RESUMEN
The stability of phosphotriester derivatives of 3'-azido-2',3'-dideoxythymidine (AZT) bearing a S-pivaloyl-2-thioethyl (tBuSATE) group and various aryl residues derived from L-tyrosine was evaluated in biological media. The results demonstrate that such compounds give rise to intracellular delivery of the parent mononucleotide through esterase and phosphodiesterase hydrolytic steps, successively.
Asunto(s)
VIH/efectos de los fármacos , Zidovudina/análogos & derivados , Zidovudina/química , Línea Celular , Didesoxinucleótidos , Estabilidad de Medicamentos , Humanos , Indicadores y Reactivos , Timidina Quinasa/deficiencia , Nucleótidos de Timina/farmacocinética , Tirosina/análogos & derivados , Zidovudina/farmacocinéticaRESUMEN
The fate of a dodecathymidine prodrug in cell extract was monitored by MALDI-TOF MS. This technique allows a facile identification and a relative quantification of metabolites produced. We showed that the relative peak intensities were similar to the relative metabolite proportions that permitted the determination of their half-lives. The oligonucleotide prodrug was fully metabolized to yield the T12 phosphorothioate likely through a carboxyesterase mediated mechanism.
Asunto(s)
Oligonucleótidos/farmacocinética , Profármacos/farmacocinética , Nucleótidos de Pirimidina/farmacocinética , Timidina/análogos & derivados , Biotransformación , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Synthesis, biological activities and decomposition kinetics of novel phosphotriester derivatives of 3'-azido-2',3'-dideoxythymidine (AZT) bearing a S-tButyl-2-thioethyl (tBuSATE) group and L-tyrosinyl residues are reported. All the derivatives appeared to be potent inhibitors of HIV-1 replication in various cell culture experiments. The proposed decomposition process of these mixed phosphotriesters may involve successively an esterase and then a phosphodiesterase activation.
Asunto(s)
Fármacos Anti-VIH/síntesis química , Fármacos Anti-VIH/farmacología , Profármacos/síntesis química , Profármacos/farmacología , Zidovudina/análogos & derivados , Fármacos Anti-VIH/química , Células Cultivadas , Diseño de Fármacos , Estabilidad de Medicamentos , Ésteres/síntesis química , Ésteres/química , Ésteres/farmacología , VIH-1/efectos de los fármacos , VIH-1/fisiología , Humanos , Profármacos/química , Replicación Viral/efectos de los fármacos , Zidovudina/síntesis química , Zidovudina/farmacologíaRESUMEN
Improvements of an "on-line cleaning" HPLC method for analysis of biological samples are presented: (i) the use of cleaning precolumns filled with hydrophobic stationary phases instead of the hydrophilic ones previously used to eliminate the biological matrix: (ii) the combination in the mobile phase of anionic and cationic pairing reagents in order to retain on the precolumn all the metabolites, whatever their hydrophilicity and ionicity are. Such modifications allowed to study the biotransformation of prodrugs of 5-Fluorouracil, designed to act as antitumoral pronucleotides.
Asunto(s)
Antimetabolitos Antineoplásicos/análisis , Cromatografía Líquida de Alta Presión/métodos , Fluorouracilo/análogos & derivados , Profármacos/análisis , Antimetabolitos Antineoplásicos/sangre , Antimetabolitos Antineoplásicos/farmacocinética , Biotransformación , Fluorouracilo/sangre , Fluorouracilo/farmacocinética , Humanos , Profármacos/farmacocinéticaRESUMEN
A new pronucleotide series is described involving a two-step degradation process mediated by, respectively, carboxylesterase and phosphoramidase. Taking AZT as nucleosidyl moiety, it is shown that most of the compounds inhibit HIV replication in TK(-) cell line, which proves 5'-AZTMP delivery.
Asunto(s)
Fármacos Anti-VIH/síntesis química , Diseño de Fármacos , Nucleótidos/química , Fármacos Anti-VIH/química , Fármacos Anti-VIH/farmacología , Cromatografía Líquida de Alta Presión , VIH/fisiología , Nucleótidos/farmacología , Profármacos/química , Profármacos/farmacología , Replicación Viral/efectos de los fármacosRESUMEN
The fate of a dodecathymidine prodrug in cell extract was monitored by MALDI-TOF MS. This technique allows a facile identification and a relative quantification of metabolites produced. We showed that the relative peak intensities were similar to the relative metabolite proportions that permitted the determination of their half-lives. We found a good fit between the calculated kinetics curves and the experimental points. The oligonucleotide prodrug was fully metabolized to yield the dodecathymidine phosphorothioate likely through a carboxyesterase mediated mechanism.
Asunto(s)
Extractos Celulares , Oligonucleótidos/farmacocinética , Profármacos/farmacocinética , Biotransformación , Oligonucleótidos/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Timidina/químicaRESUMEN
The in vitro anti-HIV activity, stability, and potential for oral absorption of a phosphotriester derivative of AZT (zidovudine; 3'-azido-2',3'-deoxythymidine) bearing a new esterase-labile S-acyl-2-thioethyl (SATE) group as transient phosphate protection are reported. The biolabile protection is characterized by the presence of a hydroxyl function in the acyl chain. In accordance with previously reported data in the bis(SATE) prodrug series, the present results demonstrate that the studied bis(hydroxytBuSATE)phosphotriester exerts its biological effects via intracellular delivery of the 5'-monophosphate of AZT. The hydroxyl function confers a high resistance against esterase hydrolysis, and the studied prodrug is able to cross the Caco-2 cell monolayers in intact form, suggesting that its further development as a possible anti-HIV pronucleotide candidate is warranted.
Asunto(s)
Fármacos Anti-VIH/farmacología , Profármacos/farmacología , Inhibidores de la Transcriptasa Inversa/farmacología , Zidovudina/farmacología , Administración Oral , Fármacos Anti-VIH/administración & dosificación , Fármacos Anti-VIH/química , Células CACO-2 , Cromatografía Líquida de Alta Presión , Estabilidad de Medicamentos , VIH-1/efectos de los fármacos , Humanos , Pruebas de Sensibilidad Microbiana , Profármacos/administración & dosificación , Profármacos/química , Inhibidores de la Transcriptasa Inversa/administración & dosificación , Inhibidores de la Transcriptasa Inversa/química , Análisis Espectral , Zidovudina/administración & dosificación , Zidovudina/químicaRESUMEN
We comparatively studied the decomposition pathways in CEM cell extract of several PHENYL phosphoramidate diesters of AZT. A correlation between anti-HIV activities in TK- cell lines and pharmacokinetic data has been observed. This study would help to design corresponding SATE phosphoramidate diesters which revealed potent anti-HIV properties.
Asunto(s)
Fármacos Anti-VIH/farmacología , Zidovudina/análogos & derivados , Fármacos Anti-VIH/química , Línea Celular , Humanos , Compuestos Organofosforados/química , Zidovudina/química , Zidovudina/farmacologíaRESUMEN
PURPOSE: To evaluate the potential of several bis-ester prodrugs of the antiviral agent 9-(2-phosphonylmethoxyethyl)adenine (PMEA, adefovir) to enhance the oral absorption of PMEA. METHODS: Caco-2 monolayers were used to estimate intestinal transport and metabolism of the bis(pivaloyloxymethyl)-ester [bis(POM)-] and a series of bis(S-acyl-2-thioethyl)-esters [bis(SATE)-] of PMEA. An LC-MS method was used for the identification of unknown metabolites which were formed from the SATE-esters. RESULTS: During transport across Caco-2 monolayers, all esters were extensively degraded as could be concluded from the appearance of the mono-ester and free PMEA in apical as well as basolateral compartments. Incubation of SATE-esters with the monolayers resulted in the formation of two additional metabolites, which were identified as 2-thioethyl-PMEA and its dimerisation product. All ester prodrugs resulted in enhanced transepithelial transport of total PMEA (i.e. the bis-esters and their corresponding metabolites, including PMEA), but significant differences could be observed between the various esters. Transport of total PMEA ranged from 0.4 +/- 0.1% for the bis[S(methyl) ATE]-ester to 15.3 +/- 0.9% for the more lipophilic bis[S(phenyl)ATE]-PMEA. A relationship between total transport of the esters and their lipophilicity (as estimated by their octanol/water partition coefficient) was established (r2 = 0.87). Incubation of prodrug esters with homogenates from Caco-2 cells showed large differences in susceptibility of the compounds to esterases, the half-lives of the bis-esters varying from 4.3 +/- 0.3 min for the bis[S(phenyl)ATE]-PMEA to 41.5 +/- 0.8 min for its methyl analogue. In addition, intracellularly formed PMEA was observed to be further converted by the cells to the diphosphorylated PMEA (PMEApp). CONCLUSIONS: Several SATE-esters of PMEA can be considered as potential alternatives to bis(POM)-PMEA, due to enhanced epithelial transport, sufficient chemical and enzymatic stability and adequate release of PMEA. Toxicological studies as well as in vivo experiments are required in order to further explore the potential of those SATE-esters as prodrugs for oral delivery of PMEA.
Asunto(s)
Adenina/análogos & derivados , Antivirales/farmacocinética , Organofosfonatos , Profármacos/farmacocinética , Adenina/farmacocinética , Células CACO-2 , Cromatografía por Intercambio Iónico , Ésteres , HumanosRESUMEN
The purpose of the present study was to compare the decomposition pathways in CEM cell extracts of various phenyl phosphoramidate derivatives of AZT. In addition, the structures of their metabolites were identified. Correlations with their anti-HIV activities in a thymidine kinase deficient (TK-) CEM cell line have been established with a rationale of designing phosphoramidate pronucleotides capable of delivering intracellularly their respective 5'-nucleoside monophosphate derivatives.
Asunto(s)
Fármacos Anti-VIH/química , VIH-1/efectos de los fármacos , Profármacos/síntesis química , Zidovudina/análogos & derivados , Zidovudina/química , Fármacos Anti-VIH/farmacología , Línea Celular , Supervivencia Celular/efectos de los fármacos , Diseño de Fármacos , VIH-1/fisiología , Semivida , Humanos , Indicadores y Reactivos , Profármacos/química , Profármacos/farmacología , Relación Estructura-Actividad , Timidina Quinasa/deficiencia , Replicación Viral/efectos de los fármacos , Zidovudina/síntesis química , Zidovudina/farmacologíaRESUMEN
MonoSATE aryl phosphotriesters of AZT are able to deliver intracellularly the corresponding 5'-mononucleotide. This process requires activation by an esterase followed by a phosphodiesterase. This finding opens the way to the design of new pronucleotide series.
Asunto(s)
Fármacos Anti-VIH/síntesis química , Diseño de Fármacos , Nucleótidos/síntesis química , Profármacos/síntesis química , Zidovudina/análogos & derivados , HumanosRESUMEN
The decomposition pathways and kinetics in various biological media and the in vitro anti-HIV-1 and anti-HIV-2 activities of four derivatives of the 5'-mononucleotide of isoddA incorporating carboxylate esterase-labile transient phosphate protecting groups are reported and compared: namely, two mononucleoside aryl phosphoramidate derivatives 1a,b and two mononucleoside phosphotriester derivatives incorporating two S-acyl-2-thioethyl groups 2a,b. All four compounds show better antiviral activity, compared to the parent nucleoside analog isoddA. The results highlight that both types of compounds act as pronucleotides, i.e. they exert their antiviral effect via intracellular delivery of the 5'-mononucleotide of isoddA. The results may give insights for the design of new more efficient pronucleotides.
Asunto(s)
Didesoxiadenosina/análogos & derivados , VIH-1/efectos de los fármacos , VIH-2/efectos de los fármacos , Línea Celular , Didesoxiadenosina/química , Didesoxiadenosina/farmacología , Humanos , Cinética , Espectroscopía de Resonancia Magnética , Nucleótidos/química , Espectrometría de Masa Bombardeada por Átomos VelocesRESUMEN
The rationale for a pronucleotide approach based on the use of phosphotriesters which incorporate enzyme-mediated bio-labile protection is discussed in detail. Among the studied bio-labile phosphate protecting groups, the S-acyl-2-thioethyl (SATE) groups appeared the most promising as exemplified in cell culture systems in the case of the pronucleotides of 3'-azido-3'-deoxythymidine, 2',3'-didehydro-3'-deoxythymidine, 2',3'-dideoxyadenosine and acyclovir In vivo implementations of such bis(SATE) pronucleotides have been planned for future animal studies.
Asunto(s)
Antivirales/síntesis química , Nucleótidos/síntesis química , Profármacos/síntesis química , Aciclovir/análogos & derivados , Animales , Antivirales/química , Antivirales/metabolismo , Diseño de Fármacos , Humanos , Indicadores y Reactivos , Modelos Biológicos , Nucleótidos/química , Nucleótidos/metabolismo , Profármacos/química , Profármacos/metabolismo , Relación Estructura-Actividad , Zidovudina/análogos & derivadosRESUMEN
The synthesis, in vitro anti-HIV-1 activity, and decomposition pathways of several mononucleoside phosphotriester derivatives of 3'-azido-2',3'-dideoxythymidine (AZT) incorporating a new kind of carboxylate esterase-labile transient phosphate-protecting group, namely, S-acyl-2-thioethyl, are reported. All the described compounds showed marked antiviral activity in thymidine kinase-deficient CEM cells in which AZT was virtually inactive. The results strongly support the hypothesis that such pronucleotides exert their biological effects via intracellular delivery of the 5'-mononucleotide of AZT. This point was corroborated by decomposition studies in cell extracts and culture medium.
Asunto(s)
Antivirales/síntesis química , VIH-1/efectos de los fármacos , Nucleótidos de Timina/farmacocinética , Zidovudina/análogos & derivados , Antivirales/metabolismo , Antivirales/farmacología , Transporte Biológico , Línea Celular , Didesoxinucleótidos , Estabilidad de Medicamentos , Zidovudina/farmacocinéticaRESUMEN
Several studies have shown that alpha-oligonucleotides (alpha-ONs) are more resistant to degradation by nucleases than are beta-oligonucleotides (beta-ONs), but a few exceptions have been reported. The present work indicates that the resistance of alpha-ONs to 3'-exonucleases contained in calf or human sera strongly depends on their 3'-terminal sequence. When the 2 last residues were ...A-G, ...C-A, or ...C-T, the degradation rates in tissue culture media with fetal calf serum were similar to those observed for beta-ONs, but stabilization factors up to 200 were observed when the 2 last residues were ...T-C, ...A-C, or ...C-C, and intermediate stabilization factors were found with ...G-A, ...T-A, or ...T-T terminal sequences. Other data confirm that alpha-ONs are significantly more resistant than beta-ONs to purified 5'-exo- and endonucleases as well as to 3'-exonucleases contained in snake venom or CEM cell lysates, but suggest that sequence specificity may also exist in these media. These findings, which are consistent with literature data and explain the behavior of the aforementioned exceptions, also emphasize that the stability observed in the presence of purified nucleases cannot be extrapolated to sera. Other results show that handling, heat inactivation, and storage conditions have little effect on the 3'-exonuclease activity of serum-containing media, but can completely modify the nuclease activities of cell extracts. This study, which was carried out by means of the accurate "on-line ISRP cleaning" HPLC technique, brings new insights to the general problem of oligonucleotide stability.
Asunto(s)
Exonucleasas/metabolismo , Oligonucleótidos Antisentido/metabolismo , Hidrolasas Diéster Fosfóricas/metabolismo , Animales , Secuencia de Bases , Cromatografía Líquida de Alta Presión , Estabilidad de Medicamentos , Humanos , Cinética , Datos de Secuencia Molecular , Oligonucleótidos Antisentido/química , Fosfodiesterasa I , Venenos de Serpiente/enzimologíaRESUMEN
The monomeric and symmetrical dimeric 5'-hydrogenphosphonate derivatives of AZT were prepared and evaluated for their inhibitory properties against HIV-1 in several cell lines. The synthesis of the compounds was achieved by reaction of AZT with in situ prepared phosphorus tris(imidazolide) or with phosphonic acid in the presence of pivaloyl chloride. The two title compounds showed in vitro anti-HIV activity similar to (but not better than) that of AZT in three cell lines which were not deficient in thymidine kinase. On the other hand they were inactive in CEM-TK- cells. Pharmacokinetic studies in several media corroborate the assumption that these compounds must not be considered as 'true antiviral agents', but that they act by releasing their nucleoside entity.
Asunto(s)
VIH-1/efectos de los fármacos , Zidovudina/análogos & derivados , Línea Celular , Didesoxinucleótidos , Estabilidad de Medicamentos , Humanos , Profármacos/metabolismo , Linfocitos T/citología , Nucleótidos de Timina/farmacología , Replicación Viral/efectos de los fármacos , Zidovudina/farmacologíaRESUMEN
On the basis of three different models (namely: ddU, AZT and PMEA), mononucleotide phosphotriester derivatives were designed to be able to liberate the corresponding monophosphate (or phosphonate) inside the cell through a reductase-mediated activation process. It was demonstrated that the use of bis[S-(2-hydroxyethylsulfidyl)-2-thioethyl] esters of ddUMP (11), AZTMP (12) and PMEA (17) resulted in intracellular delivery of the parent monophosphate (or phosphonate). This point was corroborated by observation of an anti-HIV effect of, 11 in various cell lines, for 12 in CEM TK- cells and by the enhanced activity observed for 17. Furthermore, the reported decomposition data in cell extracts fully confirm the validity of this approach and show unambiguously the potential for intracellular reductase-mediated activation of the starting drug.
Asunto(s)
Adenina/análogos & derivados , Antivirales/farmacología , Didesoxinucleósidos/farmacología , VIH-1/efectos de los fármacos , Organofosfonatos , Oxidorreductasas/metabolismo , Profármacos/metabolismo , Nucleótidos de Timina/farmacología , Zidovudina/análogos & derivados , Adenina/metabolismo , Adenina/farmacología , Antivirales/síntesis química , Antivirales/metabolismo , Linfocitos B/citología , Línea Celular , Didesoxinucleósidos/metabolismo , Didesoxinucleótidos , Ésteres/síntesis química , Ésteres/metabolismo , Humanos , Linfocitos T/citología , Nucleótidos de Timina/metabolismo , Uridina Monofosfato/análogos & derivados , Zidovudina/metabolismo , Zidovudina/farmacologíaRESUMEN
Among the 2',3'-dideoxynucleoside 5'-triphosphates containing a physiological base, 2',3'-dideoxyuridine 5'-triphosphate (ddUTP) has been reported to be among the most powerful inhibitors of human immunodeficiency virus (HIV) reverse transcriptase (RT) in cell-free systems. However, in contrast to other dideoxynucleosides, 2',3'-dideoxyuridine (ddU) is inactive in treatment of HIV-infected cells in culture, since it is a poor substrate for cellular nucleoside kinases. This problem cannot be overcome by the use of phosphorylated ddU because such compounds are unable to cross cell membranes. To promote entry and thus bypass the limiting steps of intracellular phosphorylation, we have encapsulated mono- and tri-phosphorylated ddU in liposomes coupled to monoclonal antibodies (immunoliposomes). We investigated antiviral effects in two human T cell lines (MT-4, CEM). We observed that ddU nucleotides remain phosphorylated for several weeks after encapsulation in immunoliposomes, and potent antiviral activity is obtained when these drugs are delivered into infected cells by cell-specific antibodies (ED50 < or = 1 microM on CEM). In contrast, no inhibition was observed with non-targeted liposomes containing phosphorylated ddU, or with empty liposomes, whether targeted or not.
Asunto(s)
Antivirales/farmacología , Didesoxinucleósidos/farmacología , VIH-1/efectos de los fármacos , Nucleótidos de Uracilo/farmacología , Línea Celular , Didesoxinucleósidos/síntesis química , Didesoxinucleótidos , Estabilidad de Medicamentos , VIH-1/fisiología , Humanos , Liposomas , Fosforilación , Nucleótidos de Uracilo/síntesis química , Uridina Monofosfato/análogos & derivados , Replicación Viral/efectos de los fármacosRESUMEN
12-Mer analogues, representative of seven different classes of structurally modified oligonucleotides and complementary to the same target, have been compared for their binding affinity for both single-stranded DNA and RNA, resistance to hydrolysis by nucleases in culture medium (RPMI 1640 + 10% inactivated fetal calf serum), and inhibition of HIV-1 replication in de novo infected MT4 T lymphocytes. The viral target was the splice acceptor site of the premessenger coding for the regulatory protein tat. The oligo(2'-O-alkyl)ribonucleotides (beta-2'O-allyl-RNA and beta-2'OMe-RNA) were shown to form the most stable hybrids with complementary RNA strands whereas the alpha-anomeric oligodeoxynucleoside phosphorothioate analogue displayed the highest stability in the culture medium. All the modified oligonucleotides examined in the present study exhibited a sequence-nonspecific inhibitory effect on HIV-1 replication, the phosphorothioate analogues being the most active ones (ED50 < 1 microM).
Asunto(s)
Antivirales/síntesis química , Oligonucleótidos Antisentido/síntesis química , Antivirales/química , Antivirales/farmacología , Secuencia de Bases , Línea Celular , Cromatografía Líquida de Alta Presión , VIH-1/efectos de los fármacos , Humanos , Datos de Secuencia Molecular , Oligonucleótidos Antisentido/química , Oligonucleótidos Antisentido/farmacología , Relación Estructura-Actividad , Replicación Viral/efectos de los fármacosRESUMEN
An "on-line" HPLC analysis of crude biological samples is described. A precolumn of internal surface reversed-phase material allows the passage of proteins and other unwanted products while retaining analytes which are transferred, concentrated and chromatographed on a conventional reverse-phase or ion-exchange HPLC column. This protocol allows precise kinetics of the degradation of an oligonucleotide in cell culture to be obtained without radiolabeling or sample preparation.