Biochemical modulation of cytarabine triphosphate by clofarabine

Purpose Clofarabine has proven to be effective in the treatment of adult and pediatric acute myelogenous leukemia (AML). To investigate if clofarabine could be used with success in biochemical modulation strategies, we investigated the biochemical modulation of cytarabine triphosphate (ara-CTP) by clofarabine in a myeloid leukemia cell line and the effect of this combination on cytotoxicity.Experimental design K562 cells were incubated with clofarabine and ara-C either sequentially or simultaneously to evaluate the combination effect on their phosphorylated metabolites. Clonogenic assays were used to determine the cytotoxicity of each agent alone and in combination. Deoxynucleotide analysis was performed to assess the effect of clofarabine on dNTPs.Results Clofarabine added either simultaneously or in sequence increased ara-CTP accumulation. The maximal modulation of ara-CTP accumulation occurred with 1 M clofarabine. This level was achieved at the maximum tolerated dose for adult and pediatric patients with AML. With 10 M ara-C alone, 86 M ara-CTP had accumulated after 3 h. The optimal sequence for the drug combination, i.e., clofarabine followed 4 h later by ara-C, resulted in 248 M ara-CTP at 3 h. Clofarabine accumulated maximally in the monophosphate form. Preincubation with ara-C did not affect the triphosphate form, but it lowered clofarabine monophosphate. Clofarabine resulted in the intracellular decrease of dATP and dGTP levels. Clonogenic assays revealed that the combination of clofarabine and ara-C produced synergistic killing of myeloid leukemia cells.Conclusions These findings demonstrate that combination of clofarabine followed by ara-C results in a biochemical modulation of ara-CTP and synergistic cell kill. These studies provide a compelling rationale for clinical trials using this combination regimen for adult and pediatric patients with AML.     

Pharmacodynamic and DNA methylation studies of high-dose 1-β-d-arabinofuranosyl cytosine before and after in vivo 5-azacytidine treatment in pediatric patients with refractory acute lymphocytic leukemia

Summary The primary development of clinical resistance to 1--d-arabinofuranosyl cytosine (ara-C) in leukemic blast cells is expressed as decreased cellular concentrations of its active anabolite. Correlations exist between the cellular concentrations of 1--d-arabinofuranosyl cytosine 5-triphosphate (ara-CTP) in leukemic blast cells and inhibition of DNA synthetic capacity with the clinical response to high-dose cytosine arabinoside (HDara-C). 5-Azacytidine (5-Aza-C) and its congeners are potent DNA hypomethylating agents, an action closely associated with the reexpression of certain genes such as that for deoxycytidine kinase (dCk) in ara-C-resistant mouse and human leukemic cells. Reexpression of dCk could increase the cellular ara-CTP concentrations and the sensitivity to ara-C. A total of 17 pediatric patients with refractory acute lymphocytic leukemia (ALL) received a continuous influsion of 5-Aza-C at 150 mg/m² daily for 5 days after not responding to (13/17) or relapsing from (4/17) an HDara-C regimen (3 g/m² over 3 h, every 12 h, x 8 doses). Approximately 3 days after the end of the 5-Aza-C infusion, the HDara-C regimen was given again with the idea that the induced DNA hypomethylation in the leukemic cells may have increased the dCk activity and that a reversal of the tumor drug resistance to ara-C could have occurred. Deoxycytidine kinase (expressed as cellular ara-CTP concentrations) in untreated blasts, DNA synthetic capacity (DSC), and the percentage of DNA methylcytidine levels were determined before and after 5-Aza-C administration. Cellular ara-CTP was enhanced to varying degrees in 15 of 16 patients after 5-Aza-C treatment. The average cellular concentration of ara-CTP determined in vitro by the sensitivity test was 314±390 M, 2.3-fold higher than the average value before 5-Aza-C treatment. In 12 patients in whom the DNA methylation studies were completed before and after 5-Aza-C treatment, the average DNA hypomethylation level was 55.6%+15.8% of pretreatment values (n=13; mean ± SD). DSC showed a profound decline in 2/9 evaluable patients who achieved a complete response (CR) after this regimen. The data suggest that treatment with a cytostatic but DNA-modulatory regimen of 5-Aza-C causes DNA hypomethylation in vivo, which is associated with dCk reexpression in the patients' leukemic blasts. The partial reversal of drug resistance to ara-C by 5-Aza-C yielded two CRs in this poor-prognosis, multiply relapsed patient population with refractory ALL. The data indicate that the 5-Aza-C plus HDara-C regimen may have a profound effect on controlling leukemia refractory to ara-C in patients.Abbreviations used ara-C 1--d-arabinofuranosyl cytosine - ara-CTP 1--d-arabinofuranosyl cytosine 5-triphosphate - 5-Aza-C 5-Azacytidine - HDara-C high-dose ara-C - mC 5-methyl cytosine - PCA perchloric acid - PBS phosphate-buffered saline - dCk deoxycytidine kinase - SAX strong anion exchange - SCX strong cation exchange - DSC DNA synthetic capacity - t_1/2 elimination half-life - BM bone marrow - PBC peripheral blast cells - dThd thymidine - ALL acute lymphocytic leukemia Supported by NIH-NCI research grant CA 38905, by a grant from the UpJohn Co., and by The T. J. Martell Foundation     

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

Summary Exponentially growing K562 cells incubated with 1--d-arabinofuranosylcytosine (ara-C) accumulate ara-C triphosphate (ara-CTP) at a higher rate and to a greater concentration after pretreatment with 9--d-ara-binofuranosyl-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 m/h and the rate of F-ara-ATP accumulation was 38, 47, and 33 m/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 m/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.Supported in part by grants CA 28596 and CA 16672 from the National Cancer Institute, Department of Health and Human Services, and by grant DHP-I from the American Cancer Society     

Inhibition of fludarabine metabolism by arabinosylcytosine during therapy

Summary The active 5-triphosphate of arabinosyl-2-fluoroadenine (F-ara-ATP) increases the anabolism of arabinosylcytosine (ara-C), whereas ara-C 5-triphosphate inhibits the phosphorylation of arabinosyl-2-fluoroadenine (F-ara-A) in human leukemia cells in vitro. These interactions have a potential impact on drug scheduling. Clinical trials of relapsed leukemia in which fludarabine (F-ara-A 5-monophosphate) and ara-C were given in sequence provided the opportunity to evaluate the effects of ara-C infusion on two sequelae: the pharmacokinetics of F-ara-A in plasma and that of F-ara-ATP in leukemia cells. First, F-ara-A pharmacokinetics were altered by ara-C infusion. This was visualized as a transient increase in F-ara-A plasma levels during the ara-C infusion that was given 4 h after fludarabine. The perturbation in F-ara-A plasma levels was dependent on the dose of ara-C. Second, peak F-ara-ATP concentrations were lower in leukemia cells of patients who received ara-C in addition to fludarabine as compared with those who received fludarabine alone. The terminal half-life of F-ara-A in plasma and the half-life of intracellular F-ara-ATP were reduced after the ara-C infusion in a concentration-dependent manner. Studies using purified deoxycytidine kinase support the conclusion that the increase in plasma levels of F-ara-A is in part the result of an effective competition by ara-C for phosphorylation by this enzyme, leading to a perturbation of the pharmacokinetics of intracellular F-ara-ATP.Abbreviations ara-C 9--d-arabinofuranosylcytosine - ara-CTP 9--d-arabinofuranosylcytosine 5-triphosphate - AUC area under the concentration-time curve - AUC_p inerease in the AUC caused by perturbation - CLL chronic lymphocytic leukemia - dCyd kinase deoxycytidine kinase - F-ara-A 9--d-arabinofuranosyl-2-fluoroadenine 5 - fludarabine F-ara-AMP, 9--d-arabinofuranosyl-2-fluoroadenine 5-monophosphate - F-ara-ATP 9--d-arabinofuranosyl-2-fluoroadenine 5-triphosphate - t _1/2 half-life of elimination This work was supported in part by grants CA32839, CA46452, CA53311, and CA57629 from the National Cancer Institute, Department of Health and Human Services, by the German Research Association (DFG), and by a contract from Berlex Laboratories, Inc.     

The effect of ara-C-induced inhibition of DNA synthesis on its cellular pharmacology

Summary The cytotoxicity of ara-C is believed to result from incorporation of ara-CTP into DNA and inhibition of DNA synthesis. Since complete inhibition of DNA synthesis would prevent further incorporation of ara-CTP, ara-C may have a self-limiting effect on its own cytotoxicity, particularly at the high concentrations typical of highdose ara-C clinical protocols. In this study, the incorporation of [³H]-dThd and [³H]-ara-C into DNA were compared. Within 1 h of exposure of L5178Y cells to ara-C, the rate of [³H]-dThd incorporation into the acid-insoluble fraction was reduced by 98%. Despite this nearly complete block in [³H]-dThd incorpration, DNA synthesis was not completely inhibited since [³H]-ara-C continued to be incorporated for up to 6 h, although a plateau in ara-CDNA synthesis was observed between 2 and 3 h exposure when ara-CTP levels were maximal. The effect of ara-C on [³H]-dThd incorporation into DNA was due in part to an indirect effect of ara-C on the metabolism of intracellular [³H]-dThd to [³H]-dTTP. Within 30 min exposure to 10 M ara-C, the rate of cellular [³H]-dTTP synthesis was slowed to only 15% of the control rate. This was not due to inhibition of [³H]-dThd transport, since the intracellular and extracellular concentrations of the nucleoside were equal. The effect of ara-C on [³H]-dTTP synthesis resulted from significant changes in deoxynucleoside 5-triphosphate (dNTP) pools. dTTP, dATP, and dGTP levels were increased, whereas the dCTP concentration was decreased. When dThd kinase from L5178Y cells was assayed with increased dTTP levels induced by ara-C vs the dTTP level in control cells, its activity was reduced by 72%. Thus, the [³H]-dThd incorporation experiment overestimated the extent of inhibition of DNA synthesis by ara-C due to increased feedback inhibition of dThd kinase and increased competition for DNA polymerase between the elevated unlabeled dTTP pool and the decreased levels of [³H]-dTTP. In vitro assay of DNA polymerase in the presence of the ara-CTP concentration achieved after 0.5 or 3 h exposure to 10 M ara-C (60 M and 200 M, respectively), plus the mixture of dNTPs found intracellularly at these times, resulted in 57% and 80% inhibition of the polymerase, respectively. This inhibition may account for the plateau in the accumulation of ara-CDNA that was observed at 3 h and suggests that ara-C incorporation may be self-limiting at high cellular concentrations of ara-CTP. The ara-C-induced decline in dCTP noted above was apparently a secondary effect resulting from the inhibition of ribonucleotide reductase by the elevated dTTP and dATP. CDP reductase activity in the presence of dATP and dTTP at the concentrations found in ara-C-treated cells was 58% of the activity observed in the presence of nucleotide levels found in control cells. The decrease in dCTP levels was associated with a reciprocal increase in the rate of [³H]-ara-C phosphorylation following subsequent exposure to unlabeled ara-C. Thus, ara-C self-potentiated its own uptake in these cells. These observations of the self-limiting and self-potentiating effects of high concentrations of ara-C may be relevant to the selection of the optimal dose and the duration of exposure in the clinical use of high-dose ara-C infusions.Abbreviations ara-C I--d-arabinofuranosyl, cytosine (cytosine arabinoside) - ara-CTP ara-C triphosphate - NTP unspecified nucleoside 5-triphosphate - dNTP deoxynucleoside 5-triphosphate - PBS phosphate-buffered saline This research was supported by grants from the American Cancer Society (CH35J), the National Institutes of Health (CA 12197), the Gaston County Cancer Society, and Dr. George Royer of the Upjohn Company     

Intracellular pharmacodynamic studies of the synergistic combination of 6-mercaptopurine and cytosine arabinoside in human leukemia cell lines

Selective combinations of purine and pyrimidine analogs increase remission rates in pediatric patients with relapsed leukemias. The combination of 6-mercaptopurine (6-MP) and cytosine arabinoside (ara-C) may exhibit synergism similar to that observed for fludarabine and ara-C and may diminish the potential for development of resistance since the two drugs are activated by separate enzymatic pathways. To determine the efficacy of the combination against human leukemia cells, we investigated the time-concentration relationships of the drugs given alone or in combination to the resultant cytotoxicity. To determine whether the combination leads to enhanced activity of deoxycytidine kinase (dCk), the ratelimiting enzyme in ara-C activation, we characterized the cellular dCk in CCRF/CEM/0, CCRF/CEM/ara-C/7A, and CCRF/CEM/ara-C/3A monoclonal cells before and after treatment with 6-MP. CCRF/CEM/0 (wild type), CCRF/CEM/ara-C/7A (50% ara-C-resistant as determined by ara-C sensitivity assay and dCk characterization), and CCRF/CEM/ara-C/3A (90% resistant to ara-C) human leukemia cells were incubated with various concentrations of 6-MP and ara-C given alone or in combination. Cell survival, inhibition of DNA synthetic capacity (DSC), ara-CTP anabolism, and dCk enzymatic characteristics were studied. Incubation of CEM/0 cells with 6-MP for 24 h, followed by ara-C for 48 h, increased cell-growth inhibition by approximately 0.5–1 log₁₀, corresponding to 5- to 10-fold synergism, as compared with ara-C alone after identical drug incubation in all cell lines. Simultaneous administration showed no synergism, whereas reversal of the sequence produced an antagonistic effect. The ara-CTP levels were 2-to 3.5-fold and 3- to 5-fold higher in CEM/0 and CEM/ara-C/7A cells, respectively, in cells exposed to 6-MP followed by ara-C than in those exposed to ara-C alone at the same concentrations. Furthermore, a progressive increase in ara-CTP levels was noted in CEM/0 cells exposed to increasing concentrations of 6-MP followed by 10 or 20 M ara-C. A significant decrease in DSC was observed upon treatment of wild-type and ara-Cresistant cells with 6-MP and ara-C. The combination of 6-MP and ara-C exhibits significant sequence-specific synergism in both wild-type and partially ara-C-resistant leukemia cell lines. The combination also exerts collateral sensitivity in the ara-C-resistant cell lines. 6-MP pretreatment may play a role in enhancing ara-C activation, thus producing drug synergism in sensitive and resistant leukemia cell lines.Abbreviations 6-MP 6-mercaptopurine - ara-C cytosine arabinoside - ara-CMP cytosine arabinoside monophosphate - ara-CTP cytosine arabinoside triphosphate - TIMP thioinosine monophosphate - HGPRT hypoxanthine-guanine phosphoribosyl transferase - PRPP 5-phosphoribosyl-1-pyrophosphate - AMP adenylic acid - SAMP adenylosuccinate - XMP xanthylate - TGMP thioguanylic acidDSC DNA synthetic capacity - dCk deoxycytidine kinaseRR ribonucleotide reductase - AUC area under the curvePCA perchloric acid This work was supported by the Clinical Fellowship Program, Division of Hematology/Oncology, CHLA and by the Neil Bogart Memorial Laboratories and the T.J. Martell Foundation of Cancer, Leukemia and AIDS Research     

Combination of fludarabine and arabinosylcytosine for treatment of chronic lymphocytic leukemia: clinical efficacy and modulation of arabinosylcytosine pharmacology

Previous studies have demonstrated that treatment with fludarabine 4 h prior to arabinosylcytosine (ara-C) potentiates the accumulation of the active triphosphate of ara-C (ara-CTP) in leukemic lymphocytes. The clinical efficacy of this combination was evaluated in 15 patients with chronic lymphocytic leukemia (CLL) that was advanced in their disease (median Rai stage, IV) and refractory to treatment with fludarabine. Patients received 0.5 g/m² ara-C infused i.v. over 2 h followed at 20 h by a 30-min infusion of 30 mg/m² fludarabine. At 24 h, an identical dose of ara-C was infused. To intensify the therapy and to determine the duration of fludarabine potentiation of ara-CTP accumulation, six additional patients with Rai stage III or IV CLL were treated with an amended 2-week protocol. On week 1, 30 mg/m² fludarabine was infused over 30 min, followed 4 h later by a 2-h infusion of 0.5 g/m² ara-C; on week 2, the fludarabine dose was followed 4 h later by a 4-h infusion of ara-C (1.0 g/m²). In all, 1 partial remission and 7 minor responses in 1 or more disease sites were observed in the 21 patients. The major treatment-related toxic effects were myelosuppression and infection. Comparison of the ara-CTP accumulation area under the concentration-time curve (AUC) in circulating CLL cells of patients on the amended protocol demonstrated a significant (P=0.001) 1.6-fold (range, 1.4-to 2.0-fold) increase after fludarabine administration. Although the initial rates of ara-CTP accumulation were similar for the 2-h and 4-h infusions, ara-CTP accumulation continued for up to 4 h in four of five patients who received the longer infusion. The activity of the fludarabine and ara-C combination is being evaluated in in vitro model systems and in phase II clinical trials in combination with other drugs.This study was supported in part by grants CA32839 and CA57629 from the National Cancer Institute, Department of Health and Human Services, and by Berlex Laboratories, Inc. L.E.R. is the recipient of an ASCO Young Investigator Award     

Quantitation of 1-beta-D-arabinofuranosylcytosine 5'-triphosphate in the leukemic cells from bone marrow and peripheral blood of patients receiving 1-beta-D-arabinofuranosylcytosine therapy

A method for the detection and quantitation of 1-beta-D-arabinofuranosylcytosine 5'-triphosphate (ara-CTP), the active metabolite cells and leukemic cells of the peripheral blood from patients receiving ara-C therapy is described. ara-CTP is separated from normal cellular nucleotides by high-pressure liquid chromatography and is quantitated by its absorbance of ultraviolet light at 280 nm with a lower limit of sensitivity of 25 pmol/2 x 10(7) cell equivalents. During separate courses of continuous infusion of different therapeutic doses of ara-C, ara-CTP accumulated in the leukemic bone marrow cells of a patient with acute myelogenous leukemia in proportion to the dose of ara-C. Continuous infusion of ara-C (90 mg/sq m/day) resulted in plateau levels of ara-CPT in peripheral blast cells after 24 hr (115 pmol/1 x 10(7) cell equivalents). A priming dose of ara-C(125 to 250 mg/sq m) followed by a 1-hr infusion of an equal dose of ara-C to patients with acute myelogenous leukemia facilitated the determination of ara-CTP retention in bone marrow and peripheral blood leukemic cells in vivo. This procedure should be useful for extended studies of the biochemical pharmacology of ara-CTP in vivo.     

Monitoring of Intracellular l-βD-Arabinofuranosylcytosine 5'-Triphosphate in 1-β-D-Arabinofuranosylcytosine Therapy at Low and Conventional Doses

1-β-D-Arabinofuranosylcytosine (ara-C) is used empirically at a low, conventional, or high dose. Ara-C therapy may be optimal if it is directed by the clinical pharmacokinetics of the intracellular active metabolite of ara-C, 1-β-D-arabinofuranosylcytosine 5'-triphosphate (ara-CTP). However, ara-CTP has seldom been monitored during low- and conventional-dose ara-C therapies because detection methods were insufficiently sensitive. Here, with the use of our newly established method ( Cancer Res. , 56, 1800-1804 (1996), ara-CTP was monitored in leukemic cells from acute myelog-enous leukemia patients receiving low- or conventional-dose ara-C [subcutaneous ara-C administration (10 mg/m2) (3 patients), continuous ara-C infusion (20 or 70 mg/m2/24 h) (7 patients), 2-h ara-C infusion (70 mg/m2) (4 patients), and 2-h infusion of N4-behenoyl-l-β-D-arabinofuranosylcy-tosine, a deaminase-resistant ara-C derivative (70 mg/m2) (6 patients)]. Ara-CTP could be determined at levels under 1μ M. There was a close correlation between the elimination half-life values of the plasma ara-C and the intracellular ara-CTP. The presence of ara-C in the plasma was important to maintain ara-CTP. The continuous ara-C and the 2-h N4-behenoyl-l-β-D-arabinofura-nosylcytosine infusions maintained ara-CTP and the plasma ara-C longer than the subcutaneous ara-C or the 2-h ara-C infusion. They also afforded relatively higher ara-CTP concentrations, and consequently produced ara-CTP more efficiently than the 2-h ara-C infusion. Different administration methods produced different quantities of ara-CTP even at the same dose.     

Saturation of 1-beta-D-arabinofuranosylcytosine 5'-triphosphate accumulation in leukemia cells during high-dose 1-beta-D-arabinofuranosylcytosine therapy

Twenty-seven patients with refractory leukemia were treated with 1-beta-D-arabinofuranosylcytosine (ara-C), 0.3 to 3.0 g/m2 as i.v. infusions over 1, 2, 4, or 24 h. The pharmacokinetics of ara-C in plasma and its 5'-triphosphate (ara-CTP) in leukemic cells from peripheral blood were studied after a single infusion of 3 g/m2 over 2 h in 13 patients. Accumulation of ara-CTP in leukemic cells remained linear until 1 to 2 h after the infusion. At the time when the rate of ara-CTP accumulation deviated from linearity, the plasma concentration of ara-C was 5- to 20-fold lower [8.1 +/- 4.4 (SD) microM] than the steady-state level during the infusion. Plasma ara-C and cellular ara-CTP pharmacokinetics were studied after two serial infusions in 14 additional patients. Varying the duration of infusion of an ara-C dose between 1, 2, and 4 h (corresponding to infusion rates of 3000, 1500, and 750 mg/m2/h) did not substantially change the rate of ara-CTP accumulation by leukemic cells. The peak ara-CTP concentration and the area under the concentration times time curve (AUC) of ara-CTP in leukemic cells increased with prolongation of the infusion. Although steady-state concentration of ara-C and AUC of ara-C in plasma were proportionally reduced by 1.0 or 0.5 g/m2 infusion over 2 h, ara-CTP accumulation rate and AUC in leukemic cells did not change compared with administration of 3 g/m2 over 2 h. However, when the infusion rate was further reduced to 0.4 or 0.3 g/m2 over 2 h, resulting in steady-state plasma ara-C concentrations of less than 7 microM, the accumulation rate of ara-CTP was substantially reduced as was the ara-CTP intracellular AUC. The cellular elimination rate of ara-CTP remained constant under all infusion conditions. These findings support the conclusion that high-dose ara-C therapy, as currently administered, results in plasma ara-C concentrations that saturate the accumulation of ara-CTP by circulating leukemic cells. We recommend that intermediate dose rates, 200 to 250 mg/m2/h, be evaluated in future studies as an alternative to the substantially higher ara-C dose rates currently in use.     

Modulation of the cellular pharmacokinetics of ara-CTP in human leukemic blasts by dipyridamole

Summary The effect of dipyridamole (DP) on the cellular retention of 1--d-arabinofuranosylcytosine (ara-C) and its metabolites was examined in leukemic blasts that had been isolated directly from bone marrow aspirates from patients afflicted with acute myeloid leukemia (AML). When AML cells were loaded for 2 h with 1 m [³H]-ara-C and then transferred to ara-C-free medium, total intracellular concentrations of radiolabel and [³H]-ara-C 5-triphosphate [³H]-ara-C-CTP rapidly declined. After 8 h, total intracellular levels of tritium were 4.4 times higher if 10 m was included in the washout medium; however, the majority of this intracellular radiolabel corresponded to [³H]-uridine arabinoside ([³H]-ara-U) and [³H]-ara-C. DP significantly increased the meant _1/2 for [³H]-ara-CTP from 102 to 136 min (P<0.01), but this effect was much less pronounced than that obtained for total tritium and the increase was quite variable (0–70%; median, 19%). The presence of DP in the washout medium also increased the incorporation of ara-C into DNA and the formation of ara-CDP-choline. The level of ara-CDP-choline continued to increase in both DP-containing and DP-free media for the first 4 h following drug removal and the formation of ara-CDP-choline continued during the first few hours in ara-C-free medium. At the end of the 8-h wash in DP-containing medium, the cellular concentration of ara-CDP-choline was equivalent to that found at the beginning of the washout period. Although statistically significant, the effect of DP on ara-CTP retention in AML blasts was much less pronounced than that previously observed in L5178Y leukemia. The former cells exhibited only 10% as many nucleoside transport carriers as did the L5178Y cells as measured by their capacity to bind [³H]-nitrobenzylmer-captopurine riboside (NBMPR). The effect of DP in prolonging ara-CTP retention was proportional to the number of [³H]-NBMPR binding sites. This suggests that in patients cells that exhibit extremely low transport capacity, most of the net catabolism occurs via deamination, and further inhibition of transport by DP in an effort to improve cellular retention of ara-C has little effect on ara-CTP catabolism.Abbreviations ara-C 1--d-Arabinofuranosylcytosine, cytosine arabinoside - ara-CMP ara-C 5-monophosphate - ara-CTP ara-C 5-triphosphate - ara-C-DNA ara-C incorporated into DNA - DP dipyridamole - Persantine 2,2,2,2-[4,8-dipiperidinopyrimido(5,4-d)-pyrimidine-2,6-dinitrilo] tetraethanol - NBMPR nitrobenzylmercaptopurine riboside Present address:US Bioscience Inc., One Tower Bridge, 100 Front Streen, West Conshohocken, PA 19428, USASupported by grant CH-471L from the American Cancer Society. This work was also supported in part by the Leukemia Cell Distribution Laboratory of the Comprehensive Cancer Center of Wake Forest University (grant CA 12 197). One of the authors (J. C. W.) is a Leukemia Society of America Scholar     

Close Correlation of 1-β-D-Arabinofuranosylcytosine 5'-Triphosphate, an Intracellular Active Metabolite, to the Therapeutic Efficacy of N4-Behenoyl-1-β-D-arabinofuranosylcytosine Therapy for Acute Myelogenous Leukemia

N ⁴-Behenoyl-l-β-D-arabinofuranosylcytosine (BHAC), a prodrug of 1-β -D-arabinofuranosylcy-tosine, 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-β -D-arabinofuranosylcytosine 5'-tri-phosphate (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/m²) in combination remission induction therapy. ara-CTP could be monitored at levels under 1 μ M. 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 tune, 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.     

Fludarabine infusion potentiates arabinosylcytosine metabolism in lymphocytes of patients with chronic lymphocytic leukemia.

Our previous work has shown that incubation of K562 cells or lymphocytes from patients with advanced chronic lymphocytic leukemia (CLL) with arabinosyl-2-fluoroadenine (F-ara-A) potentiates the rate of arabinosylcytosine 5'-triphosphate (ara-CTP) synthesis during subsequent treatment with arabinosylcytosine (ara-C). To test the biochemical modulation of ara-CTP in a clinical setting, we designed a protocol to administer fludarabine (Fludara, F-ara-AMP) and ara-C in a pharmacologically directed sequence for patients with CLL refractory to conventional fludarabine therapy. ara-C was infused in seven patients with progressive CLL at a dose rate that maximizes ara-CTP accumulation (0.5 g/m2 during 2 h). Fludarabine (30 mg/m2 during 30 min) was infused 20 h later, followed by a second, identical dose of ara-C at 24 h, when the concentration of F-ara-A 5'-triphosphate (F-ara-ATP) was maximal in CLL cells. Comparison of ara-CTP pharmacokinetics in circulating CLL cells demonstrated that the ara-CTP area under the curve increased by a median of 1.5-fold (range, 1.1- to 1.7-fold) after fludarabine infusion. Plasma pharmacokinetics indicated that neither the median steady-state ara-C concentrations nor the levels of its deamination product arabinosyluracil were significantly affected by fludarabine infusion. The median rate of ara-CTP elimination was slightly faster after fludarabine treatment (t1/2, 6.7 versus 5.8 h), suggesting that catabolism of ara-CTP was not responsible for the increased ara-CTP area under the curve. The rate of ara-CTP accumulation by CLL cells after fludarabine infusion, however, was increased by a median of 1.3-fold in seven of the eight patients (range, 1.2- to 1.8-fold); the peak occurred within 1 h of the end of the infusion. In vitro incubation of leukemic lymphocytes with F-ara-A before ara-C also showed a median 1.3-fold increase in the rate of ara-CTP accumulation. Thus, infusion of fludarabine before ara-C augments ara-CTP metabolism in leukemic lymphocytes. This knowledge should be considered in the design of combination chemotherapy.     

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.     

Intracellular pharmacodynamic studies of the synergistic combination of 6-mercaptopurine and cytosine arabinoside in human leukemia cell lines

Selective combinations of purine and pyrimidine analogs increase remission rates in pediatric patients with relapsed leukemias. The combination of 6-mercaptopurine (6-MP) and cytosine arabinoside (ara-C) may exhibit synergism similar to that observed for fludarabine and ara-C and may diminish the potential for development of resistance since the two drugs are activated by separate enzymatic pathways. To determine the efficacy of the combination against human leukemia cells, we investigated the time-concentration relationships of the drugs given alone or in combination to the resultant cytotoxicity. To determine whether the combination leads to enhanced activity of deoxycytidine kinase (dCk), the rate-limiting enzyme in ara-C activation, we characterized the cellular dCk in CEM/CEM/0, CEM/CEM/ara-C/7A, and CEM/CEM/ara-C/3A monoclonal cells before and after treatment with 6-MP. CEM/CEM/0 (wild type), CEM/CEM/ara-C/7A (˜50% ara-C-resistant as determined by ara-C sensitivity assay and dCk characterization), and CEM/CEM/ara-C/3A (˜90% resistant to ara-C) human leukemia cells were incubated with various concentrations of 6-MP and ara-C given alone or in combination. Cell survival, inhibition of DNA synthetic capacity (DSC), ara-CTP anabolism, and dCk enzymatic characteristics were studied. Incubation of CEM/0 cells with 6-MP for 24 h, followed by ara-C for 48 h, increased cell-growth inhibition by approximately 0.5–1 log ₁₀, corresponding to 5- to 10-fold synergism, as compared with ara-C alone after identical drug incubation in all cell lines. Simultaneous administration showed no synergism, whereas reversal of the sequence produced an antagonistic effect. The ara-CTP levels were 2- to 3.5-fold and 3- to 5-fold higher in CEM/0 and CEM/ara-C/7A cells, respectively, in cells exposed to 6-MP followed by ara-C than in those exposed to ara-C alone at the same concentrations. Furthermore, a progressive increase in ara-CTP levels was noted in CEM/0 cells exposed to increasing concentrations of 6-MP followed by 10 or 20 µ M ara-C. A significant decrease in DSC was observed upon treatment of wild-type and ara-C-resistant cells with 6-MP and ara-C. The combination of 6-MP and ara-C exhibits significant sequence-specific synergism in both wild-type and partially ara-C-resistant leukemia cell lines. The combination also exerts collateral sensitivity in the ara-C-resistant cell lines. 6-MP pretreatment may play a role in enhancing ara-C activation, thus producing drug synergism in sensitive and resistant leukemia cell lines.     

Monitoring of intracellular 1-beta-D-arabinofuranosylcytosine 5'-triphosphate in 1-beta-D-arabinofuranosylcytosine therapy at low and conventional doses.

1-beta-D-Arabinofuranosylcytosine (ara-C) is used empirically at a low, conventional, or high dose. Ara-C therapy may be optimal if it is directed by the clinical pharmacokinetics of the intracellular active metabolite of ara-C, 1-beta-D-arabinofuranosylcytosine 5'-triphosphate (ara-CTP). However, ara-CTP has seldom been monitored during low- and conventional-dose ara-C therapies because detection methods were insufficiently sensitive. Here, with the use of our newly established method (Cancer Res., 56, 1800 -- 1804 (1996)), ara-CTP was monitored in leukemic cells from acute myelogenous leukemia patients receiving low- or conventional-dose ara-C [subcutaneous ara-C administration (10 mg / m(2) ) (3 patients), continuous ara-C infusion (20 or 70 mg / m(2) / 24 h) (7 patients), 2-h ara-C infusion (70 mg / m(2) ) (4 patients), and 2-h infusion of N(4)-behenoyl-1-beta-D-arabinofuranosylcytosine, a deaminase-resistant ara-C derivative (70 mg / m(2) ) (6 patients)]. Ara-CTP could be determined at levels under 1 microM. There was a close correlation between the elimination half-life values of the plasma ara-C and the intracellular ara-CTP. The presence of ara-C in the plasma was important to maintain ara-CTP. The continuous ara-C and the 2-h N(4)-behenoyl-1-beta-D-arabinofuranosylcytosine infusions maintained ara-CTP and the plasma ara-C longer than the subcutaneous ara-C or the 2-h ara-C infusion. They also afforded relatively higher ara-CTP concentrations, and consequently produced ara-CTP more efficiently than the 2-h ara-C infusion. Different administration methods produced different quantities of ara-CTP even at the same dose.     

Efficient formation of cytosine arabinoside-5'-triphosphate in leukemic blasts of human T-cell acute lymphoblastic leukemia

The potential usefulness of cytosine arabinoside (ara-C) in the treatment of T-cell acute lymphoblastic leukemia (T-ALL) was studied by measuring the influx of ara-C into the lymphoblasts and the conversion of ara-C to its triphosphate form (ara-CTP) in lymphoblasts from the bone marrow of eighteen patients of T-ALL. Intracellular accumulation of ara-CTP was 3.5 times greater in T-cell lymphoblasts than in non-T or AML blasts. The increased formation of ara-CTP in T-ALL cell may be correlated with the good clinical response of T-ALL patients to the treatment with ara-C in combination with mitoxantrone.     

Cellular pharmacology ofN 4-hexadecyl-1-β-D-arabinofuranosylcytosine in the human leukemic cell lines K-562 and U-937

The mechanisms of cytotoxicity, cellular drug uptake, intracellular drug distribution, cellular pharmacokinetics, formation of arabinofuranosylcytosine triphosphate (ara-CTP), and DNA incorporation ofN ⁴-hexadecyl-1--d-arabinofuranosylcytosine (NHAC), a new lipophilic derivative of arabinofuranosylcytosine (ara-C) formulated in small unilamellar liposomes, were determined in vitro in the human leukemic cell lines K-562 and U-937. Furthermore, the induction of erythroid differentiation by NHAC was tested in K-562 cells. The cytotoxicity of NHAC in both cell lines was not influenced by the deoxycytidine (dCyd) concentration or the presence of the nucleoside-transport-blocking agent dipyridamole as demonstrated in coincubations with dCyd and/or dipyridamole, where-as in contrast, the cytotoxicity of ara-C was decreased additively by both drugs. As compared with ara-C, the uptake of NHAC displayed up to 16- and 5-fold increases in K-562 and U-937 cells, respectively, depending on the drug concentration. Studies of the drug distribution and pharmacokinetics of NHAC revealed a depot effect for NHAC in the cell membranes, resulting in half-lives 2.6 and 1.4 times longer than those of ara-C in the two cell lines. The ara-CTP concentrations derived from NHAC were 150- and 75-fold lower at a drug concentration of 1 M in K-562 and U-937 cells, respectively. The DNA incorporation of the drugs observed after incubation with 2 M NHAC was 60- and 30-fold lower as compared with that seen at 2 M ara-C in the two cell lines. Furthermore, NHAC was capable of inducing irreversible erythroid differentiation to a maximum of only 22% of K-562 cells, where-as ara-C induced differentiation at a drug concentration 100-fold lower in 50% of the cells. These results indicate a mechanism of cytotoxicity for NHAC that is independent of the nucleoside transport mechanism and the phosphorylation pathway and suggest that the mechanisms of action of NHAC are significantly different from those of ara-C. Therefore, NHAC might be used for the treatment of ara-C-resistant malignancies.This work was supported by the Sassella Foundation, Stiftung für experimentelle Krebsforschung, Stiftung für Krebsbekämpfung, and the Swiss National Science Foundation (grant 32-29979.90)     

Deoxypyrimidine-induced inhibition of the cytokinetic effects of 1-β-d-arabinofuranosyluracil

Summary Ara-U-induced S-phase accumulation and the interaction between high concentrations of ara-U (HiCAU) and ara-C were investigated in L1210 leukemia cells in vitro. Treatment of exponentially growing L1210 murine leukemia cells with ara-U (200–1000 m) for 48 h caused a dose-dependent accumulation of cells in the S-phase. The extent of this ara-U-induced S-phase accumulation correlated with ara-U incorporation into DNA and with increases of up to 172% and 464% in the specific activities of deoxycytidine kinase and thymidine kinase, respectively, over control values. Metabolism of 1 m ara-C following the exposure of cells to ara-U (1mm) resulted in 4.5 pmol ara-C DNA/mg protein vs 2.1 pmol/mg protein in control cells. Although 48-h exposure of cells to 200 and 400 m ara-U is not cytotoxic, it enhances the cytotoxicity of ara-C (10–100 m) 4- to 10-fold. Ara-U-induced S-phase accumulation is inhibited by deoxypyrimidine nucleosides but not by pyrimidine or deoxypurine nucleosides. Some of the ara-U and ara-C concentrations used in this study are achievable in clinical practice, and ara-U/ara-C interactions may explain in part the unique therapeutic utility of high-dose ara-C.Abbreviations ara-C 1--d-arabinofuranosylcytosine - ara-U 1--d-arabinofuranosyluracil - ara-CTP 1--d-arabinofuranosylcytidine triphosphate - HiDAC high-dose ara-C - HiCAU high concentrations of ara-U - dCTP deoxycytidine triphosphate - HiDAU high-dose ara-U - FiTC Fluoroisothiocyanate - dUDP deoxyuridine diphosphate - dUTP deoxyuridine triphosphate - dTTP thymidine triphosphate - BrdUrd bromodeoxyuridine - dCyd kinase deoxycytidine kinase Supported in part by grant CH-35H from the American Cancer Society, by Public Health Service grant CA-12197 from the National Cancer Institute, National Institutes of Health, and by the Gaston Cancer Society     

Mechanism of synergistic cell killing by hydroxyurea and cytosine arabinoside

Synergistic cell killing of human lymphoblastic leukemia cell line, CCRF-CEM, occurred when hydroxyurea (HU) was administered before 1-beta-D-arabinofuranosylcytosine (ara-C). At the optimal dose of HU (1mM), the ara-CTP concentration increased 4-fold and the intracellular accumulation of ara-C increased 4-fold, while the dCTP concentration decreased by more than 50%. Increased intracellular accumulation of ara-C after HU treatment was also observed in human acute myelogenous leukemic cells in circulating blood. Therefore, the synergistic cell kill of HU and ara-C may be the consequence of greater inhibition of DNA polymerase by the increased level of ara-CTP in the presence of the decreased concentration of the natural substrate of this enzyme, dCTP. This synergism was not due to an increased incorporation of ara-C into DNA since the treatment of cells with HU did not enhance the ara-C incorporation into DNA but rather suppressed it.