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Biomedical subjects

W Plunkett

Publications and source records attributed to W Plunkett.

At least 145 records · Page 8Linked to original sources
PubMed1990

Inhibition of ribonucleotide reduction in CCRF-CEM cells by 2',2'-difluorodeoxycytidine.

The new deoxycytidine analogue 2',2'-difluorodeoxycytidine (dFdC) is a specific inhibitor of DNA synthesis that has marked cytotoxicity and therapeutic activity. A 2-hr incubation with 0.1-10 microM dFdC decreased cellular viability 78-97%. This treatment reduced deoxynucleoside triphosphate pools, similar to the action of the ribonucleotide reductase inhibitor hydroxyurea. The most pronounced decrease occurred in the dCTP pool, quantitatively followed by the decrease of dATP, dGTP, and dTTP. In contrast, inhibition of DNA synthesis by arabinosylcytosine did not affect the dCTP level, whereas dATP, dGTP, and dTTP pools increased, but less than 2-fold. The incorporation of [5-3H]cytidine into the dCTP pool, a measure of ribonucleotide reductase activity in whole cells, was reduced to 3% of controls by 0.1 microM dFdC, but to only 40% by 0.1 microM ara-C. Each drug decreased incorporation of [5-3H]cytidine into DNA to a similar extent (greater than 94%), suggesting limitation by a reaction proximal to this step. The cellular concentration of dFdC 5'-diphosphate was 0.3 microM at 50% inhibition of the in situ activity of ribonucleotide reductase. Direct assays of partially purified ribonucleoside diphosphate reductase (EC 1.17.4.1) demonstrated 50% inhibition by 4 microM dFdC 5'-diphosphate; dFdC 5'-triphosphate was much less inhibitory. We conclude that dFdC 5'-diphosphate acts as an inhibitor of ribonucleoside diphosphate reductase.

Cell Line↗
PubMed1990

Cellular ara-CTP pharmacokinetics, response, and karyotype in newly diagnosed acute myelogenous leukemia.

We evaluated relationships between ara-CTP pharmacokinetics in myeloblasts, response, and karyotype in 147 patients with newly diagnosed AML treated on three protocols each initiated by a 3 g/m2 ara-C dose given over 2 h. Area under the curve of ara-CTP concentration times time (AUC) or peak ara-CTP concentration after this dose did not predict response or response duration, suggesting that inability to form ara-CTP is an unlikely mechanism of ara-C resistance. Following high-dose bolus ara-C therapy patients with INV [16] or del [16q] had long remissions despite low AUC and peak values while patients with -5, del [5q], -7, or del [7q] were frequently resistant despite average AUC and peak values. In 55 patients treated with high-dose continuous infusion ara-C as a single agent, steady-state ara-CTP concentrations were significantly lower in resistant patients (who again were disproportionately those with -5, del [5q], -7, or del [7q]) although there was no correlation with CR duration.

Amsacrine↗
PubMed1989

Modulation of deoxynucleotide metabolism by the deoxycytidylate deaminase inhibitor 3,4,5,6-tetrahydrodeoxyuridine.

Tetrahydrodeoxyuridine (dTHU) inhibits deoxycytidine deaminase and, after intracellular phosphorylation to the active 5'-monophosphate, also inhibits deoxycytidylate deaminase (dCMPD). Because in vitro studies have shown that dCMPD may regulate pyrimidine deoxynucleotide metabolism, the objective of this study was to investigate the effects of dTHU on deoxynucleotide metabolism in whole cells. Nearly complete inhibition of dCMPD, measured in intact CCRF-CEM cells by incorporation of [14C]dCyd into dTTP, occurred after a 45-min incubation with 100 microM dTHU. This was accompanied by an 8-fold dCTP pool expansion, although dATP, dTTP, dGTP, and ribonucleoside triphosphate pools were unaffected. Tetrahydrouridine, which inhibits deoxycytidine deaminase exclusively, had no effect on nucleotide pools. The dCTP pool expansion was directly proportional to the dTHU concentration (3-100 microM) and reached a maximum after 2 hr. Inhibition of ribonucleotide reductase by hydroxyurea completely prevented the dTHU-induced dCTP pool expansion, indicating that the substrate of dCMPD was derived from the ribonucleotide pool and that CDP was the predominant precursor of dCTP. dTHU-mediated inhibition of dCMPD appeared reversible. Exposure of cells to 100 microM dTHU followed by washing into fresh medium resulted in a linear decrease of the dCTP pool and an increase in the dTTP pool. The increased dCTP concentration after preincubation with dTHU was associated with an inhibition of deoxycytidine kinase, as indicated by a reduced capacity of cells to phosphorylate ara-C. dTHU is a useful new tool for investigating the role of dCMPD in the regulation of deoxynucleotide metabolism in whole cells.

Cell Line↗
PubMed1989

Modulation of arabinosylcytosine metabolism by arabinosyl-2-fluoroadenine in lymphocytes from patients with chronic lymphocytic leukemia: implications for combination therapy.

Our previous studies indicated that K562 cells loaded with arabinosyl-2-fluoroadenine 5'-triphosphate (F-ara-ATP) accumulated arabinosylcytosine 5'-triphosphate (ara-CTP) at a threefold higher rate compared to the control cells. In the present study lymphocytes were obtained from patients with chronic lymphocytic leukemia before and after F-ara-A monophosphate therapy. The rate of ara-CTP accumulation after in vitro ara-C incubation was compared in lymphocytes obtained prior to therapy without any other manipulation, after ex vivo F-ara-ATP (100 mumol/L) treatment, and after in vivo F-ara-A monophosphate therapy. Lymphocytes showed a 2.2-fold (n = 23) and 1.7-fold (n = 23) median increase in the cellular concentration of ara-CTP after an ex vivo incubation with 100 mumol/L F-ara-A and 20 to 24 hours after the first dose (25 or 30 mg/m2) of F-ara-A monophosphate in vivo treatment, respectively. Although the rates of F-ara-ATP and ara-CTP accumulation varied among patients, a relationship was observed in individuals between the cellular concentration of F-ara-ATP at the beginning of the ara-C incubation and ara-CTP accumulation. These studies strongly suggest that a protocol designed to administer F-ara-A monophosphate prior to ara-C infusion will augment ara-CTP accumulation by leukemia cells.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1989

Interaction of arabinosyl nucleotides in K562 human leukemia cells.

The objective of this investigation was to evaluate the ability of arabinosyl nucleotides to modulate the cellular metabolism of different arabinosyl nucleosides in K562 cells. The maximum rate of accumulation of the respective 5'-triphosphate (TP) was observed in cells incubated with 10 microM arabinosylcytosine (ara-C), 10 microM arabinosylguanine (ara-G), 300 microM arabinosyl-2-fluoroadenine (F-ara-A), and greater than 1000 microM arabinosyladenine (ara-A). Cell extract fractionation studies demonstrated that ara-C and F-ara-A were phosphorylated by dCyd kinase, whereas ara-A was phosphorylated by dCyd kinase and Ado kinase; ara-G phosphorylation was attributed to dGuo kinase. When nucleoside kinase was rate limiting to arabinosyl nucleotide accumulation, cells preloaded with F-ara-ATP showed increased rates of ara-CTP and ara-GTP accumulation, whereas cells preloaded with ara-CTP had decreased rates of F-ara-ATP and ara-GTP accumulation. Preloading cells with ara-GTP had little effect on arabinosyl nucleoside triphosphate accumulation. F-ara-ATP accumulation was inhibited in cells containing all other arabinosyl nucleotides, whereas ara-ATP metabolism was not affected by preincubation with any other nucleoside. Cells incubated with ara-C and ara-G had a general rise in dNTP, whereas F-ara-A incubation was associated with a decrease in cellular dNTP. The differential effects of arabinosyl nucleotides and cellular metabolism of other arabinosyl nucleosides are due to phosphorylation by distinct nucleoside kinases that likely have characteristic sensitivities to cellular dNTP levels.

Arabinofuranosylcytosine Triphosphate↗
PubMed1989

Gene deletion, a mechanism of induced mutation by arabinosyl nucleosides.

9-beta-D-Arabinofuranosyl-2-fluoroadenine (F-ara-A) and 9-beta-D-arabinofuranosyladenine (ara-A) are purine nucleoside analogues which are incorporated into nucleic acids. This study demonstrates the mutagenic properties of F-ara-A and ara-A and provides evidence for mechanisms by which the arabinosyl nucleosides induce mutation. At the drug dosages that evoked exponential cell killing, F-ara-A and ara-A caused a significant increase in the number of 6-thioguanine-resistant mutants in Chinese hamster ovary cells. Southern analyses showed that 15 of 16 drug-induced mutants had lost all or part of the HPRT gene, whereas no loss of the gene was found in 4 spontaneous mutants. We conclude that both F-ara-A and ara-A induced mutation predominantly by causing deletion of genetic material. The remarkable frequency of gene deletion among these drug-induced mutations is discussed with respect to possible mechanisms of action of arabinosyl nucleosides in mutational studies.

Animals↗
PubMed1989

Patient-specific dose rate for continuous infusion high-dose cytarabine in relapsed acute myelogenous leukemia.

We hypothesized that the steady-state concentration of intracellular cytarabine 5'-triphosphate (ara-CTPss) in leukemia cells is proportional to the dose rate of cytarabine (ara-C) during continuous infusion. To evaluate this possibility, patients with acute myelogenous leukemia in relapse were treated with two sequential schedules of serially increasing ara-C dose rates over a total of 36 hours. Schedule I consisted of serial infusions of 250, 500, and 750 mg/m2 each over 12 hours. Subsequently, patients entered on schedule II received 500, 1,000, and 1,500 mg/m2 serially, each over 12 hours. Steady-state levels of ara-CTP were achieved within four hours after beginning ara-C infusion and, in separate studies of a single ara-C dose rate, were shown to be maintained beyond 36 hours. Four patients treated with schedule I and two patients treated with schedule II showed a linear dose rate-dependent increase-of ara-CTPss at all three dose rates. The cells of one patient on schedule I and two patients on schedule II had a dose rate-dependent ara-CTPss increase only over the first two dose levels, while no increase or lower ara-CTPss was observed at the third dose rate. The ara-CTPss of one patient on schedule II did not change. These results suggest that there is a proportionality between the continuous infusion dose rate of ara-C and the ara-CTPss in circulating leukemia cells within the dose range of 250 to 1,000 mg/m2 over 12 hours. This opens the possibility that pharmacologic determinations may be used to redirect the ara-C dose rate to achieve a desired ara-CTPss level in leukemia blasts during therapy.

Adult↗
PubMed1989

Fludarabine: a new agent with major activity against chronic lymphocytic leukemia.

Fludarabine was used to treat 68 patients with previously treated chronic lymphocytic leukemia (CLL). Nine (13%) patients achieved a complete remission and 30 (44%) a partial remission. The response rates for Rai stages 0 to 2, 3, and 4 were 64%, 58%, and 50% respectively. Seventeen (43%) of the 40 Rai stage 1 to 3 patients and four (19%) of the Rai stage 4 patients returned to Rai stage 0. Survival was strongly correlated with the final Rai stage achieved. The survival of the 11 partial responders with residual disease consisting only of residual bone-marrow nodules was similar to the complete responders (36+ months) and superior to the other partial response patients (16 months). The response to fludarabine was rapid, with 36 (92%) of the 39 responders having achieved at least a partial response following the first three courses. Complete responses occurred in the blood, liver, spleen, and lymph nodes in 48% to 69% of the patients. Eradication of all disease in the bone marrow occurred in only 13% of the cases. Neutropenia and thrombocytopenia occurred in 56% and 25% of evaluable courses. Major infections occurred in 9% of evaluable courses and fevers of unknown origin or minor infections in 12% of courses respectively. Myelosuppression and infection were more common in patients with initial Rai stages 3 and 4 and in nonresponding patients. Other toxicity was mild. No CNS toxicity was noted.

Antimetabolites, Antineoplastic↗
PubMed1988

Comparison of the cellular pharmacokinetics and toxicity of 2',2'-difluorodeoxycytidine and 1-beta-D-arabinofuranosylcytosine.

2',2'-Difluorodeoxycytidine (dFdC) is a new deoxycytidine analogue with good activity against human leukemic cell lines and murine solid tumors, while the activity of 1-beta-D-arabinofuranosylcytosine (ara-C) is established in experimental systems and for the treatment of human adult leukemia. This study compared the cellular metabolism and cytotoxic properties of dFdC and ara-C in Chinese hamster ovary cells. In wild-type cells, dFdC was significantly more cytotoxic than ara-C after both 4- and 18-h incubations. The 5'-triphosphate of dFdC (dFdCTP) was the major cellular metabolite (85-90%), reaching cellular concentrations up to 20-fold greater than those observed for ara-C 5'-triphosphate at equimolar concentrations of the parent drug. A deoxycytidine kinase-deficient mutant neither accumulated dFdCTP nor showed any cytotoxic response up to drug concentrations of 100 microM. The cytotoxicity of dFdC could be competitively reversed by deoxycytidine further suggesting that dFdC, like ara-C, required phosphorylation by deoxycytidine kinase for biological activity. Several explanations for the different cellular accumulation of the drug triphosphates were established: (a) nucleoside transport studies demonstrated that the membrane permeation of dFdC was 65% more rapid than that of ara-C; (b) deoxycytidine kinase had a higher affinity for dFdC (Km = 3.6 microM) than for ara-C (Km = 8.8 microM), while the Km for deoxycytidine was 1.4 microM; (c) the elimination of intracellular dFdCTP was biphasic with t1/2 alpha = 3.9 and t1/2 beta greater than 16 h while the degradation of ara-CTP was monophasic and significantly faster (t1/2 = 0.7 h). The comparatively long half-life of dFdCTP was related to the prolonged inhibition of DNA synthesis after removal of exogenous nucleoside. Together these factors contribute to the more potent cytotoxicity of dFdC compared with ara-C.

Animals↗
PubMed1988

Modulation of arabinosylnucleoside metabolism by arabinosylnucleotides in human leukemia cells.

Previous studies have indicated that deoxycytidine kinase (dCK) is requisite and rate limiting in the phosphorylation of 1-beta-D-arabinofuranosylcytosine (ara-C) and 9-beta-D-arabinofuranosyl-2-fluoroadenine (F-ara-A) on the pathway to their respective cytotoxic 5'-triphosphates. In K562 cells, the rate of triphosphate accumulation was maximal during incubation with 10 microM ara-C (35 microM/h) and 300 microM F-ara-A (102 microM/h). Under these conditions, accumulation of cellular ara-CTP plateaued at about 110 microM after 3 h, whereas in separate cultures, F-ara-ATP continued to accumulate at a linear rate to cellular concentrations greater than 500 microM after 5 h. Other laboratories have demonstrated that dCK activity in cell-free extracts was inhibited by ara-CTP. To determine whether ara-CTP exhibited the same activity in whole cells, K562 cells were preincubated with ara-C to accumulate 110 microM ara-CTP. After washing into medium containing F-ara-A, the rate of F-ara-ATP accumulation was significantly decreased (37 microM/h). However, cells loaded with F-ara-ATP exhibited an increased rate of ara-CTP accumulation (110 microM/h) that resulted in cellular ara-CTP concentrations in excess of 400 microM after 5 h. This stimulation was proportional to the cellular concentration of F-ara-ATP, achieving a maximum effect between 75 and 100 microM. Phosphorylation of ara-C by cell-free extracts supplemented with physiological levels of ribo- and deoxyribonucleoside 5'-triphosphates was stimulated by addition of F-ara-ATP. The decreased rate of accumulation of products of dCK in intact cells containing 110 microM ara-CTP suggests that this active triphosphate may limit its own synthesis and phosphorylation of other substrates. In contrast, stimulation of the accumulation of ara-CTP in cells containing F-ara-ATP suggests new possibilities for the design of combination chemotherapy regimens.

Arabinofuranosylcytosine Triphosphate↗
PubMed1988

Mitoxantrone-induced DNA damage in leukemia cells is enhanced by treatment with high-dose arabinosylcytosine.

In a phase II study, patients with chronic myelogenous leukemia in blast crisis (CML-BC) were treated with intravenous (IV) mitoxantrone (5 mg/m2 per day given over 30 min x 5 days and high-dose arabinosylcytosine (ara-C) (3 g/m2 IV q 12 h x 6). The effect of this treatment on DNA damage was studied in the leukemia cells of four patients using the alkaline elution technique modified to measure DNA in unlabeled human cells. A fluorescence assay using Hoechst 33258 dye was applied for the determination of eluted DNA. After a single infusion of mitoxantrone, neither frank nor protein-associated single-strand breaks (SSB) were observed. Even repeated treatment with mitoxantrone on 3 consecutive days did not induce significant SSB. However, after the combined sequential infusion of ara-C and mitoxantrone the DNA elution pattern changed, showing significant DNA damage. SSB remained apparent after 24 h and increased with subsequent doses of ara-C and mitoxantrone. Studies of other patients treated with ara-C alone did not reveal significant SSB (n = 5). Following mitoxantrone infusion the median peak concentrations of intracellular ara-CTP (the triphosphate of ara-C) exceeded 900 microM, a value greater than that observed in CML-BC patients receiving ara-C alone (230 microM, n = 15, P less than 0.02). The present study shows the applicability of the alkaline elution method for the assay of DNA damage in vivo. The enhanced DNA damage after combined treatment with mitoxantrone and high-dose ara-C suggests a synergistic drug effect.

Adult↗
PubMed1987

Phosphorolytic cleavage of 2-fluoroadenine from 9-beta-D-arabinofuranosyl-2-fluoroadenine by Escherichia coli. A pathway for 2-fluoro-ATP production.

2-Fluoroadenine (F-Ade) is a metabolite of 9-beta-D-arabinofuranosyl-2-fluoroadenine (F-ara-A) that may be involved in the development of toxic side effects from this anticancer drug. The liberation of F-Ade from F-ara-A has been examined in different biological systems. Extracts of Escherichia coli but not mammalian cells or tissues catalyzed the conversion of F-ara-A to F-Ade with apparent Km and Vmax values of 1350 microM and 7.7 nmol/min/mg protein respectively. This reaction depended on the presence of phosphate and was inhibited by purine nucleosides in a competitive manner, indicating that the enzyme responsible for the conversion is purine nucleoside phosphorylase. After incubation of intact bacteria with 100 microM [3H]F-ara-A, [3H]F-Ade was the same percentage of cellular radioactivity as in the medium, but it was only one-tenth the concentration of F-ara-A in the cells. In contrast, the cellular concentration of 2-fluoro-ATP was 20-fold greater than that of F-ara-A-5'-triphosphate. These results suggest that F-ara-A entered the bacteria intact and was phosphorolytically cleaved to liberate F-Ade, which would have been either anabolized to the toxic triphosphate or excreted. The latter pathway would provide a route by which F-Ade might be absorbed into the host circulation.

Adenine↗
PubMed1987

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.

Arabinofuranosylcytosine Triphosphate↗
PubMed1987

Comparison between the plasma and intracellular pharmacology of 1-beta-D-arabinofuranosylcytosine and 9-beta-D-arabinofuranosyl-2-fluoroadenine 5'-monophosphate in patients with relapsed leukemia.

The pharmacokinetics of 1-beta-D-arabinofuranosylcytosine (ara-C) and 9-beta-D-arabinofuranosyl-2-fluoroadenine (F-ara-A) and their respective 5'-triphosphates, ara-CTP and F-ara-ATP, were compared in human plasma and circulating leukemic blasts (CLB) since initial phosphorylation of ara-C and F-ara-A is catalyzed by the same enzyme, deoxycytidine kinase. These investigations were conducted in 4 patients after the first infusion of high-dose ara-C therapy (3 g/m2 i.v. infused over 2 hr) and, following the failure of each to respond, after the initial bolus of F-ara-A monophosphate (50-100 mg/m2 i.v. over 30 min) in a subsequent treatment regimen. The median terminal rate of elimination (t1/2) of F-ara-A was 8.4 hr compared to 2.2 hr for ara-C. The median t1/2 for F-ara-ATP in CLB was 12.2 hr relative to 1.9 hr for ara-CTP. To evaluate the possibility that diminished deoxycytidine kinase was a mechanism of drug resistance, the relative area under the concentration X time curve (AUC) of the active triphosphate of each prodrug in the CLB of individuals was compared. The intracellular nucleotide AUC was normalized by dividing it by the AUC of the respective nucleoside in plasma. A value of 1.0 for the resulting ratio would be expected if the accumulation and retention of F-ara-ATP and ara-CTP were identical. In these patients, however, this ratio ranged between 0.2 and 68.2. When a similar analysis was performed in vitro using four established human leukemia cell lines, a 150-fold variation was found in the normalized nucleotide AUC ratio. Thus, the metabolic characteristics of ara-CTP in CLB of patients who fail to respond to high-dose ara-C may not predict the cellular metabolism of F-ara-ATP in the same patient at a later disease stage.

Adult↗
PubMed1987

Effect of amsacrine on ara-CTP cellular pharmacology in human leukemia cells during high-dose cytarabine therapy.

The combination of high-dose cytarabine (ara-C) and amsacrine (m-AMSA) is effective treatment for relapsed adult acute leukemia. Studies were performed to determine if m-AMSA affected the pharmacokinetics of the active triphosphate ara-CTP in HL-60 and K562 cells in culture. No significant differences were observed in accumulation, rate of elimination, or total intracellular exposure to ara-CTP in cultures treated with 100 microM ara-C alone or in combination with 1 microM m-AMSA. In clinical investigations, the accumulation and retention of ara-CTP in circulating leukemic cells were studied in five patients after two serial doses of ara-C (3 g/m2 infused over 2 hours) and in six additional patients in whom the second dose of ara-C was accompanied by an infusion of m-AMSA (30 mg/m2 infused over 1 hour). While substantial differences were observed in the cellular pharmacokinetics of ara-CTP among patients, the rate of ara-CTP elimination and the total intracellular exposure to ara-CTP in individuals were remarkably similar after each ara-C infusion. Infusion of m-AMSA with the second dose of ara-C did not significantly affect the cellular pharmacokinetics of ara-CTP. These studies demonstrate the feasibility and utility of conducting investigations of the cellular pharmacology of drug-drug interactions in human leukemic cells during therapy.

Acute Disease↗