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Biomedical subjects

W Plunkett

Publications and source records attributed to W Plunkett.

At least 127 records · Page 7Linked to original sources
PubMed1992

Membrane-permeable dideoxyuridine 5'-monophosphate analogue inhibits human immunodeficiency virus infection.

2',3'-Dideoxyuridine (ddU) is ineffective at controlling human immunodeficiency virus type 1 (HIV-1) infection in human T cells, because it is not biotransformed to the active 5'-triphosphate. The metabolic block resides in the poor substrate affinity of ddU for cellular nucleoside kinases. This problem cannot be overcome by supplying the preformed nucleotides, because such compounds are unable to penetrate cells. To circumvent the requirement of ddU for enzymic phosphorylation, we have prepared bis(pivaloyloxymethyl) 2',3'-dideoxyuridine 5'-monophosphate (piv2 ddUMP), as a potential membrane-permeable prodrug of ddUMP, and investigated its metabolism and anti-HIV activity in two human T cell lines, one with wild-type thymidine kinase activity (MT-4) and the other deficient in thymidine kinase activity (CEM-tk-). The 5'-mono-, di-, and triphosphates of ddU were formed in both cell lines after exposure to piv2-ddUMP. In contrast, phosphorylated metabolites were not observed in cells treated with ddU or ddUMP alone. piv2-ddUMP also reduced the cytopathic effects of HIV-1 in MT-4 cells (ED50, 4.75 microM) and inhibited virus production in culture fluid (ED50, 20 microM). In addition, piv2-ddUMP protected CEM-tk- cells from HIV-1 infection, as demonstrated by inhibition of intracellular p24 antigen levels (ED50, 3 microM) and reverse transcriptase activity in culture medium (Ed50, 2.5 microM). Based on these findings, we propose that the "masked nucleotide" strategy may make available for development nucleoside analogues hitherto considered inactive because of failure to undergo biotransformation to the corresponding 5'-monophosphates. Moreover, by circumventing metabolic dependency on nucleoside kinases, the strategy may overcome acquired resistance to nucleoside analogues caused by the loss or depletion of nucleoside kinases.

Acquired Immunodeficiency Syndrome↗
PubMed1991

Action of 2',2'-difluorodeoxycytidine on DNA synthesis.

The action of the new deoxycytidine analogue 2',2'-difluorodeoxycytidine (dFdC) on DNA synthesis was investigated in whole cells and in vitro assay systems with purified DNA polymerases. DNA synthesis in human lymphoblastoid CEM cells was inhibited by dFdC in a concentration-dependent manner that could not be reversed by exogenous deoxynucleosides. The analogue was incorporated into cellular DNA; most of the incorporated dFdC 5'-monophosphate (dFdCMP) residues were in internucleotide linkage. In vitro DNA primer extension assays demonstrated that dFdC 5'-triphosphate (dFdCTP) competed with deoxycytidine triphosphate for incorporation into the C sites of the growing DNA strand. The ratios of the apparent Km values for the incorporation of dFdCTP and dCTP into a C site of M13mp19 DNA were 21.8 and 22.9 for DNA polymerases alpha and epsilon, respectively. The apparent Ki values of dFdCTP were 11.2 microM for DNA polymerase alpha and 14.4 microM for polymerase epsilon. After dFdCMP incorporation, the primer was extended by one deoxynucleotide before a major pause in the polymerization process was observed. This was in contrast to the action of arabinosylcytosine 5'-triphosphate, which caused both DNA polymerases alpha and epsilon to pause at the site of incorporation. The 3'----5' exonuclease activity of DNA polymerase epsilon was essentially unable to excise nucleotides from DNA containing dFdCMP at either the 3'-end or at an internal position, whereas arabinosylcytosine monophosphate was removed from the 3'-terminus at 37% the rate for deoxynucleotides. The cytotoxic activity of dFdC was strongly correlated with the amount of dFdCMP incorporated into cellular DNA. Our results demonstrate qualitative and quantitative differences in the molecular actions of dFdC and arabinosylcytosine on DNA metabolism, but are consistent with an important role for such incorporation in the toxicity of dFdC.

Antimetabolites, Antineoplastic↗
PubMed1991

A sensitive fluorescence assay for quantitation of fludarabine and metabolites in biological fluids.

Rapid and quantitative dephosphorylation of the new anticancer nucleotide analogue fludarabine phosphate to its nucleoside 9-beta-D-arabinofuranosyl-2-fluoroadenine (F-ara-A) renders this metabolite the target for pharmacologic investigations. At clinically effective doses of fludarabine phosphate (18-30 mg/m2 per day) comprehensive pharmacokinetic analysis of F-ara-A has been limited by the sensitivity of UV based HPLC assays. To address this problem we developed a sensitive test based on the condensation of F-ara-A with chloroacetaldehyde to form the fluorescent derivative, arabinosyl-1,N6-etheno-isoguanine. Combined with a solid-phase extraction step prior to derivatization and separation of the reaction products by reverse-phase HPLC, this assay had a quantitation limit of 2 pmol F-ara-A per ml plasma. Slightly modified, the system was also applicable to urine specimens, with a quantitation limit of 1 nmol F-ara-A per ml urine.

Antimetabolites, Antineoplastic↗
PubMed1991

Plasma and cerebrospinal fluid pharmacokinetics of 1-beta-D-arabinofuranosylcytosine and 1-beta-D-arabinofuranosyluracil following the repeated intravenous administration of high- and intermediate-dose 1-beta-D-arabinofuranosylcytosine.

We examined the plasma and cerebrospinal fluid (CSF) pharmacokinetics of 1-beta-D-arabinofuranosylcytosine (ara-C) and 1-beta-D-arabinofuranosyluracil (ara-U) in 19 patients with acute leukemia in order to determine whether ara-C or ara-U accumulate in these fluid compartments over time. Plasma and CSF samples were obtained just prior to the conclusion of the first and seventh, and immediately before the second and eighth, 2-h, twice-daily i.v. infusions of 3 g/m2/dose of ara-C (n = 10), 2 g/m2/dose of ara-C (n = 3), and 0.75 g/m2/dose of ara-C (n = 6). There was no accumulation of ara-C in the plasma or CSF, or of ara-U in the plasma following repeated ara-C infusions, ara-U did accumulate in the CSF; the end-dose 1/end-dose 7 CSF ara-U ratio was 0.35 +/- 0.12 and significantly different from this ratio for CSF ara-C (2.10 +/- 3.01; P = 0.004). The end-dose 7 CSF ara-U level was greater than the end-dose 1 CSF ara-U level in all paired specimens. There was a significant correlation between the dose of ara-C administered and the end-dose plasma ara-C and the end-dose CSF ara-U levels (P less than 0.02). One patient who received 3 g/m2/dose of ara-C developed neurotoxicity; his plasma and CSF ara-C and ara-U levels were not extraordinary during the period of ara-C administration, but he had persistent CSF ara-U demonstrable 75 h after his final ara-C dose. CSF ara-U accumulation might underlie the pathophysiology of ara-C-induced neurotoxicity. Intermediate doses of ara-C given i.v. (0.75 g/m2/dose over 2 h) appeared to generate therapeutic CSF ara-C levels and cleared CSF leukemia in one patient.

Adult↗
PubMed1991

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

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 high-pressure 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 microM 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 microM 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.

Antimetabolites, Antineoplastic↗
PubMed1991

Metabolism and action of purine nucleoside analogs.

Recent investigations have identified many new purine nucleoside analogs that act as antimetabolites. This article focuses on the metabolism and mechanisms of action of tiazofurin, 3-deazaguanosine, neplanocin A, arabinosyladenine in combination with inhibitors of adenosine deaminase, arabinosyl-2-fluoroadenine, and 2-chloro-2'-deoxyadenosine, drugs that are either currently being evaluated in clinical trials or are close to that stage. The diverse metabolic requirements for activation, unique mechanisms of action, and differential biological activities of these compounds are characterized and evaluated for prospective therapeutic application.

2-Chloroadenosine↗
PubMed1991

Action of 9-beta-D-arabinofuranosyl-2-fluoroadenine on RNA metabolism.

The action of 9-beta-D-arabinofuranosyl-2-fluoroadenine (F-ara-A) on RNA metabolism was evaluated both in whole cells and in cell-free systems. F-ara-A was converted to its 5'-triphosphate, F-ara-ATP, in cells and then incorporated into RNA as well as DNA. F-ara-A inhibited RNA synthesis in cultured cells in a concentration-dependent manner. This inhibition was mediated mainly by F-ara-ATP. Experiments using isolated nuclei demonstrated that RNA polymerases I, II, and III accounted for 24, 73, and 3% of the total RNA synthesis activity, respectively. About 88% of the total inhibition was attributed to the suppression of RNA polymerase II activity. In cultured cells, F-ara-A was preferentially incorporated into the poly(A)+ RNA fraction. Approximately 78% of the incorporated F-ara-A monophosphate residues were located at the terminal position of the RNA chain. The incorporation of F-ara-A monophosphate into mRNA resulted in premature termination of the RNA transcript and impaired its functioning as a template for protein synthesis. The inhibitory action of F-ara-A on RNA metabolism is a unique property of this compound, differing from the action of arabinosylcytosine and arabinosyladenine.

Antineoplastic Agents↗
PubMed1990

Pharmacologically directed design of the dose rate and schedule of 2',2'-difluorodeoxycytidine (Gemcitabine) administration in leukemia.

The objective of this study was to determine the dose rate of 2',2'-difluorodeoxycytidine (dFdC) that maximizes the accumulation of the active 5'-triphosphate (dFdCTP) in circulating leukemia cells during therapy. The investigational approach was to evaluate the relationship between plasma dFdC and the accumulation of dFdCTP by circulating leukemia cells during infusion of different dFdC dose rates in the same individuals. Four patients with relapsed leukemia were treated weekly with two or three consecutive infusions of 800 mg/m2, the first administered over 1 h, the second over 2 h, and the third over 3 h. Two patients, one with acute myelogenous leukemia and one with acute lymphocytic leukemia, received all three infusions, but thrombocytopenia prohibited infusion of the third dose to two patients with chronic lymphocytic leukemia. The average steady-state plasma dFdC levels, achieved within 15 min after the infusion began, were 43.8 microM during infusion of 800 mg/m2/h, 9.4 microM during infusion of 400 mg/m2/h, and 5.6 microM at 267 mg/m2/h. The median area under the concentration times time curve of dFdCTP in leukemia cells during infusion was increased 2.3- and 5.1-fold for the 2- and 3-h infusions, respectively. In vitro incubations of leukemia cells from the four patients with 2.5-100 microM dFdC for 1 h showed that the maximum cellular accumulation of dFdCTP was produced by 15-20 microM dFdC. We conclude that a dose rate of greater than 400 mg/m2/h was required to achieve plasma dFdC levels that supported the maximum rate of dFdCTP accumulation in leukemia cells.

Adult↗
PubMed1990

Termination of DNA synthesis by 9-beta-D-arabinofuranosyl-2-fluoroadenine. A mechanism for cytotoxicity.

The action of 9-beta-D-arabinofuranosyl-2-fluoroadenine (F-ara-A) on DNA synthesis was evaluated both in whole cells and in vitro. 9-beta-D-Arabinofuranosyl-2-fluoroadenine was converted to its 5'-triphosphate 9-beta-D-arabinofuranosyl-2-fluoroadenine 5'-triphosphate (F-ara-ATP) in cells and then incorporated into DNA in a self-limiting manner. More than 94% of the analogue was incorporated into DNA at the 3' termini, indicating a chain termination action. In vitro DNA primer extension experiments further revealed that F-ara-ATP compared with dATP for incorporation into the A site of the extending DNA strand. The incorporation of F-ara-AMP into DNA resulted in termination of DNA strand elongation. Human DNA polymerase alpha incorporated more F-ara-AMP into DNA than polymerase epsilon (proliferating cell nuclear antigen-independent DNA polymerase delta) and was more sensitive to inhibition by F-ara-ATP. On the other hand, DNA polymerase epsilon was able to excise the incorporated F-ara-AMP from DNA in vitro. The incorporation of F-ara-AMP into DNA was linearly correlated both with inhibition of DNA synthesis and with loss of clonogenicity; thus it may be the mechanism of cytotoxicity.

Aphidicolin↗
PubMed1990

Selective action of 3'-azido-3'-deoxythymidine 5'-triphosphate on viral reverse transcriptases and human DNA polymerases.

The action of 3'-azido-3'-deoxythymidine 5'-triphosphate (N3dTTP) on DNA strand elongation catalyzed by human immunodeficiency virus type 1 reverse transcriptase was evaluated in comparison with human DNA polymerase alpha and proliferating cell nuclear antigen-independent DNA polymerase delta. Sequencing gel analysis demonstrated that the human immunodeficiency virus 1 reverse transcriptase preferentially incorporated N3dTTP into the T sites of the growing DNA strands and caused chain termination in a dose-dependent manner. This effect was observed even when the N3dTTP concentration was 0.3 microM, 100-fold less than dTTP. Studies with reverse transcriptases from avian myeloblastosis virus and Moloney murine leukemia virus showed that N3dTTP was also efficiently incorporated into DNA by these enzymes and terminated DNA strand elongation. In contrast, human DNA polymerases alpha and delta did not incorporate detectable amounts of N3dTTP into the DNA and were not inhibited by 300 microM N3dTTP. The selective incorporation of the chain-terminating nucleotide by the viral reverse transcriptases appears to be a molecular basis for the positive therapeutic index of 3'-azido-3'-deoxythymidine.

Antiviral Agents↗
PubMed1990

Modulatory activity of 2',2'-difluorodeoxycytidine on the phosphorylation and cytotoxicity of arabinosyl nucleosides.

This investigation analyzed the metabolism of 2',2'-difluorodeoxycytidine (dFdC) in K562 human leukemia cells and evaluated it as a biochemical modulator for the phosphorylation of several arabinosyl nucleosides. The rate of accumulation of dFdC triphosphate was linear up to 3 h and maximal during incubation with 10 microM dFdC (92 microM/h). Deoxynucleotides analyzed at this time showed a decrease in dCTP, dATP, and dGTP levels, indicating an inhibitory role of dFdC nucleotides in ribonucleotide reduction. We evaluated the hypothesis that dFdC-mediated deoxyribonucleoside triphosphate perturbation enhances the phosphorylation of substrates that use deoxycytidine kinase or deoxyguanosine kinase, because these enzymes are inhibited by dCTP or dGTP, respectively. When the activity of these nucleoside kinases was rate limiting to triphosphate formation, the accumulation of triphosphates of deoxycytidine, 1-beta-D-arabinofuranosylcytosine, and 1-beta-D-arabinofuranosylguanine was potentiated in cells pretreated with dFdC. In contrast, the phosphorylation of 9-beta-D-arabinofuranosyladenine was not affected, since it is mainly phosphorylated by adenosine kinase, which is not influenced by deoxyribonucleoside triphosphates. Treatment of cells with dFdC followed by 1-beta-D-arabinofuranosylcytosine resulted in greater cytotoxicity than sum effects of each drug alone. The data indicate that an enhanced cytotoxicity could be obtained by administering dFdC as a modulator followed by 1-beta-D-arabinofuranosylcytosine or 1-beta-D-arabinofuranosylguanine in optimal sequence, suggesting that these results should be considered in the design of combination clinical protocols.

Arabinonucleosides↗
PubMed1990

Pharmacologically directed design of leukemia therapy.

The ability to accumulate and retain the active metabolite of Ara-C varies widely among patients. Our studies demonstrate a significant correlation between clinical response and the pharmacokinetics of Ara-CTP in leukemia cells during therapy. Knowledge of the cellular pharmacology of Ara-CTP has been used to optimize dose rates and to design combination treatment schedules. An understanding of the cellular pharmacodynamics of other drugs is likely to be a useful parameter for planning treatment protocols.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1990

Metabolism and action of fludarabine phosphate.

Fludara I.V. (fludarabine phosphate) (9-beta-D-arabinosyl-2-fluoroadenine, F-ara-A) is an adenine nucleoside analogue resistant to adenosine deaminase that shows promising therapeutic activity in the clinical treatment of lymphocytic hematologic malignancies. F-ara-A is transported into cells, where it is converted to its 5'-triphosphate (F-ara-ATP), the principal active metabolite. Deoxycytidine kinase is the enzyme responsible for the initial step of this activation metabolism. The differential transport and phosphorylation of F-ara-A and accumulation of F-ara-ATP by normal and cancer cells may constitute the metabolic basis of its positive therapeutic index. The major action of F-ara-A is the inhibition of DNA synthesis. F-ara-ATP competes with deoxyadenosine triphosphate for incorporation into the A sites of the elongating DNA strand by DNA polymerases and terminates DNA synthesis at the incorporation sites. That action is potentiated by the decrease of cellular dATP that results from inhibition of ribonucleotide reductase by F-ara-ATP. In vitro experiments demonstrated that DNA polymerase delta is able to excise the incorporated F-ara-AMP residues from DNA with its 3' to 5' exonuclease activity. The terminal incorporation of F-ara-AMP into DNA results in deletion of genetic material. That mechanism may be responsible for the observed mutagenicity of Fludara I.V., and ultimately its cytotoxic action.

Animals↗