with relapsed AML to test the effectiveness of this combination therapy. In the phase I study 27 patients (25 AML and two MDS) with a median first CR duration of 21 weeks, received 2CdA at doses ranging from 5 to 13 mg/m2/day by continuous infusion (CI) for 7 days. In vitro and ex vivo pharmacologic studies performed to determine the effect of pretreatment with 2CdA on Ara-CTP accumulation in leukemic blasts demonstrated a 50-65% increase in the rate of Ara-CTP accumulation. Based on this biochemical modulation, 2CdA (12 mg/m2/day x 5 days by CI) was combined with Ara-C (1 g/m2/day over 2 h) in a phase II study. Seventeen patients (15 AML, two MDS) with relapsed AML (median 1st CR of 19 weeks) were treated. In the phase I study two patients died before the day 14 marrow (ED). Marrow hypoplasia developed in 16 of the remaining 25. Leukemic regrowth occurred in nine after a median hypoplastic period of 2 weeks (range 1-3 weeks). The other seven patients died with aplastic marrows, median duration of hypoplasia was 2 weeks, range 1-4 weeks. None achieved CR and the median survival was 10.5 weeks. Toxicity generally was mild except for three late occurring cases of grade III or IV renal dysfunction and two cases of tumor lysis syndrome. The MTD was 10.8 mg/m2/day x 7 days. In the phase II study two patients, both with AML, achieved CR (95% CI 1-33%). In both cases leukemia relapsed after 10 weeks and 17 weeks. There was one ED. Most (11/16) cleared their marrow although leukemic infiltrate regrew in six cases. Toxicity was generally mild, with two episodes of grade 2 GI bleeding, one episode of severe renal dysfunction and one case of grade 2 CNS toxicity. We conclude that as a single agent 2CdA at the MTD is a cytoreductive agent but is not sufficient to achieve CR in adults with relapsed AML. While combination of Ara-C with 2CdA increases the Ara-CTP uptake in these heavily treated patients this regimen does not appear to be an improvement over existing modalities.

[Intracellular retention of cytarabine-triphosphate (Ara-CTP) in blasts of children with acute lymphoblastic leukemia. Correlation with clinical course parameters]. / Intrazelluläre Retention von Cytarabin-Triphosphat (Ara-CTP) in Blasten von Kindern mit akuter lymphoblastischer Leukämie. Korrelation zu klinischen Verlaufsparametern.

BACKGROUND: Cellular uptake and intracellular phosphorylation to the nucleotide cytosine arabinoside-triphosphate (Ara-CTP) is the precondition for the cytostatic effect of cytarabine. The pharmacokinetics of Ara-CTP in leukemic cells was reported to be of clinical importance in adult nonlymphoblastic leukemia. Therefore, the role of intracellular Ara-CTP formation and retention was investigated in blast cells from children with acute lymphoblastic leukemia (ALL). PATIENTS AND METHODS: At the time of diagnosis, peripheral or bone marrow blast cells from 41 children with ALL and 13 with relapsed ALL were incubated in Ara-C containing medium (1 hour, 1 or 3 micrograms/ml) followed by reincubation in Ara-C free medium (3 h). Intracellular Ara CTP formation and Ara-CTP retention were determined. MAIN RESULTS: Ara-CTP formation did not show marked differences between the different immunological subtypes. Ara-CTP retention, however, was significantly lower in T-ALL (37 +/- 15%, n = 8) compared to non-T-ALL (67 +/- 25%, n = 33; p < 0.003). Ara-CTP retention was also significantly different in children with and without persistence of peripheral blast cells after one week of prednison treatment (71 +/- 30%, n = 9 and 53 +/- 19%, n = 21; p = 0.031) as well as in children with and without complete bone marrow remission on day 15 of the ALL-BFM treatment protocol (66 +/- 17%, n = 19 and 43 +/- 18%, n = 11; p = 0.018). Ara-CTP retention was inversely correlated with the risk groups defined by the ALL-BFM treatment protocols (standard 79 +/- 29, intermediate 59 +/- 25, high risk 47 +/- 21%). A trend towards lower Ara-CTP retention was observed in relapsed leukemias (relapsed non-T-ALL 51 +/- 17%, p = 0.061). The difference in the probability of event free survival (following risk group adapted treatment according to ALL-BFM trials) between children with high (> or = 72%; 0.92 +/- 0.08) and low (< 72%: 0.58 +/- 0.15) Ara-CTP retention up to now did not reach statistical significance (p = 0.12). CONCLUSIONS: The more rapid decrease of cellular Ara-CTP in T-cell leukemia and children at higher clinical risk groups provide a pharmacokinetic rationale for prolonged infusion duration as an alternative to the intensification by dose escalation alone.

Intracellular retention of cytosine arabinoside triphosphate in blast cells from children with acute myelogenous and lymphoblastic leukemia.

The importance of the cellular pharmacokinetics of cytarabine triphosphate (ara-CTP) with regard to therapeutic efficacy is well established. In vitro and in vivo monitoring of pharmacokinetic parameters of leukemic blast cells were initiated in order to contribute to the pharmacological basis of optimal ara-C treatment strategies. Peripheral or bone marrow blast cells from 66 leukemic patients [51 acute myelogenous leukemia (ALL), 15 acute lymphoblastic leukemia (AML) were separated and incubated with ara-C for 1 hour and in ara-C-free medium for another 3 hours, and the intracellular formation and retention of ara-CTP was measured. In eight children who received continuous ara-C infusion for induction treatment, the ara-CTP concentration in circulating blast cells was monitored in vivo. The in vitro values observed in this assay corresponded to the cellular levels monitored in vivo. The ara-CTP retention differed clearly among the individual groups, as classified by immunophenotype at the time of the initial diagnosis: non-T-ALL 67+/-25% (x+/-SD, n=33), T-ALL 37+/-15% (n=8), and AML 34+/-18% (n=14). The difference in ara-CTP retention between non-T-All and AML (P<0.05) as well as T-ALL (P<0.05) was significant. There was a tendency toward lower ara-CTP retention in relapsed as compared with newly diagnosed ALL, but the difference was not significant. The maximal accumulation of ara-CTP (after 1 hour incubation) was comparable in AML, T-ALL, non-T-ALL, and blast cells from children in relapse. The observed similarity of cellular accumulation in all groups and the significantly more rapid decrease in T-ALL and AML provide the pharmacokinetic rationale supporting the prolonged infusion duration for ara-C in these subgroups as an alternative to the intensification by high-dose ara-C schedules with short-term infusion.

Characterization of a multidrug resistant human erythroleukemia cell line (K562) exhibiting spontaneous resistance to 1-beta-D-arabinofuranosylcytosine.

We have assessed the response of a previously characterized multidrug resistant (MDR) human erythroleukemia cell line (K562R) to the nucleoside analog antimetabolite 1-beta-D-arabinofuranosylcytosine (ara-C). This cell line has been subjected to selection pressure by intermittent exposure to daunorubicin, but not ara-C, since its initial isolation. In comparison to the parental line (K562S), K562R were approximately 15-fold more resistant to ara-C as determined by 3H-dThd incorporation, MTT dye reduction and clonogenicity. Following a 4-h exposure to 10 microM ara-C, K562S accumulated approximately seven times more ara-CTP, and incorporated approximately 250% more ara-C into DNA than their resistant counterparts. The intracellular generation of ara-CTP was not significantly influenced by the cytidine deaminase inhibitor THU or the deoxycytidylate deaminase inhibitor dTHU (1 mM each) in either cell line. Rates of dephosphorylation of ara-CTP were equivalent in sensitive and resistant cells, as were intracellular levels of both ribonucleotide and deoxyribonucleotide triphosphates. However, K562R displayed a significant (ie 70%) reduction in the level of activity of the pyrimidine salvage pathway enzyme, deoxycytidine kinase (dCK), compared to K562S cells. In contrast to U937 leukemic cells, DNA extracted from K562S and K562R cells following exposure to 10 microM ara-C for 6 h did not exhibit the characteristic internucleosomal DNA cleavage on agarose gel electrophoresis typical of drug-induced apoptosis. Lastly, Northern analysis revealed equivalent levels of dCK message in the two cell lines. K562R represents an unusual example of a classical multidrug resistant human leukemic cell line exhibiting spontaneous cross-resistance to the antimetabolite ara-C, and may prove of value in attempts to understand the mechanism(s) by which human leukemic myeloblasts survive in vivo exposure to combination chemotherapeutic regimens containing drugs that are not classically associated with the multidrug resistance phenomenon.

Effect of granulocyte-macrophage colony-stimulating factor on the metabolism of arabinosylcytosine triphosphate in blasts during therapy of patients with chronic myelogenous leukemia.

Because in vitro studies have indicated that granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulates arabinosylcytosine (ara-C) metabolism in leukemia blasts, we analyzed the pharmacokinetics of ara-C triphosphate (ara-CTP) in the blasts of patients with chronic myelogenous leukemia who were undergoing therapy with GM-CSF and ara-C. Patients received a 2-h infusion of 1.0 g/m2 ara-C followed by daily infusions of GM-CSF (125 micrograms/m2/day i.v. over 6 h) for 2-4 days. After the last GM-CSF infusion, a second, identical dose of ara-C was administered. The cellular pharmacokinetics of ara-CTP in circulating blasts were determined during and after each ara-C dose, and the area under the accumulation and elimination curve (AUC) measured over 12 h was compared before and after GM-CSF. Ara-CTP accumulation peaked within 1 h after the end of each ara-C infusion. Comparison of the AUC of ara-CTP before and after GM-CSF administration suggested that in the blasts of three of four patients, GM-CSF treatment decreased the ara-CTP AUC; the AUC values were altered only slightly in a fourth patient. Studies of these patients' blasts incubated in vitro with ara-C before and after clinical infusion of GM-CSF revealed similar ara-CTP accumulation patterns. Together, these studies suggest that 2-4 days of GM-CSF administration does not increase the accumulation of ara-CTP in the circulating blasts from patients in the blastic phase of chronic myelogenous leukemia.

Long-term outcome of patients with acute myelogenous leukemia: the role of maintenance therapy, consolidation therapy and the predictive value of two in vitro assays.

Three successive strategies used to treat acute myelogenous leukemia (AML) patients who entered complete remission are compared with respect to their long term efficacy (> 5 years). Additionally, the ability of two in vitro assays to identify patients who would be long term remitters was assessed. With respect to the proportion of patients in long term remission, 5 years of maintenance therapy, intensive consolidation therapy plus 3 years of additional therapy, and 4 courses of consolidation therapy produced equivalent results with 18 to 22% of patients in remission at 7 years. Patients whose leukemia cells retained high levels of cytosine arabinoside triphosphate (araCTP) and who received maintenance therapy were less likely to experience early relapse than comparably treated patients whose leukemia cells failed to retain araCTP. The failure of leukemia cells to clone in vitro was also associated with a lower early relapse rate for patients who received maintenance therapy. Neither the ability of leukemia cells to retain araCTP nor their ability to clone in vitro were of prognostic significance for patients treated with 4 courses of intensive consolidation therapy.

Enhancement of cytosine arabinoside cytotoxicity by granulocyte/macrophage colony-stimulating factor and granulocyte colony-stimulating factor in a human myeloblastic leukemia cell line.

Enhancement of the cytotoxicity of cytosine arabinoside (ara-C) by granulocyte/macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF), and the mechanisms involved, were studied in the AML-193 human leukemia cell line. AML-193 cells require GM-CSF and G-CSF(CSFs) for optimum growth, and 24 h deprivation of CSFs decreased DNA synthesis measured in terms of 3H-thymidine incorporation. The DNA synthesis gradually recovered upon addition of CSFs. To examine the sensitivity to ara-C under different growth conditions, two groups of cell suspensions, one pretreated with CSFs after 24 h deprivation (CSFs(+) cells), and the other held continuously under CSFs-free conditions (CSFs(-) cells), were exposed to 1.0 microgram/ml of ara-C for 16 h. In clonogenic assays, CSFs(+) cells showed higher sensitivity to ara-C than CSFs(-) cells. These cell groups showed no significant difference in ara-C triphosphate accumulation or retention, though the amount of ara-C incorporated into the acid-insoluble fraction was two times greater in CSFs(+) cells than CSFs(-) cells, and that difference became even clearer in the retention pools. These data suggest that the enhancement of cytotoxicity by CSFs was due to the promotion of ara-C incorporation into DNA as a result of an increase of the cell fraction in the S phase.

Clinical pharmacokinetics of anti-metabolites.

Anti-metabolites are among the most important agents used in cancer chemotherapy. Ara-C, the thiopurines and MTX are active drugs for both induction and maintenance chemotherapy of childhood and adult leukaemia, while the new adenosine analogues are active against hairy cell leukaemia, with promising activity against other malignancies such as malignant lymphomas. Methotrexate and 5FU are being used in the treatment of several solid malignancies. Recent advances in the clinical pharmacology of widely used antimetabolites have shown a relationship among dose, plasma concentrations and clearance with the toxicity and anti-tumour activity. Thus, it has been shown that adaptative control of 5FU administration is possible, limiting the toxicity of this drug. Recent advances in the pharmacogenetics of, for example, 6MP and 5FU will possibly enable researchers to identify patients who may have an increased risk of toxicity. For ara-C, some evidence has been obtained to identify populations at risk of no response. In addition, for most anti-metabolites, convincing evidence of their intracellular (intratumour) metabolism has been obtained, thus making it possible to identify patients who are likely to respond to treatment. These studies (eg accumulation of active metabolites such as ara-CTP, thioguanine nucleotides, FdUMP, MTX-polyglutamates; and inhibition of target enzymes such as thymidylate synthase) have made it possible to develop the basis of biochemical modulation--that is, specific manipulation of intracellular metabolism of the drug. It is anticipated that new technical developments in molecular biology, biochemistry, cell biology and immunology will make it possible to improve the identification of resistant patients in order to modulate specifically drug metabolism in the tumour cells. Biochemical modulation has been successful in achieving significant improvements in treatment and currently is a keystone in cancer chemotherapy. Together with the development of promising new anti-metabolites, biochemical modulation (with other drugs, biologicals) will be a major strategy for the future.

Cell cycle-specific metabolism of arabinosyl nucleosides in K562 human leukemia cells.

Exponentially growing K562 cells incubated with 1-beta-D-arabinofuranosylcytosine (ara-C) accumulate ara-C triphosphate (ara-CTP) at a higher rate and to a greater concentration after pretreatment with 9-beta-D-arabinofuranosyl-2-fluoroadenine (F-ara-A) than do cells treated with ara-C alone. Potentiation of ara-C metabolism is due in part to an indirect effect of F-ara-A triphosphate (F-ara-ATP)-mediated reduction in deoxynucleotide pools and consequent activation of deoxycytidine kinase. Because the levels of deoxynucleotide pools and the activity of deoxycytidine kinase are cell cycle-specific, we investigated the effect of cell cycle phases on the accumulation of ara-CTP and the influence of F-ara-A pretreatment on such accumulation. Exponentially growing K562 cells were fractionated into G1, S, and G2+M phase-enriched subpopulations (each enriched by > 60%) by centrifugal elutriation. The rate of ara-CTP accumulation was 22, 25, and 14 microM/h and the rate of F-ara-ATP accumulation was 38, 47, and 33 microM/h in the G1, S, and G2+M subpopulations, respectively. The rate of elimination of arabinosyl triphosphates was similar among the different phases of the cell cycle. After pretreatment with F-ara-A, the rate of ara-CTP accumulation in the G1, S, and G2+M phase-enriched subpopulations was 43, 37, and 26 microM/h, indicating a 1.7-, 1.5-, and 1.9-fold increase, respectively. These results suggest that a combination of F-ara-A and ara-C may effectively potentiate ara-CTP accumulation in all phases of the cell cycle. This observation is consistent with the results of studies on the modulation of ara-C metabolism by F-ara-A in lymphocytes and leukemia blasts obtained from patients with chronic lymphocytic leukemia and acute myelogenous leukemia, respectively.

Membrane transport of 1-beta-D-arabinofuranosylcytosine and accumulation of 1-beta-D-arabinofuranosylcytosine 5'-triphosphate in P388 murine leukemic cells resistant to vincristine.

The membrane transport of ara-C and intracellular ara-CTP accumulation were investigated in P388 murine leukemic cells resistant to vincristine (P388/VCR) and its parent cell line. The transport of ara-C in P388/VCR cell line was a 1.4-fold increase at 30 sec compared to that in P388 parent cell line (P less than 0.01). The increase of the transport of ara-C in P388/VCR cell line, however, was not completely abolished by the nucleoside transport inhibitor, nitrobenzylthioinosine (NBTI) to the level in parent cell line. Scatchard analyses revealed that the resistant cells had significantly less NBTI binding sites than the parent cells had. These results suggested that the changes in ara-C transport in P388/VCR cells were due, in part, to increase of NBTI-insensitive transport sites in the membrane. The measurement of the intracellular ara-CTP concentration by high-performance liquid chromatography revealed that the intracellular ara-CTP level in P388/VCR cells was also significantly higher than that in parent cells (1.4-fold, P less than 0.01). As the transport of ara-C is rate limiting at a concentration of 1 microM in the both cell lines, we concluded that the accumulation of ara-CTP in P388/VCR cells might have partially resulted from the enhancement of the ara-C transport.

High-dose versus intermediate-dose cytosine arabinoside in combination with mitoxantrone for the treatment of relapsed and refractory acute myeloid leukemia--preliminary clinical and pharmacological data of a randomized comparison.

The present randomized trial addressed the pending question whether cytosine arabinoside (AraC) should be given at high or intermediate dose to patients with relapsed or refractory acute myeloid leukemia. Based upon the previously established regimen of the sequential administration of AraC and mitoxantrone (S-HAM) patients below 60 years of age were randomized to receive AraC at either 3.0 g/m2 vs. 1.0 g/m2 per dose while older patients were randomly assigned to either 1.0 g/m2 or 0.5 g/m2 AraC. Concurrent pharmacokinetic analyses were performed to determine the plasma AraC pharmacokinetics as well as the intracellular AraCTP peak concentrations and retention times. At the present stage 65 patients are evaluable for response and toxicity. Complete remissions were achieved at similar frequencies for patients treated with 3.0 g/m2 or 1.0 g/m2 AraC with 56% and 50%, respectively. Reasons for failure were different, however, with a higher incidence of resistant disease in patients treated with 1.0 g/m2 AraC and more early deaths in the higher dose treatment group. Pharmacokinetic studies indicated a homogeneous distribution of AraC plasma concentrations but a substantial interpatient variability for intracellular AraCTP peak concentrations and retention times.

[Studies on intracellular kinetics of ara-C triphosphate in HL-60, human leukemia cells in relation to reasonable administration of ara-C].

To study the pharmacokinetics of 1-beta-D-arabinofuranosylcytosine (ara-C), which is one of the main drugs used in chemotherapy for acute leukemia, its intracellular metabolism was investigated using HL-60 cells derived from human acute non-lymphocytic leukemia. The concentration of the drug and its metabolites in the cells were serially determined and the following results were obtained. 1) The uptake of ara-C into HL-60 cell (1 X 10(7)/ml) was very rapid when they were incubated with 2 microM ara-C. The total intracellular ara-C content per 10(9) cells exceeded the ara-C concentration in the extracellular fluid at about 7 minutes after the start of incubation. It reached about 4 times higher than the extracellular concentration after 60 minutes. 2) Conversion of ara-C to the active form, ara-CTP, was also rapid. The intracellular concentration of ara-CTP was about 3 times higher than the ara-C concentration in the extracellular fluid after incubation for 60 minutes. 3) Total accumulation of ara-C in the cells was dependent on the extracellular ara-C concentration up to a concentration of 100 microM. The production of ara-CTP occurred in such a way that, when the extracellular ara-C concentration was lower than 10 microM, more than 90% of the uptake of ara-C was converted to ara-CTP, while at concentrations above 10 microM the efficiency at production (the ratio of total ara-C to ara-CTP production) was decreased. The maximum intracellular ara-CTP concentration was estimated to reach to 45 microM.(ABSTRACT TRUNCATED AT 250 WORDS)