RESUMO
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.
Assuntos
Antivirais/síntese química , HIV-1/efeitos dos fármacos , Nucleotídeos de Timina/farmacocinética , Zidovudina/análogos & derivados , Antivirais/metabolismo , Antivirais/farmacologia , Transporte Biológico , Linhagem Celular , Didesoxinucleotídeos , Estabilidade de Medicamentos , Zidovudina/farmacocinéticaRESUMO
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).
Assuntos
Antivirais/síntese química , Oligonucleotídeos Antissenso/síntese química , Antivirais/química , Antivirais/farmacologia , Sequência de Bases , Linhagem Celular , Cromatografia Líquida de Alta Pressão , HIV-1/efeitos dos fármacos , Humanos , Dados de Sequência Molecular , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/farmacologia , Relação Estrutura-Atividade , Replicação Viral/efeitos dos fármacosRESUMO
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.
Assuntos
Didesoxiadenosina/análogos & derivados , HIV-1/efeitos dos fármacos , HIV-2/efeitos dos fármacos , Linhagem Celular , Didesoxiadenosina/química , Didesoxiadenosina/farmacologia , Humanos , Cinética , Espectroscopia de Ressonância Magnética , Nucleotídeos/química , Espectrometria de Massas de Bombardeamento Rápido de ÁtomosRESUMO
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.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Meios de Cultura/química , Oligonucleotídeos/química , CinéticaRESUMO
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.
Assuntos
Adenina/análogos & derivados , Antivirais/farmacologia , Didesoxinucleosídeos/farmacologia , HIV-1/efeitos dos fármacos , Organofosfonatos , Oxirredutases/metabolismo , Pró-Fármacos/metabolismo , Nucleotídeos de Timina/farmacologia , Zidovudina/análogos & derivados , Adenina/metabolismo , Adenina/farmacologia , Antivirais/síntese química , Antivirais/metabolismo , Linfócitos B/citologia , Linhagem Celular , Didesoxinucleosídeos/metabolismo , Didesoxinucleotídeos , Ésteres/síntese química , Ésteres/metabolismo , Humanos , Linfócitos T/citologia , Nucleotídeos de Timina/metabolismo , Uridina Monofosfato/análogos & derivados , Zidovudina/metabolismo , Zidovudina/farmacologiaRESUMO
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.
Assuntos
HIV-1/efeitos dos fármacos , Zidovudina/análogos & derivados , Linhagem Celular , Didesoxinucleotídeos , Estabilidade de Medicamentos , Humanos , Pró-Fármacos/metabolismo , Linfócitos T/citologia , Nucleotídeos de Timina/farmacologia , Replicação Viral/efeitos dos fármacos , Zidovudina/farmacologiaRESUMO
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.
Assuntos
Antivirais/farmacologia , Didesoxinucleosídeos/farmacologia , HIV-1/efeitos dos fármacos , Nucleotídeos de Uracila/farmacologia , Linhagem Celular , Didesoxinucleosídeos/síntese química , Didesoxinucleotídeos , Estabilidade de Medicamentos , HIV-1/fisiologia , Humanos , Lipossomos , Fosforilação , Nucleotídeos de Uracila/síntese química , Uridina Monofosfato/análogos & derivados , Replicação Viral/efeitos dos fármacosRESUMO
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.
Assuntos
Antivirais/síntese química , Nucleotídeos/síntese química , Pró-Fármacos/síntese química , Aciclovir/análogos & derivados , Animais , Antivirais/química , Antivirais/metabolismo , Desenho de Fármacos , Humanos , Indicadores e Reagentes , Modelos Biológicos , Nucleotídeos/química , Nucleotídeos/metabolismo , Pró-Fármacos/química , Pró-Fármacos/metabolismo , Relação Estrutura-Atividade , Zidovudina/análogos & derivadosRESUMO
A rapid procedure for extraction and partial purification of ciguatoxin has been achieved and compared to one of the routine methods. Fish from two species which provided extracts of differing purity by the routine method were used. From 8 g of raw flesh, 1.0 +/- 0.2 mg of a semi-purified extract of ciguatoxin of homogeneous quality was obtained within 1 hr, whatever the species or toxicity of the fish. The results were reproducible, making the procedure very promising.
Assuntos
Ciguatoxinas/isolamento & purificação , Peixes Venenosos/metabolismo , Toxinas Marinhas/isolamento & purificação , Acetona , Animais , Ciguatoxinas/toxicidade , Dose Letal MedianaRESUMO
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.
Assuntos
Fármacos Anti-HIV/farmacologia , Pró-Fármacos/farmacologia , Inibidores da Transcriptase Reversa/farmacologia , Zidovudina/farmacologia , Administração Oral , Fármacos Anti-HIV/administração & dosagem , Fármacos Anti-HIV/química , Células CACO-2 , Cromatografia Líquida de Alta Pressão , Estabilidade de Medicamentos , HIV-1/efeitos dos fármacos , Humanos , Testes de Sensibilidade Microbiana , Pró-Fármacos/administração & dosagem , Pró-Fármacos/química , Inibidores da Transcriptase Reversa/administração & dosagem , Inibidores da Transcriptase Reversa/química , Análise Espectral , Zidovudina/administração & dosagem , Zidovudina/químicaRESUMO
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.
Assuntos
HIV/efeitos dos fármacos , Zidovudina/análogos & derivados , Zidovudina/química , Linhagem Celular , Didesoxinucleotídeos , Estabilidade de Medicamentos , Humanos , Indicadores e Reagentes , Timidina Quinase/deficiência , Nucleotídeos de Timina/farmacocinética , Tirosina/análogos & derivados , Zidovudina/farmacocinéticaRESUMO
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.
Assuntos
Oligonucleotídeos/farmacocinética , Pró-Fármacos/farmacocinética , Nucleotídeos de Pirimidina/farmacocinética , Timidina/análogos & derivados , Biotransformação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
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.
Assuntos
Antimetabólitos Antineoplásicos/análise , Cromatografia Líquida de Alta Pressão/métodos , Fluoruracila/análogos & derivados , Pró-Fármacos/análise , Antimetabólitos Antineoplásicos/sangue , Antimetabólitos Antineoplásicos/farmacocinética , Biotransformação , Fluoruracila/sangue , Fluoruracila/farmacocinética , Humanos , Pró-Fármacos/farmacocinéticaRESUMO
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.
Assuntos
Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/farmacologia , Pró-Fármacos/síntese química , Pró-Fármacos/farmacologia , Zidovudina/análogos & derivados , Fármacos Anti-HIV/química , Células Cultivadas , Desenho de Fármacos , Estabilidade de Medicamentos , Ésteres/síntese química , Ésteres/química , Ésteres/farmacologia , HIV-1/efeitos dos fármacos , HIV-1/fisiologia , Humanos , Pró-Fármacos/química , Replicação Viral/efeitos dos fármacos , Zidovudina/síntese química , Zidovudina/farmacologiaRESUMO
A reconstituted mixture of five cross-linked dinucleosides possibly involved in DNA-nitrosourea interactions, and of their degradation products (nucleobases, deoxynucleosides and mono- or disubstituted deoxynucleosides), was analysed by reversed-phase high-performance liquid chromatography using C18 columns and an diode-array detector. The chromatographic conditions for separating the twenty-one investigated compounds were optimized, and the compounds were identified by both their retention times and their UV spectra. A structure-retention time relationship was observed under suitable conditions and is discussed. Its validity was confirmed by the prediction of the retention time of a new cross-linked dinucleoside synthesized for this purpose.
Assuntos
DNA/análise , Compostos de Nitrosoureia/análise , Nucleosídeos/análise , Cromatografia Líquida de Alta Pressão , Concentração de Íons de Hidrogênio , Espectrofotometria UltravioletaRESUMO
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.
Assuntos
Exonucleases/metabolismo , Oligonucleotídeos Antissenso/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Animais , Sequência de Bases , Cromatografia Líquida de Alta Pressão , Estabilidade de Medicamentos , Humanos , Cinética , Dados de Sequência Molecular , Oligonucleotídeos Antissenso/química , Fosfodiesterase I , Venenos de Serpentes/enzimologiaRESUMO
Reversed-phase high-performance liquid chromatography using a C18 column was applied to the analysis of reconstituted mixtures of previously synthesized alpha, beta D-xylo- and D-lyxofuranonucleosides as well as a number of commercially available D-ribo- and D-arabinofuranonucleosides. From a detailed study of various parameters (size of support particles, nature and pH of the mobile phase, temperature), optimized conditions were established. Correlations between the retention times and structures of the bases, the orientations of the secondary hydroxyl groups of the sugar moiety and the anomeric configurations of the nucleosides are also reported.
Assuntos
Nucleosídeos/análise , Fenômenos Químicos , Química , Cromatografia Líquida de Alta Pressão , Concentração de Íons de Hidrogênio , Tamanho da Partícula , Temperatura , Xilose/análogos & derivados , Xilose/análiseRESUMO
For the first time, carbo-oligodeoxynucleotides, namely c-dT4 and c-dT12, have been synthesized. As compared to the natural oligomers these carbo-oligodeoxynucleotides are at least 5 times more stable toward enzymatic degradation and bind more strongly to complementary DNA. These preliminary data indicate that such oligomers fulfill the requirements to be considered as potential antisense agents.
Assuntos
Carboidratos , Oligodesoxirribonucleotídeos/síntese química , Composição de Bases , Indicadores e Reagentes , Cinética , Espectroscopia de Ressonância Magnética/métodos , Conformação de Ácido Nucleico , Desnaturação de Ácido Nucleico , Diester Fosfórico Hidrolases/metabolismo , TermodinâmicaRESUMO
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.
Assuntos
Extratos Celulares , Oligonucleotídeos/farmacocinética , Pró-Fármacos/farmacocinética , Biotransformação , Oligonucleotídeos/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Timidina/químicaRESUMO
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.