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     

Protection against fludarabine neurotoxicity in leukemic mice by the nucleoside transport inhibitor nitrobenzylthioinosine

Summary Fludarabine phosphate (F-ara-AMP, Fludara) is rapidly converted in the circulation to fludarabine (F-ara-A) and is among the most effective single agents in the treatment of chronic lymphocytic leukemia. Although current treatment protocols are well tolerated, severe neurotoxicity was a consequence of high-dose F-ara-AMP regimens used in early phase I trials against adult acute leukemia. The present study showed that in mice implanted with leukemia L1210, fatal neurotoxicity, which initially manifested as hind-limb paralysis, was a consequence of high-dose-F-ara-AMP treatment. However, the incidence of neurotoxicity was reduced by the coadministration of NBMPR-P, the 5-phosphate of nitrobenzylthioinosine, a potent inhibitor of thees equilibrative nucleoside transport (NT) system. NBTGR-P, the 5-phosphate of nitrobenzylthioguanosine (also a potent NT inhibitor) similarly prevented F-ara-AMP neurotoxicity in this experimental system. Treatment with F-ara-AMP/NBMPR-P combinations was more effective with respect to the fractional yield of cured mice than were the same treatment regimens without NBMPR-P.Abbreviations ara-A 9--d-arabinofuranosyladenine - F-ara-A 9--d-arabinofuranosyl-2-fluoroadenine (fludarabine) - F-ara-AMP fludarabine 5-monophosphate (Fludara) - NBMPR 6-[(4-nitrobenzyl)thio]-9--d-ribofuranosylpurine - NBMPR-P NBMPR 5-monophosphate - NBTGR 2-amino-6[(4-nitrobenzyl)thio]-9--d-ribofuranosylpurine - NBTGR-P NBMPR 5-monophosphate - NBdAdo-P N⁶-(4-nitrobenzyl)-9--d-2-deoxyribofuranosyladenine 5-monophosphate This study was supported by the National Cancer Institute of Canada     

9-β-D-Arabinofuranosyl-2-fluoroadenine 5′-monophosphate pharmacokinetics in plasma and tumor cells of patients with relapsed leukemia and lymphoma

Summary The pharmacokinetics of 9--D-arabinofuranosyl-2-fluoroadenine (F-ara-A) in plasma and its biologically active 5-triphosphate (F-ara-ATP) in leukemic cells obtained from the peripheral blood and bone marrow was evaluated in patients with hematologic malignancies subsequent to the first dose of 20–125 mg/m² per day for 5 days of F-ara-A 5-monophosphate (F-ara-AMP) administered as an IV bolus over 30 min. The terminal half-lives of elimination of both F-ara-A (8 h) in plasma and intracellular F-ara-ATP (15 h) were not dependent upon the dose of F-ara-AMP. The area under the concentration x time curves for F-ara-A and F-ara-ATP, on the other hand, were increased in proportion to the prodrug dose. There was a high correlation between F-ara-ATP levels in circulating leukemic cells and those in bone marrow cells aspirated at the same time. DNA-synthetic capacity of leukemic cells was inversely related to the associated F-ara-ATP concentration. A linear trend was noted when F-ara-ATP levels in pretreatment peripheral blood leukemic cells incubated with F-ara-A in vitro were compared with the amount of F-ara-A that was incorporated into nucleic acids. Finally, F-ara-ATP concentrations were three times higher in bone marrow cells from patients with lymphomatous bone marrow involvement than from those without evidence of marrow disease.Supported in part by grants CA28153 and CA32839 from the National Cancer Institute, United States Department of Health and Human Services     

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     

Mechanistic implications of alterations in HL-60 cell nascent DNA after exposure to 1-β-d-arabinofuranosylcytosine

Summary To improve our understanding of the mechanism of 1--d-arabinofuranosylcytosine (ara-C) incorporation into DNA, we investigated the physical properties (size, position of nucleoside incorporation) of small fragments of nascent DNA (nDNA) obtained by pH-step alkaline elution of intact HL-60 cells following their exposure to ara-C. In the pH-step alkaline elution procedure, the smallest fragments of nDNA elute at pH 11. Anion-exchange high-performance liquid chromatography (HPLC) of nDNA obtained by 1 h elution at pH 11.0 of lysed HL-60 cells revealed a preponderance of nDNA fragments ranging from 0.5 to 40 kb in control ([³H]-dThd-labeled) cells. Exposure of cells to ara-C (0.8–1 m) resulted in a loss of the preponderance of radiolabel in fragments of 0.5–40 kb along with redistribution of the radiolabel (from [³H]-dThd or [³H]-ara-C) into smaller nDNA fragments (predominantly <100 bases in length) as determined by HPLC. We used the ability of pH-step alkaline elution to provide these small nDNA fragments produced by ara-C to investigate the paradoxical behavior of ara-C as a chain terminator in cell-free DNA synthetic systems while being incorporated into an internucleotide position in intact cells. Following the digestion of purified nDNA with micrococcal nuclease and spleen phosphodiesterase II, the proportion of radiolabel in 3-dNMP (indicating an internucleotide position) or free nucleoside (indicating a chain terminus position) was determined by reverse-phase HPLC. In digests of prelabeled genomic DNA, as expected, <90% of the radiolabel from [¹⁴C]-dThd or [³H]-ara-C was found to exist in an internucleotide position (as determined by co-chromatography with authentic 3-dTMP or 3-ara-CMP). In contrast, digests of nDNA that eluted at pH 11.0 revealed a significantly higher proportion of radiolabel in the chain terminus position (29%–35%) when the nDNA was obtained from cells exposed to 1 m [³H]-ara-C as compared with cells exposed to [³H]-dThd or [³H]-dCyd alone (<10%). These data obtained from pH-step alkaline elution of intact cells suggest that by causing the inhibition of chain elongation while failing to inhibit the formation of new nDNA replication intermediates, ara-C exposure leads to the production of very small nDNA fragments. This relative chain-terminating effect of ara-C is most apparent in the small nDNA replication fragments that elute at pH 11.0. Since ara-C is accumulated predominantly in an internal position, even in the small nDNA fragments that elute at pH 11.0, ligation and gap-filling mechanisms in the intact cell must rapidly transform ara-C from a chain terminus to an internal position.Abbreviations ara-C 1--d-arabinofuranosylcytosine ara-CMP, 1--d-arabinofuranosylcytosine monophosphate - ara-CTP 1--d-arabinofuranosylcytosine 5-triphosphate - dNMP deoxynucleoside monophosphate - nDNA nascent DNA - dThd dCyd, 2-deoxycytosine - PBS phosphatebuffered saline (0.85% NaCl, 6.7mm potassium phosphate, pH 7.4); nuclease buffer, 0.5mm TRIS, 2mm CaCl₂, pH 8 - HPLC high-performance liquid chromatography - EDTA ethylenediaminetetraacetic acid Supported in part by grant RO-1-CA40188 from the NIH, National Cancer Institute. Presented in part at the 81st and 82nd Annual Meetings of the American Association for Cancer Research in Washington, D. C. (May 1990), and Houston, Texas (May 1991), respectively     

Phenotypic analysis of 1-B-d-arabinofuranosylcytosine deamination in patients treated with high doses and correlation with response

Summary Two phenotypes for 1-B-d-arabinofuranosylcytosine (ara-C) deamination corresponding to a ratio of distribution for slow (ratio, 14) vs fast (ratio, >14) deaminators of 70%30%, have been determined on the basis of studies on plasma ratios of 1-B-d-arabinofuranosyluracil/ara-C (ara-U/ara-C) in 56 subjects treated with high-dose ara-C (3 g/m² infused i.v. over 3 h). A positive correlation of age with the concentration of ara-U was observed. In a subgroup of 36 patients with leukemia, the ara-U/ara-C pattern was similar to that observed for all 56 subjects. In these leukemic patients, who were treated with combinations of ara-C plus other conventional agents, a tendency toward a positive response (complete response +partial response) was found for those showing low ara-U/ara-C ratios (slow deaminators). The phenotypic effect of deamination in acute leukemia needs to be evaluated prospectively.This work was supported by the Don Monti Memorial Research Foundation     

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     

Cellular metabolism of 1-β-d-arabinofuranosyl-5-azacytosine and incorporation into DNA and RNA of human lymphoid CEM/0 and CEM/dCk(−) cells

Summary 1--d-arabinosyl-5-azacytosine (ara-AC) is a relatively new antitumor agent under clinical investigation, which has the 2- arabinosyl configuration found in the tumoricidal drug ara-C and the nitrogen substitution in the 5-position of the pyrimidine ring found in 5-azacytidine (5-aza-C). The present study examined the cellular metabolism and the effect on DNA methylation of ara-AC in human CCRF/CEM cells sensitive and resistant to ara-C. The triphosphate anabolite of the drug, ara-ACTP, was the major anabolite in the CEM cellular extracts, peaking at 50.6±23 M 4 h after incubation with IC₅₀ concentrations (0.25 M) of [³H]ara-AC. The mono- and diphosphate anabolites accumulated 10-fold lower cellular concentrations than ara-ACTP. The nucleoside triphosphate (NTP) pools and, especially, cellular ATP declined significantly by 9 h after the initiation of drug treatment and remained depleted for the 24-h treatment. The drug anabolite was gradually incorporated into both RNA and DNA, peaking in CEM/0 at 3.44 and 0.14 nmol/10⁷ cells, respectively. The DNA methylation levels in these cells declined rapidly after treatment with ara-AC, attaining a nadir plateau at 29% of control methylation value. The deoxycytidine kinase (dCk) mutant CEM cell line [CEM/dCk(–)] neither activated ara-AC at appreciable levels nor induced DNA hypomethylation at low concentrations (0.25–1 M). However, the drug was activated at 0.2–1 M extracellular concentrations of ara-AC, probably by an as yet unknown nucleoside kinase at approximately 10% of the amount in CEM/0 cells. Ara-AC appears to mediate its cytotoxic action through the accumulation of its triphosphate anabolite, ara-ACTP, and the subsequent incorporation into nucleic acids. DNA methylation may also contribute to its cytotoxicity.Abbreviations 5-aza-C 5-azacytidine - ara-AC 1--d-arabinofuranosyl-5-azacytosine - ara-ACTP 1--d-arabinofuranosyl-5-azacytosine 5-triphosphate - dCk deoxycytidine kinase - PCA perchloric acid - SAX strong anion exchange - PBS phosphate-buffered saline Supported by research grant CA 38905 from the National Institute of Health, NCI, and the T. J. Martell Foundation for Leukemia and Cancer Research, The Neil Bogart Memorial Laboratories     

Epirubicin and doxorubicin comparative metabolism and pharmacokinetics

Summary The pharmacokinetics and metabolism of doxorubicin (DX) and epirubicin (epiDX) were investigated in eight cancer patients who received 60 mg/m² of both drugs independently by intravenous (i.v.) bolus at 3-week intervals according to a balanced cross-over design. Unchanged DX and epiDX plasma levels followed a triexponential decay. Half-lives (t/2) of the three decay phases were longer for DX (t/2: 4.8 vs. 3 min; t/2 2.57 h vs. 1.09 h; t/2 48.4 vs. 31.2 h). According to a model-independent analysis, the different plasma disposition kinetics of the two compounds appears to be related to a higher plasma clearance (PlCl) and to a lower mean residence time (MRT) of epiDX (PlCl: 75.0 l/h, range: 35.6–133.4 l/h; MRT: 31.6 h, range: 7.0–41.5 h;) compared to DX (PlCl: 56.8 l/h, range: 24.4–119.5; MRT: 45.6 h, range: 26.0–83.1 h). No statistically significant differences could be detected for the volume of distribution at steady state (Vss) (epiDX, 31.8 l/kg; DX, 33.3 l/kg). Metabolites common to both compounds were detected in plasma: the 13-dihydro derivatives doxorubicinol (DXol) and epirubicinol (epiDXol), together with monor amounts of four aglycones (7-deoxy adriamycinone, adriamycinone, 7-deoxy 13-dihydro adriamycinone, and 13-dihydro adriamycinone). Following epiDX administration, two additional major metabolites were detected: the glucuronic acid conjugates of epiDX (4-O--d-glucuronyl-4-epiDX) and epiDXol (4-O--d-glucuronyl 13-dihydro-4-epiDX). This additional detoxication route appears to account for the more efficient and faster elimination of epiDX than of DX. In the urine collected in the 6 days after treatment, 12.2% of the DX and 11.9% of the epiDX dose was excreted as unchanged drug and fluorescent metabolites. A comparable renal clearance was calculated for DX (4.7 l/h, range 1.4–7.0 l/h) and epiDX (4.4 l/h, range 1.7–7.0 l/h). One patient with hepatic metastates and abnormal bilirubin serum level had percutaneous biliary drainage because of extrahepatic obstruction. The elimination of both drugs was significantly impaired in this patient; nevertheless, elimination of epiDX was still more efficient and faster than that of DX (PlCl: 35.6 vs. 24.4 l/h; MRT: 39.0 vs. 83.1 h; t/2: 47 vs. 74 h). This patient's biliary excretion accounted for 35.4% of the epiDX dose and 18.2% of the DX dose.Supported in part by contract CNR, No. 85.02282.44 (Progetto Finalizzato Oncologia)     

Stereoselective metabolism of ftorafur (R,S-1-(tetrahydro-2-furanyl)-5-fluorouracil)

Summary The metabolism of R and S isomers of ftorafur (FT) was studied in vivo and in vitro. FT and its metabolites, i.e., hydroxylated derivatives, (OH-FT), -butyrolactone (GBL), 5-fluorouracil (FU), and dehydro-FT, were analyzed by gas chromatography and high-pressure liquid chromatography. Although previous results did not demonstrate any differences in the biological activity of the FT isomers, R- and S-FT were metabolized to different extents by individual pathways. Cleavage of R-FT at the N₁-C₂ position to form GBL by non-microsomal soluble enzymes in mouse and rabbit liver homogenates was greater than that of S-FT. Following IP administration of S-FT, both cis- and trans-4-OH-FT were recovered in 24-h rat urine; however, significantly less trans-4-OH-FT and no cis-4-OH-FT was detected following R_FT administration. There were no glucuronide or sulfate conjugates of these OH-FT metabolites. These results indicate that urinary hydroxylated metabolites generated from racemic FT consist predominantly of -l-4-OH-FT and -l-4-OH-FT, and only a small fraction of the trans-4-OH-FT appears in the -d configuration of natural nucleosides, confirming earlier reports. The low extent of urinary excretion of hydroxylated FT metabolites in the -configuration suggests that either there is stereoselective hydroxylation of S-FT or the hydroxylated metabolites with the natural -d configuration generated from R-FT were preferentially further metabolized prior to excretion into urine. A sample of chemically synthesized -d-4-OH-FT was quantitatively converted to FU by thymidine phosphorylase in vitro; this compound may represent a potential FU prodrug.     

The role of deoxycytidine-metabolizing enzymes in the cytotoxicity induced by 3′-amino-2′,3′-dideoxycytidine and cytosine arabinoside

Summary The cellular metabolism of 3-amino-2,3-dideoxycytidine (3-NH₂-dCyd), a cytotoxic agent previously reported to be a poor substrate for purified Cyd/dCyd deaminase (dCydD), was compared with that of cytosine arabinoside (ara-C) in cells that displayed dCydD activity (HeLa) and in cells that did not (L1210). Growth inhibition induced by 3-NH₂-dCyd was dependent on the levels of anabolic enzymes, particularly dCyd kinase (dCydK), whereas cytotoxicity induced by ara-C was dependent on the expression of both anabolic and catabolic enzyme activities. Competition kinetics using purified enzyme revealed that the binding affinity of ara-C to dCydK was 5-fold that of the amino analog. However, this binding advantage is apparently offset in cells that contain high levels of dCydD, since the K_i values for this enzyme were 0.2 and 23mm for ara-C and 3-NH₂-dCyd, respectively. This was reflected in the decrease in analog sensitivity observed between the two cell lines, whereby the concentrations of ara-C and 3-NH₂-dCyd required to inhibit growth by 50% were 200 and 7 times higher, respectively, in the dCydD-containing HeLa cells as compared with the dCydD-deficient L1210 cells. The metabolic stability and cytotoxicity of 3-NH₂-dCyd was independent of cell number. An unexpected finding was the extent to which the effectiveness of ara-C could be mitigated by the number of dCydD-containing cells. A completely cytotoxic concentration of ara-C was rendered nontoxic by a 10-fold increase in cell number. This observation was supported by an increase in I--d-arabinofuranosyluracil (ara-U) formation, a decrease in ara-C 5-triphosphate (ara-CTP) accumulation, and a rise in cell viability with increasing cell number. These findings indicate that unlike ara-C, the effectiveness of 3-NH₂-dCyd is independent of the level of deaminase, which suggests its possible utility in situations in which high levels of deaminase are manifest.Supported by grant CH-452 from the American Cancer Society     

Cellular pharmacology of 1-β-d-arabinofuranosylcytosine in human myeloid,B-lymphoid and T-lymphoid leukemic cells

Summary The in vitro inhibitory action and metabolism of 1--d-arabinofuranosylcytosine (ara-C) on human myeloid (HL-60), B-lymphoid (RPMI-8392), and T-lymphoid (Molt-3) leukemic cells was compared. Ara-C produced greater inhibitory effects in Molt-3 cells than in either HL-60 or RPMI-8392 cells. At a 48 h exposure, ara-C was 7 and 10 times more cytotoxic to Molt-3 cells than to HL-60 and RPMI-8392 cells, respectively. The total ara-C uptake to nucleotides and the formation of 1--d-arabinofuranosylcytosine 5-triphosphate (ara-CTP) was about 5 times greater in Molt-3 cells than in either HL-60 or RPMI-8392 cells. The incorporation of ara-C into DNA was also higher in Molt-3 cells than in either HL-60 or RPMI-8392 cells. The mean intracellular half-life of ara-CTP was 31.7, 59.4, and 155 min for RPMI-8392, HL-60, and Molt-3 leukemic cells, respectively. The Km and V_max values of ara-C for deoxycytidine kinase and the feedback inhibition of this enzyme by ara-CTP in the different leukemic cell lines could not explain the differences in metabolism of this analogue in these cells. These data indicate that the increased sensitivity of T-lymphoid leukemic cells to ara-C than as compared with B-lymphoid and myeloid leukemic cells was due to an ireased rate of formation and a longer half-life of ara-CTP in the T-cells.Supported by a grant from the Cancer Research Society. N.O.-P. is a Fellow of the Leukemia Society of America. To whom requests for reprints should be addressed, at Centre de recherche pédiatrique, Hôpital Ste-Justine, 3175 Ch. Côte Ste-Catherine, Montreal, Quebec H3T 1C5, Canada     

Growth inhibitory effect of recombinant α and β interferon on human glioma cells

Summary Growth inhibitory activity of recombinant and interferon on two human glioma cell lines, EFC-2 and KE cells, was determined by two different growth assays. Recombinant interferon showed slight growth inhibitory effect on EFC-2 cells at day 3, and maximum inhibition was seen on day 6 with an ID50 of 50 U/ml. Recombinant interferon showed no significant growth inhibition at any concentration. KE cells were resistant to both recombinant and interferon. The growth inhibitory activity of recombinant interferon on EFC-2 cells was not blocked by recombinant interferon, although recombinant and interferons shared same receptors on EFC-2 cells. Addition of DFMO (-difluoromethylornithine) to interferon in the media showed additive effect rather than synergistic effect in growth inhibition of glioma cells. Out of 7 glioma cell lines tested, 4 showed heterogeneous sensitivity to recombinant interferon, and all were resistant to recombinant interferon. These results suggest differential sensitivity of EFC-2 cells to recombinant interferon, as well as a heterogeneous sensitivity to recombinant interferon among different glioma cell lines.     

Phase II trial of VP16-213 in non-small cell lung cancer (NSCLC)

Summary Fifty-one patients with non-small cell lung cancer (NSCLC) were treated, during a phase II trial, with 4 demethylepipodophyllotoxin--d-ethylidene glucoside (VP16-213). Forty-nine were evaluable for response, and of these two (4%) had partial responses lasting 5 and 6 months. Prior treatment with chemotherapy may have adversely affected response rate; none of the 24 previously treated patients had a major response. Myelosuppression was the dose limiting toxicity. Anorexia, nausea and vomiting, partial alopecia, and chills plus hypotension during drug infusion were the other toxic effects. We conclude that VP16-213 has only minimal activity as a single agent in NSCLC.Supported in part by NIH Grant No. CA-05826 and CA-09027, and by NCI Contract NO-1-CM 972744Demethylepipodophyllotoxin--d-Ethylidene Glucoside (NSC141540) was supplied by the Drug Evaluation Branch of the National Cancer Institute     

Clinical pharmacology of N4-palmitoyl-1-β-d-arabinofuranosylcytosine in patients with hematologic malignancies

Summary The pharmacokinetics of oral N⁴-palmitoyl-1--d-arabinofuranosylcytosine (PLAC), a lipophilic and deaminase-resistant derivative of 1--d-arabinofuranosylcytosine (ara-C), were determined in patients with hematologic malignancies. The concentration of ara-C and 1--d-arabinofuranosyluracil (ara-U), metabolites of PLAC, were measured by radioimmunoassay and gas chromatography-mass spectrometry-mass fragmentography, respectively. The concentration of PLAC was determined by measuring ara-C, which was derived from PLAC by hydrolyzation. In six patients given an oral bolus of PLAC (300 mg/m²), the plasma-disappearance curve of PLAC corresponded to a one-compartment open model, including first-order absorption. The peak plasma level was 22.9±6.4 ng/ml, and the predicted time to reach the peak level was 2.5±1.0 h. The elimination half-life was 3.8±2.7 h. The plasma ara-C level increased slowly to 6.9 ng/ml during the 1st 2–3 h after administration and remained over 1.0 ng/ml for 12 h. Plasma ara-U was detectable for at least 24 h, with a peak concentration of 376 ng/ml at 6 h. Urinary PLAC excretion was below the limit of detection (5 ng/ml) in all cases. Prolonged urinary ara-C and ara-U excretion was detected, but the total recovery rate was low (6.7% in 24 h) and varied between patients. In spite of the lipophilic nature of the drug, the PLAC concentration in the cerebrospinal fluid, measured at 3 or 6 h, was below the limit of detection in all four patients with no meningeal involvement. This study showed low but persistent levels of PLAC in plasma and tissues, with a continuous release of small amounts of ara-C, which demonstrated antitumor activity in patients with hematologic malignancies.This study was supported in part by Grants-in-Aid from the Ministry of Health and Welfare (62-18 and 63-3), Japan     

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     

Characteristics of in vitro antiproliferation activity of human interferon-β

Summary We compared the in vitro antiproliferative activity of highly purified interferon (IFN)- (>10⁷ U protein/mg in antiviral activity) with that of IFNs- and lymphoblastoid, using human cells of malignant and non-malignant origin. IFN- was the least active of three IFNs in suppressing Daudi cell proliferation. Three hematological cells other than Daudi cells cultivated in suspension were insensitive to each of three IFNs. IFN- was more active than IFNs- and lymphoblastoid in suppressing all eight epithelioid cells tested and, particularly with respect to five epithelioid cells sensitive to IFN, IFN- was seven to 49 times as active as IFN-. These results indicate that suppression of cell proliferation by IFN depends not only on the target cell species but also on the IFN species, and emphasize the need for careful selection of the most appropriate IFN species in therapy.We found that the antiproliferative characteristics of the present IFN- preparation were consistent with those reported previously, supporting the idea that IFN- molecules in the present preparation were responsible for suppressing cell proliferation. The antiproliferation activity of our preparation was species-specific but not selective for cells of malignant origin; it was absorbable by IFN-sensitive but not by IFN-insensitive cells; and it was achieved by a cytostatic effect.     

Potentiation of the antitumor activity of 5-trifluoromethyl-2′-deoxyuridine by the use of depot forms of the parent compound

Summary 5-Trifluoromethyl-2-deoxyuridine (CF₃dUrd), an antitumor agent, is known to be short-lived in human plasma. Since its rapid elimination from the blood-stream seems to have descouraged the clinical evaluation of this drug, we explored the potential use of masked derivatives of CF₃dUrd as depot forms of the parent compound. First, we observed that the toxicity of CF₃dUrd against HeLA cells in culture was 10⁴ times greater for a 24-h treatment as compared with a 1-h treatment at identical concentrations of the drug, which suggests the importance of using a prolonged treatment period. In fact, the divided dosing of CF₃dUrd to L1210-bearing mice was markedly more effective than its single administration. 5-O-Hexanoyl-,N ³-p-butylbenzoyl-, 5-O-benzyloxymethyl-, and 3-O-benzyl-CF₃dUrd were found to be effective in maitaining the CF₃dUrd concentration in plasma. The oral doses of these agents required to achieve 50% growth inhibition (ED₅₀) in mice bearing sarcoma 180 tumors were 19, 34, 10, and 13 mg kg^–1 day^–1, respectively, whereas that of CF₃dUrd was 63 mg kg^–1 day^–1. The ED₅₀ values for these compounds were inversely correlated with the residence time of CF₃dUrd in plasma. The therapeutic indices of these compounds, calculated as the dose producing a 50% inhibition of body-weight gain (IB₅₀) divided by the ED₅₀ value (1.89, 1,21, 1.40, and 2.15, respectively), were significantly higher than that of CF₃dUrd (0.78). Consequently, these depot forms of CF₃dUrd, particularly 3-O-benzyl-CF₃dUrd, are expected to be more useful than the parent compound as antitumor agents.Abbreviations CF₃dUrd 5-trifluoromethyl-2-deoxyuridine - CF₃dUMP 5-trifluoromethyl-2-deoxyuridine-5-monophospate - S180 sarcoma 180 - L1210 L1210 leukemia - k_el elimination rate constant - T1/2 half-life time - AUC area under the curve - ILS increase in life span - TS thymidylade synthase - FdUMP 5-fluoro-2-deoxyuridine-5 monophosphate - FUra 5-fluorouracil     

Saturation of 2′, 2′-difluorodeoxycytidine 5′-triphosphate accumulation by mononuclear cells during a phase I trial of gemcitabine

Summary The plasma and cellular pharmacology of 2, 2-difluorodeoxycytidine (dFdC, Gemcitabine) was studied during a phase I trial. The steady-state concentration of dFdC in plasma was directly proportional to the dFdC dose, which ranged between 53 and 1,000 mg/m² per 30 min. The cellular pharmacokinetics of an active metabolite, dFdC 5-triphosphate (dFdCTP), were determined in mononuclear cells of 22 patients by anion-exchange highpressure liquid chromatography. The rate of dFdCTP accumulation and the peak cellular concentration were highest at a dose rate of 350 mg/m² per 30 min, during which steady-state dFdC levels of 15–20 M were achieved in plasma. A comparison of patients infused with 800 mg/m² over 60 min with those receiving the same dose over 30 min demonstrated that the dFdC steady-state concentrations were proportional to the dose rate, but that cellular dFdCTP accumulation rates were similar at each dose rate. At the lower dose rate, the AUC for dFdCTP accumulation was 4-fold that observed at the higher dose rate. Consistent with these observations, the accumulation of dFdCTP by mononuclear cells incubated in vitro was maximal at 10–15 M dFdC. These studies suggest that the ability of mononuclear cells to use dFdC for triphosphate formation is saturable. In the design of future protocols, a dose rate should be considered that produces maximal nucleotide analogue formation, with increased intensity being achieved by prolonging the duration of infusion.Abbreviations ara-C I--d-arabinosylcytosine - ara-C _ss steady-state concentration of ara-C - ara-CTP 5-triphosphate of ara-C - dFdC 2, 2-difluorodeoxycytidine, Gemcitabine - dFdC _ss steady-state concentration of dFdC - dFdCTP 5-triphosphate of dFdC Supported in part by grants CA28596 and CA32839 from the National Cancer Institute, Department of Health and Human Services, and by grant CH-130 from the American Cancer Society     

Coexpression of c-kit and stem cell factor in breast cancer results in enhanced sensitivity to members of the EGF family of growth factors

Insulinlike growth factor (IGF)I protects many cell types from apoptosis. As a result, it is possible that IGFIresponsive cancer cells may be resistant to apoptosisinducing chemotherapies. Therefore, we examined the effects of IGFI on paclitaxel and doxorubicininduced apoptosis in the IGFIresponsive breast cancer cell line MCF7. Both drugs caused DNA laddering in a dosedependent fashion, and IGFI reduced the formation of ladders. We next examined the effects of IGFI and estradiol on cell survival following drug treatment in monolayer culture. IGFI, but not estradiol, increased survival of MCF7 cells in the presence of either drug. Cell cycle progression and counting of trypanblue stained cells showed that IGFI was inducing proliferation in paclitaxeltreated but not doxorubicintreated cells. However, IGFI decreased the fraction of apoptotic cells in doxorubicin but not paclitaxeltreated cells. Recent work has shown that mitogenactivated protein kinase (MAPK) and phosphotidylinositol3 (PI3) kinase are activated by IGFI in these cells. PI3 kinase activation has been linked to antiapoptotic functions while MAPK activation is associated with proliferation. We found that IGFI rescue of doxorubicininduced apoptosis required PI3 kinase but not MAPK function, suggesting that IGFI inhibited apoptosis. In contrast, IGFI rescue of paclitaxelinduced apoptosis required both PI3 kinase and MAPK, suggesting that IGFImediated protection was due to enhancement of proliferation. Therefore, IGFI attenuated the response of breast cancer cells to doxorubicin and paclitaxel by at least two mechanisms: induction of proliferation and inhibition of apoptosis. Thus, inhibition of IGFI action could be a useful adjuvant to cytotoxic chemotherapy in breast cancer.