RESUMEN

Thiarabine has demonstrated exceptional antitumor activity against numerous human tumor xenografts in mice, being superior to gemcitabine, clofarabine, or cytarabine. Unlike cytarabine, thiarabine demonstrated excellent activity against solid tumor xenografts, suggesting that this agent has the kind of robust activity in animal models that leads to clinical utility. Thiarabine is effective orally (bioavailability of approximately 16%) and with once per day dosing: Two characteristics that distinguish it from cytarabine. Although both the structure and basic mechanism of action of thiarabine are similar to that of cytarabine, there are many quantitative differences in the biochemical pharmacology of these two agents that can explain the superior antitumor activity of thiarabine. Two important attributes are the long retention time of the 5'-triphosphate of thiarabine in tumor cells and its potent inhibition of DNA synthesis. The biochemical pharmacology of thiarabine is also different from that of gemcitabine. Thiarabine has been evaluated in three phase I clinical trials, where it has demonstrated some activity in heavily pretreated patients with hematologic malignancies and solid tumors. Because of its impressive activity against numerous human tumor xenografts in mice, its unique biochemical activity, and encouraging clinical results in phase I clinical trials, we believe thiarabine should continue to be evaluated in the clinic for treatment of hematologic and/or solid tumors. The preclinical results to date (superior in vivo antitumor activity, oral bioavailability, and once per day dosing), suggest that thiarabine could replace cytarabine in the treatment of acute myelogenous leukemia.

Asunto(s)

Antineoplásicos/química , Arabinonucleósidos/química , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Arabinonucleósidos/farmacología , Arabinonucleósidos/uso terapéutico , Supervivencia Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Farmacorresistencia Bacteriana , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Neoplasias/patología , Nucleósidos/química , Nucleósidos/farmacología , Nucleósidos/uso terapéutico , Trasplante Heterólogo

RESUMEN

BACKGROUND: The use of Escherichia coli purine nucleoside phosphorylase (PNP) to activate fludarabine has demonstrated safety and antitumor activity during preclinical analysis and has been approved for clinical investigation. PATIENTS AND METHODS: A first-in-human phase I clinical trial (NCT 01310179; IND 14271) was initiated to evaluate safety and efficacy of an intratumoral injection of adenoviral vector expressing E. coli PNP in combination with intravenous fludarabine for the treatment of solid tumors. The study was designed with escalating doses of fludarabine in the first three cohorts (15, 45, and 75 mg/m(2)) and escalating virus in the fourth (10(11)-10(12) viral particles, VP). RESULTS: All 12 study subjects completed therapy without dose-limiting toxicity. Tumor size change from baseline to final measurement demonstrated a dose-dependent response, with 5 of 6 patients in cohorts 3 and 4 achieving significant tumor regression compared with 0 responsive subjects in cohorts 1 and 2. The overall adverse event rate was not dose-dependent. Most common adverse events included pain at the viral injection site (92%), drainage/itching/burning (50%), fatigue (50%), and fever/chills/influenza-like symptoms (42%). Analysis of serum confirmed the lack of systemic exposure to fluoroadenine. Antibody response to adenovirus was detected in two patients, suggesting that neutralizing immune response is not a barrier to efficacy. CONCLUSIONS: This first-in-human clinical trial found that localized generation of fluoroadenine within tumor tissues using E. coli PNP and fludarabine is safe and effective. The pronounced effect on tumor volume after a single treatment cycle suggests that phase II studies are warranted. CLINICALTRIALSGOV IDENTIFIER: NCT01310179.

Asunto(s)

Escherichia coli/enzimología , Terapia Genética , Vectores Genéticos/uso terapéutico , Neoplasias/genética , Neoplasias/terapia , Purina-Nucleósido Fosforilasa/administración & dosificación , Vidarabina/análogos & derivados , Adenoviridae/genética , Anciano , Anciano de 80 o más Años , Terapia Combinada , Relación Dosis-Respuesta a Droga , Femenino , Estudios de Seguimiento , Humanos , Inyecciones Intralesiones , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Neoplasias/patología , Pronóstico , Purina-Nucleósido Fosforilasa/genética , Células Tumorales Cultivadas , Vidarabina/uso terapéutico

RESUMEN

The use of E. coli purine nucleoside phosphorylase (PNP) to activate prodrugs has demonstrated excellent activity in the treatment of various human tumor xenografts in mice. E. coli PNP cleaves purine nucleoside analogs to generate toxic adenine analogs, which are activated by adenine phosphoribosyl transferase (APRT) to metabolites that inhibit RNA and protein synthesis. We created tumor cell lines that encode both E. coli PNP and excess levels of human APRT, and have used these new cell models to test the hypothesis that treatment of otherwise refractory human tumors could be enhanced by overexpression of APRT. In vivo studies with 6-methylpurine-2'-deoxyriboside (MeP-dR), 2-F-2'-deoxyadenosine (F-dAdo) or 9-ß-D-arabinofuranosyl-2-fluoroadenine 5'-monophosphate (F-araAMP) indicated that increased APRT in human tumor cells coexpressing E. coli PNP did not enhance either the activation or the anti-tumor activity of any of the three prodrugs. Interestingly, expression of excess APRT in bystander cells improved the activity of MeP-dR, but diminished the activity of F-araAMP. In vitro studies indicated that increasing the expression of APRT in the cells did not significantly increase the activation of MeP. These results provide insight into the mechanism of bystander killing of the E. coli PNP strategy, and suggest ways to enhance the approach that are independent of APRT.

Asunto(s)

Adenina Fosforribosiltransferasa/metabolismo , Escherichia coli/enzimología , Profármacos/farmacología , Purina-Nucleósido Fosforilasa/metabolismo , Animales , Línea Celular Tumoral , Escherichia coli/metabolismo , Terapia Genética , Vectores Genéticos/genética , Humanos , Ratones , Profármacos/uso terapéutico , Nucleósidos de Purina/metabolismo , Trasplante Heterólogo , Fosfato de Vidarabina/análogos & derivados , Fosfato de Vidarabina/metabolismo

RESUMEN

Gene transfer of the Escherichia coli purine nucleoside phosphorylase (PNP) results in potent cytotoxicity after administration of the prodrug fludarabine phosphate (F-araAMP). Here, we have tested whether application of this strategy in the context of replication-competent retrovirus (RCR) vectors, which can achieve highly efficient tumor-restricted transduction as well as persistent expression of transgenes, would result in effective tumor inhibition, or, alternatively, would adversely affect viral replication. We found that RCR vectors could achieve high levels of PNP expression concomitant with the efficiency of their replicative spread, with significant cell killing activity in vitro and potent therapeutic effects in vivo. In U-87 xenograft models, replicative spread of the vector resulted in progressive transmission of the PNP transgene, as evidenced by increasing PNP enzyme activity with time after vector inoculation. On F-araAMP administration, high efficiency gene transfer of PNP by the RCR vector resulted in significant suppression of tumor growth and extended survival time. As the RCR mediates stable integration of the PNP gene and continuous expression, an additional round of F-araAMP administration resulted in further survival benefit. RCR-mediated PNP suicide gene therapy thus represents a highly efficient form of intracellular chemotherapy, and may achieve effective antitumor activity with less systemic toxicity.

Asunto(s)

Escherichia coli/enzimología , Vectores Genéticos , Glioma/terapia , Profármacos/farmacología , Purina-Nucleósido Fosforilasa/genética , Retroviridae/genética , Fosfato de Vidarabina/análogos & derivados , Animales , Antimetabolitos Antineoplásicos/farmacología , Terapia Genética , Glioma/genética , Glioma/virología , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Inyecciones Intralesiones , Inyecciones Subcutáneas , Ratones , Ratones Desnudos , Fosfato de Vidarabina/farmacología

RESUMEN

As an extension of previous work with bis(POM) nucleotide prodrugs, we report the synthesis and biological evaluation in tumor cell culture of the bis(pivaloyloxymethyl) phosphotriester prodrug of slightly cytotoxic 2'-deoxy-4'-thioadenosine and its alpha-anomer. We have experienced need for an alternative phosphate masking group, particularly with purine nucleosides. Accordingly, we report synthesis and biological evaluation of the bis(tBuSA TE) phosphotriester prodrugs of 8-azaguanosine and 6-methylpurine riboside, nucleoside analogs with moderate to significant cytotoxicity. All four prodrugs were examined in tumor cell culture in parallel with the parent nucleosides. Synthetic routes and biological data are presented.

Asunto(s)

Adenosina/análogos & derivados , Guanosina/análogos & derivados , Profármacos/farmacología , Tionucleósidos/química , Zidovudina/análogos & derivados , Adenosina/química , Antineoplásicos/farmacología , Antivirales/química , Antivirales/farmacología , Cromatografía , Cromatografía en Capa Delgada , Didesoxinucleótidos , Diseño de Fármacos , Guanosina/química , Guanosina/farmacología , Humanos , Concentración 50 Inhibidora , Modelos Químicos , Nucleósidos/química , Organofosfonatos/química , Fosfatos/química , Profármacos/química , Estereoisomerismo , Zidovudina/química , Zidovudina/farmacología

RESUMEN

A novel series of 6-methylpurine nucleoside derivatives with substitutions at 5-position have been synthesised These compounds bear a 5'-heterocycle such as triazole or a imidazole with a two carbon chain, and an ether, thio ether or amine. To extend the SAR study of 2-fluoroadenine and 6-methyl purine nucleosides, their corresponding alpha-linker nucleosides with L-xylose and L-lyxose were also synthesized. All of these compounds have been evaluated for their substrate activity with E. coli PNP.

Asunto(s)

Adenina/análogos & derivados , Terapia Genética/métodos , Neoplasias/tratamiento farmacológico , Neoplasias/terapia , Nucleósidos/síntesis química , Profármacos/farmacología , Purinas/química , Adenina/farmacología , Antineoplásicos/farmacología , Carbono/química , Escherichia coli/enzimología , Humanos , Modelos Químicos , Mutación , Nucleósidos/química , Profármacos/química , Nucleósidos de Purina/química , Purina-Nucleósido Fosforilasa/química , Especificidad por Sustrato , Xilosa/química

RESUMEN

Because of the excellent in vivo activity of 4'-thio-beta-D-arabinofuranosylcytosine (T-araC) against a variety of human solid tumors, we have studied its metabolism in CEM cells to determine how the biochemical pharmacology of this compound differs from that of beta-D-arabinofuranosylcytosine (araC). Although there were many quantitative differences in the metabolism of T-araC and araC, the basic mechanism of action of T-araC was similar to that of araC: it was phosphorylated to T-araC-5'-triphosphate (T-araCTP) and inhibited DNA synthesis. The major differences between these two compounds were: (i) T-araC was phosphorylated to active metabolites at 1% the rate of araC; (ii) T-araCTP was 10- to 20-fold more potent as an inhibitor of DNA synthesis than was the 5'-triphosphate of araC (araCTP); (iii) the half-life of T-araCTP was twice that of araCTP; (iv) the catalytic efficiency of T-araC with cytidine deaminase was 10% that of araC; and (v) the 5'-monophosphate of araC was a better substrate for deoxycytidine 5'-monophosphate deaminase than was the 5'-monophosphate of T-araC. Of these differences in the metabolism of these two compounds, we propose that the prolonged retention of T-araCTP is a major factor contributing to the activity of T-araC against solid tumors. The data in this study represent another example of how relatively small structural changes in nucleoside analogs can profoundly affect the biochemical activity.

Asunto(s)

Antineoplásicos/metabolismo , Arabinonucleósidos/metabolismo , Desoxicitidina/análogos & derivados , Desoxicitidina/metabolismo , Tionucleósidos/metabolismo , Animales , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Arabinonucleósidos/farmacocinética , Arabinonucleósidos/farmacología , Transporte Biológico , División Celular/efectos de los fármacos , Citarabina/metabolismo , Citarabina/farmacocinética , Citarabina/farmacología , Citidina Desaminasa/metabolismo , ADN/biosíntesis , ADN/efectos de los fármacos , Desaminación , Desoxicitidina/farmacología , Desoxicitidina Quinasa/metabolismo , Nucleótidos de Desoxicitosina/metabolismo , Humanos , Masculino , Ratones , Ratones Desnudos , Tionucleósidos/farmacología , Células Tumorales Cultivadas

RESUMEN

This report examines a major barrier to suicide gene therapy in cancer and other diseases: namely, bystander cell killing. Existing vectors for in vivo gene delivery are inefficient and often transduce or transfect less than 1% of target cells. The E. coli PNP gene brings about cellular necrosis under conditions when 1 in 100 to 1 in 1000 cells express the gene product in vitro. In vivo bystander killing at or near this magnitude has not been reported previously. In the present experiments, transfection of cells with the E. coli PNP gene controlled by a SV40 promoter resulted in 30 nmol 6-methyl purine deoxyriboside (MeP-dR) converted per milligram tumor cell extract per hour (or conversion units (CU)). This level of expression led to elimination of entire populations of tumor cells in vitro after treatment with MeP-dR. Much earlier killing was observed using a tat transactivated E. coli PNP vector (approximately seven-fold higher activity, 230 CU). In vivo effects on tumor growth were next examined. Human ovarian tumors transfected with E. coli PNP were excised 5 days after i.p. implantation from the peritoneal cavities of mice in order to determine both E. coli PNP enzymatic activity and the fraction of cells expressing the gene. PNP activity at 5 days after gene transfer was approximately 170 CU and was expressed in approximately 0.1% of the tumor cells as judged by in situ hybridization. The expression of E. coli PNP at this level produced a 30% increase in life span (P < 0.001) and 49% reduction in tumor size (P < 0.005) after MeP-dR treatment, as compared with control tumors. Our observations lead to the conclusion that pronounced bystander killing by E. coli PNP is conferred in vivo, and that vectors capable of transgene expression in as few as one in 1000 cells can produce substantial antitumor effects if expression on a per cell basis is very high.

Asunto(s)

Escherichia coli/genética , Terapia Genética/métodos , Vectores Genéticos/uso terapéutico , Neoplasias Ováricas/terapia , Purina-Nucleósido Fosforilasa/metabolismo , Animales , Muerte Celular , Femenino , Expresión Génica , Humanos , Ratones , Ratones SCID , Trasplante de Neoplasias , Neoplasias Ováricas/enzimología , Neoplasias Ováricas/patología , Profármacos/uso terapéutico , Nucleósidos de Purina/uso terapéutico , Purina-Nucleósido Fosforilasa/genética , Activación Transcripcional , Células Tumorales Cultivadas

RESUMEN

The metabolism of O6-propyl-carbovir and N6-propyl-carbovir, two selective inhibitors of HIV replication, has been evaluated in CEM cells. Both compounds were phosphorylated in intact cells to carbovir-5'-triphosphate. The metabolism of these two agents was inhibited by deoxycoformycin and mycophenolic acid, but not erythro-9-(2-hydroxy-3-nonyl)adenine. No evidence of the 5'-triphosphate of either compound was detected in CEM cells.

Asunto(s)

Fármacos Anti-VIH/metabolismo , Línea Celular/metabolismo , Didesoxinucleósidos/metabolismo , Alquilación , Fármacos Anti-VIH/antagonistas & inhibidores , Fármacos Anti-VIH/síntesis química , Didesoxinucleósidos/antagonistas & inhibidores , Didesoxinucleósidos/síntesis química , VIH-1/efectos de los fármacos , VIH-1/metabolismo , Ácido Micofenólico/farmacología , Pentostatina/farmacología , Fosforilación/efectos de los fármacos , Replicación Viral/efectos de los fármacos

RESUMEN

4'-thio-beta-D-arabinofuranosylcytosine was synthesized by a facile route in high yields. It was evaluated for antitumor activity against a panel of human tumors, both in vitro and in vivo.

Asunto(s)

Antimetabolitos Antineoplásicos/farmacología , Antineoplásicos/síntesis química , Arabinonucleósidos/síntesis química , Citarabina/farmacología , Animales , Antimetabolitos Antineoplásicos/química , Antineoplásicos/química , Antineoplásicos/farmacología , Arabinonucleósidos/química , Arabinonucleósidos/farmacología , Citarabina/química , Replicación del ADN/efectos de los fármacos , ADN de Neoplasias/metabolismo , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Concentración 50 Inhibidora , Ratones , Ratones Desnudos , Fosforilación , Células Tumorales Cultivadas

RESUMEN

To maintain the telomeres at the ends of the chromosomes, telomerase in human cells adds a repeating sequence of nucleotides (TTAGGG) to the 3'-end of each chromosome using an RNA component of the enzyme as the template for DNA synthesis. Because of the selective expression of this enzyme in cancer cells, we have evaluated the interaction of human telomerase with several deoxyguanosine nucleotides of clinical importance. 2',3'-dideoxyguanosine 5'-triphosphate, 6-thio-2'-deoxyguanosine 5'-triphosphate (T-dGTP), carbovir 5'-triphosphate, and D-carbocyclic-2'-deoxyguanosine 5'-triphosphate (D-CdG-TP) inhibited telomerase activity by 50% when these analogs were present at only 2 to 9 times the dGTP concentration. The L-enantiomer of CdG-TP was far less inhibitory, thereby demonstrating the stereoselectivity of telomerase for nucleotide substrates. T-dGTP was incorporated into the DNA by telomerase in the absence of dGTP, but unlike dGTP there was little extension of the DNA chain after its incorporation. These results indicate that the metabolites of three clinically useful agents (6-mercaptopurine, 6-thioguanine, and Abacavir) can inhibit human telomerase activity, and it is possible that the effect of these nucleotides on telomerase activity or telomere function could contribute to the mechanism of action of these agents.

Asunto(s)

ADN/metabolismo , Nucleótidos de Desoxiguanina/metabolismo , Telomerasa/metabolismo , División Celular/efectos de los fármacos , Extractos Celulares , Nucleótidos de Desoxiguanina/química , Nucleótidos de Desoxiguanina/farmacología , Células HeLa , Humanos

RESUMEN

Four 5-substituted (chloro, fluoro, bromo, methyl) 1-(4-thio-beta-D-arabinofuranosyl)cytosines and their alpha anomers were synthesized by a facile route in high yields. All of these nucleosides were evaluated for cytotoxicity against a panel of human tumor cell lines in vitro. Only 5-fluoro-1-(4-thio-beta-D-arabinofuranosyl)cytosine was found to be highly cytotoxic in all the cell lines and was further evaluated in vivo.

Asunto(s)

Arabinonucleósidos/síntesis química , Arabinonucleósidos/farmacología , Arabinonucleósidos/química , Línea Celular , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Espectroscopía de Resonancia Magnética , Relación Estructura-Actividad

RESUMEN

During the last few years, many gene therapy strategies have been developed for various disease targets. The development of anticancer gene therapy strategies to selectively generate cytotoxic nucleoside or nucleotide analogs is an attractive goal. One such approach involves the delivery of herpes simplex virus thymidine kinase followed by the acyclic nucleoside analog ganciclovir. We have developed another gene therapy methodology for the treatment of cancer that has several significant attributes. Specifically, our approach involves the delivery of E. coli purine nucleoside phosphorylase, followed by treatment with a relatively non-toxic nucleoside prodrug that is cleaved by the enzyme to a toxic compound. This presentation describes the concept, details our search for suitable prodrugs, and summarizes the current biological data.

Asunto(s)

Escherichia coli/enzimología , Terapia Genética , Neoplasias/terapia , Profármacos/farmacocinética , Purina-Nucleósido Fosforilasa/metabolismo , Animales , Biotransformación , Flucitosina/farmacocinética , Ganciclovir/farmacocinética , Ratones , Ratones Desnudos , Purina-Nucleósido Fosforilasa/genética , Simplexvirus/enzimología , Timidina Quinasa/genética

RESUMEN

In an effort to understand biochemical features that are important to the selective antitumor activity of 2-chloro-9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)adenine [Cl-F( upward arrow)-dAdo], we evaluated the biochemical pharmacology of three structurally similar compounds that have quite different antitumor activities. Cl-F( upward arrow)-dAdo was 50-fold more potent as an inhibitor of CEM cell growth than were either 2-chloro-9-(2-deoxy-2-fluoro-beta-D-ribofuranosyl)adenine [Cl-F( downward arrow)-dAdo] or 2-chloro-9-(2-deoxy-2, 2-difluoro-beta-D-ribofuranosyl)adenine [Cl-diF( upward arrow downward arrow)-dAdo]. The compounds were similar as substrates of deoxycytidine kinase. Similar amounts of their respective triphosphates accumulated in CEM cells, and the rate of disappearance of these metabolites was also similar. Cl-F( upward arrow)-dAdo was 10- to 30-fold more potent in its ability to inhibit the incorporation of cytidine into deoxycytidine nucleotides than either Cl-F( downward arrow)-dAdo or Cl-diF( upward arrow downward arrow)-dAdo, respectively, which indicated that ribonucleotide reductase was differentially inhibited by these three compounds. Thus, the differences in the cytotoxicity of these agents toward CEM cells were not related to quantitative differences in the phosphorylation of these agents to active forms but can mostly be accounted for by differences in the inhibition of ribonucleotide reductase activity. Furthermore, the inhibition of RNA and protein synthesis by Cl-F( downward arrow)-dAdo and Cl-diF( upward arrow downward arrow)-dAdo at concentrations similar to those required for the inhibition of DNA synthesis can help explain the poor antitumor selectivity of these two agents because all cells require RNA and protein synthesis.

Asunto(s)

Antineoplásicos/farmacología , Arabinonucleósidos/farmacología , Desoxiadenosinas/farmacología , Nucleótidos de Adenina , División Celular/efectos de los fármacos , Clofarabina , ADN/biosíntesis , ADN/efectos de los fármacos , Desoxicitidina/metabolismo , Desoxicitidina Quinasa/antagonistas & inhibidores , Desoxicitidina Quinasa/aislamiento & purificación , Desoxicitidina Quinasa/metabolismo , Inhibidores Enzimáticos/farmacología , Humanos , Sustancias Macromoleculares , Fosforilación/efectos de los fármacos , Especificidad por Sustrato , Tritio , Células Tumorales Cultivadas

RESUMEN

Activation of purine nucleoside analogs by Escherichia coli purine nucleoside phosphorylase (PNP) is being evaluated as a suicide gene therapy strategy for the treatment of cancer. Because the mechanisms of action of two toxic purine bases, 6-methylpurine (MeP) and 2-fluoroadenine (F-Ade), that are generated by this approach are poorly understood, mechanistic studies were initiated to learn how these compounds differ from agents that are being used currently. The concentration of F-Ade, MeP, or 5-fluorouracil required to inhibit CEM cell growth by 50% after a 4-hr incubation was 0.15, 9, or 120 microM, respectively. F-Ade and MeP were also toxic to quiescent MRC-5, CEM, and Balb 3T3 cells. Treatment of CEM, MRC-5, or Balb 3T3 cells with either F-Ade or MeP resulted in the inhibition of protein, RNA, and DNA syntheses. CEM cells converted F-Ade and MeP to F-ATP and MeP-ribonucleoside triphosphate (MeP-R-TP), respectively. The half-life for disappearance of HeP-ribonucleoside triphosphate from CEM cells was approximately 48 hr, whereas the half-lives of F-ATP and ATP were approximately 5 hr. Both MeP and F-Ade were incorporated into the RNA and DNA of CEM cells. These studies indicated that the mechanisms of action of F-Ade and MeP were quite different from those of other anticancer agents, and suggested that the generation of these agents in tumor cells by E. coli PNP could result in significant advantages over those generated by either herpes simplex virus thymidine kinase or E. coli cytosine deaminase. These advantages include a novel mechanism of action resulting in toxicity to nonproliferating and proliferating tumor cells and the high potency of these agents during short-term treatment.

Asunto(s)

Adenina/análogos & derivados , Purinas/metabolismo , Células 3T3 , Adenina/metabolismo , Animales , División Celular/efectos de los fármacos , Línea Celular , Cicloheximida/farmacología , ADN/efectos de los fármacos , ADN/metabolismo , Fluorouracilo/farmacología , Humanos , Ratones , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Inhibidores de la Síntesis de la Proteína/farmacología , ARN/efectos de los fármacos , ARN/metabolismo

RESUMEN

The carbocyclic analog of 2'-deoxyguanosine (CdG) has broad-spectrum antiviral activity. Because of recent observations with other nucleoside analogs that biological activity may be associated the L enantiomer rather than, as expected, with the D enantiomer, we have studied the metabolism of both enantiomers of CdG to identify the enzymes responsible for the phosphorylation of CdG in noninfected and virally infected human and duck cells. We have examined the enantiomers as substrates for each of the cellular enzymes known to catalyze phosphorylation of deoxyguanosine. Both enantiomers of CdG were substrates for deoxycytidine kinase (EC 2.7.1.74) from MOLT-4 cells, 5'-nucleotidase (EC 3.1.3.5) from HEp-2 cells, and mitochondrial deoxyguanosine kinase (EC 2.7.1.113) from human platelets and CEM cells. For both deoxycytidine kinase and mitochondrial deoxyguanosine kinase, the L enantiomer was the better substrate. Even though the D enantiomer was the preferred substrate with 5'-nucleotidase, the rate of phosphorylation of the L enantiomer was substantial. The phosphorylation of D-CdG in MRC-5 cells was greatly stimulated by infection with human cytomegalovirus. The fact that the phosphorylation of D-CdG was stimulated by mycophenolic acid and was not affected by deoxycytidine suggested that 5'-nucleotidase was the enzyme primarily responsible for its metabolism in virally infected cells. D-CdG was extensively phosphorylated in duck hepatocytes, and its phosphorylation was not affected by infection with duck hepatitis B virus. These results are of importance in understanding the mode of action of D-CdG and related analogs and in the design of new biologically active analogs.

Asunto(s)

5'-Nucleotidasa/metabolismo , Desoxicitidina Quinasa/metabolismo , Desoxiguanosina/análogos & derivados , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Animales , Antibióticos Antineoplásicos/farmacología , Células Cultivadas/enzimología , Células Cultivadas/virología , Citomegalovirus/efectos de los fármacos , Desoxiguanosina/química , Desoxiguanosina/metabolismo , Desoxiguanosina/farmacología , Patos , Humanos , Mitocondrias/efectos de los fármacos , Mitocondrias/enzimología , Ácido Micofenólico/farmacología , Nucleósidos/farmacología , Fosforilación/efectos de los fármacos , Estereoisomerismo , Especificidad por Sustrato

RESUMEN

Mason-Pfizer monkey virus (M-PMV), the prototype type D retrovirus, differs from most other retroviruses by assembling its Gag polyproteins into procapsids in the cytoplasm of infected cells. Once assembled, the procapsids migrate to the plasma membrane, where they acquire their envelope during budding. Because the processes of M-PMV protein transport, procapsid assembly, and budding are temporally and spatially unlinked, we have been able to determine whether cellular proteins play an active role during the different stages of procapsid morphogenesis. We report here that at least two stages of morphogenesis require ATP. Both procapsid assembly and procapsid transport to the plasma membrane were reversibly blocked by treating infected cells with sodium azide and 2-deoxy-D-glucose, which we show rapidly and reversibly depletes cellular ATP pools. Assembly of procapsids in vitro in a cell-free translation/assembly system was inhibited by the addition of nonhydrolyzable ATP analogs, suggesting that ATP hydrolysis and not just ATP binding is required. Since retrovirus Gag polyproteins do not bind or hydrolyze ATP, these results demonstrate that cellular components must play an active role during retrovirus morphogenesis.

Asunto(s)

Adenosina Trifosfato/metabolismo , Cápside/fisiología , Virus del Mono Mason-Pfizer/fisiología , Precursores de Proteínas/fisiología , Ensamble de Virus , Adenosina Trifosfato/análogos & derivados , Animales , Antimetabolitos/farmacología , Transporte Biológico , Línea Celular , Membrana Celular/metabolismo , Supervivencia Celular , Desoxiglucosa/farmacología , Macaca mulatta , Azida Sódica/farmacología

RESUMEN

Expression of Escherichia coli purine nucleoside phosphorylase (PNP) activates prodrugs and kills entire populations of mammalian cells, even when as few as 1% of the cells express this gene. This phenomenon of bystander killing has been previously investigated for herpes simplex virus-thymidine kinase (HSV-TK) and has been shown to require cell to cell contact. Using silicon rings to separate E. coli PNP expressing cells from non-expressing cells sharing the same medium, we demonstrate that bystander cell killing by E. coli PNP does not require cell-cell contact. Initially, cells expressing E. coli PNP convert the non-toxic prodrug, 6-methylpurine-2'-deoxyriboside (MeP-dR) to the highly toxic membrane permeable toxin, 6-methylpurine (MeP). As the expressing cells die, E. coli PNP is released into the culture medium, retains activity, and continues precursor conversion extracellularly (as determined by reverse phase high performance liquid chromatography of both prodrug and toxin). Bystander killing can also be observed in the absence of extracellular E. coli PNP by removing the MeP-dR prior to death of the expressing cells. In this case, 100% of cultured cells die when as few as 3% of the cells of a population express E. coli PNP. Blocking nucleoside transport with nitrobenzylthioinosine reduces MeP-dR mediated cell killing but not MeP cell killing. These mechanisms differ fundamentally from those previously reported for the HSV-TK gene.

Asunto(s)

Comunicación Celular , Escherichia coli/enzimología , Purina-Nucleósido Fosforilasa/farmacología , Purinas/farmacología , Marcadores de Afinidad , Animales , Muerte Celular , División Celular , Medios de Cultivo , Humanos , Ratones , Profármacos/metabolismo , Profármacos/farmacología , Nucleósidos de Purina/metabolismo , Nucleósidos de Purina/farmacología , Purina-Nucleósido Fosforilasa/metabolismo , Purinas/metabolismo , Tioinosina/análogos & derivados , Tioinosina/farmacología , Células Tumorales Cultivadas

RESUMEN

We have developed a new strategy for the gene therapy of cancer based on the activation of purine nucleoside analogs by transduced E. coli purine nucleoside phosphorylase (PNP, E.C. 2.4.2.1). The approach is designed to generate antimetabolites intracellularly that would be too toxic for systemic administration. To determine whether this strategy could be used to kill tumor cells without host toxicity, nude mice bearing human malignant D54MG glioma tumors expressing E. coli PNP (D54-PNP) were treated with either 6-methylpurine-2'-deoxyriboside (MeP-dR) or arabinofuranosyl-2-fluoroadenine monophosphate (F-araAMP, fludarabine, a precursor of F-araA). Both prodrugs exhibited significant antitumor activity against established D54-PNP tumors at doses that produced no discernible systemic toxicity. Significantly, MeP-dR was curative against this slow growing solid tumor after only 3 doses. The antitumor effects showed a dose dependence on both the amount of prodrug given and the level of E. coli PNP expression within tumor xenografts. These results indicated that a strategy using E. coli PNP to create highly toxic, membrane permeant compounds that kill both replicating and nonreplicating cells is feasible in vivo, further supporting development of this cancer gene therapy approach.

Asunto(s)

Antimetabolitos Antineoplásicos/uso terapéutico , Terapia Genética/métodos , Glioma/tratamiento farmacológico , Profármacos/farmacología , Purina-Nucleósido Fosforilasa/fisiología , Animales , Antimetabolitos Antineoplásicos/toxicidad , Escherichia coli/enzimología , Escherichia coli/genética , Vectores Genéticos/genética , Humanos , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Nucleósidos de Purina/uso terapéutico , Nucleósidos de Purina/toxicidad , Purina-Nucleósido Fosforilasa/genética , Retroviridae/genética , Fosfato de Vidarabina/análogos & derivados , Fosfato de Vidarabina/uso terapéutico , Fosfato de Vidarabina/toxicidad

RESUMEN

Carbovir (CBV) is a guanine nucleoside analog with potent in vitro anti-HIV activity. A prodrug of CBV is currently being evaluated in clinical trials as a potential agent for the treatment of AIDS. The ability of CBV to inhibit mitochondrial DNA synthesis in intact CEM cells was evaluated in the present study, because most of the currently available anti-HIV nucleoside analogs have significant toxicities that result from their inhibition of mitochondrial DNA synthesis. No delayed cytotoxicity was observed in CEM cells treated with 50 microM CBV for 4 weeks. In addition, CBV at concentrations as high as 1 mM did not cause a decline in mitochondrial DNA levels and only minimally increased the concentration of lactic acid in the medium. In contrast to these results with CBV, treatment of CEM cells with 0.5 microM 2',3'-dideoxycytidine resulted in delayed cytotoxicity, a decrease in mitochondrial DNA content and increases in lactic acid levels in the medium. These results indicated that treatment of CEM cells with CBV did not result in the inhibition of mitochondrial DNA synthesis and suggested that treatment of AIDS patients with CBV, or a prodrug of CBV, would not result in some of the toxicities seen with the other anti-HIV nucleoside analogs.

Asunto(s)

Fármacos Anti-VIH/toxicidad , Didesoxinucleósidos/toxicidad , Mitocondrias/efectos de los fármacos , Síndrome de Inmunodeficiencia Adquirida/tratamiento farmacológico , Síndrome de Inmunodeficiencia Adquirida/virología , Línea Celular , ADN Mitocondrial/biosíntesis , Evaluación Preclínica de Medicamentos , Humanos , Ácido Láctico/metabolismo , Mitocondrias/metabolismo , Profármacos/toxicidad , Zalcitabina/toxicidad