RESUMO
GS-9191 is a novel double prodrug of the nucleotide analog 9-(2-phosphonylmethoxyethyl)guanine (PMEG) designed as a topical agent to permeate skin and be metabolized to the active nucleoside triphosphate analog in the epithelial layer. The prodrug was shown to be metabolized intracellularly to 9-(2-phosphonylmethoxyethyl)-N(6)-cyclopropyl-2,6,diaminopurine (cPrPMEDAP) and subsequently deaminated to PMEG. The active form, PMEG diphosphate, was shown to be a potent inhibitor of DNA polymerase alpha and beta while showing weaker activity against mitochondrial DNA polymerase gamma (50% enzyme inhibition observed at 2.5, 1.6, and 59.4 microM, respectively). GS-9191 was markedly more potent than PMEG or cPrPMEDAP in a series of human papillomavirus (HPV)-positive cell lines, with effective concentrations to inhibit 50% cell growth (EC(50)) as low as 0.03, 207, and 284 nM, respectively. In contrast, GS-9191 was generally less potent in non-HPV-infected cells and primary cells (EC(50)s between 1 and 15 nM). DNA synthesis was inhibited by GS-9191 within 24 h of treatment; cells were observed to be arrested in S phase by 48 h and to subsequently undergo apoptosis (between 3 and 7 days). In an animal model (cottontail rabbit papillomavirus), topical GS-9191 was shown to decrease the size of papillomas in a dose-related manner. At the highest dose (0.1%), cures were evident at the end of 5 weeks, and lesions did not recur in a 30-day follow-up period. These data suggest that GS-9191 may have utility in the treatment of HPV-induced lesions.
Assuntos
Antivirais/farmacologia , Guanina/análogos & derivados , Guanina/farmacologia , Fenilalanina/análogos & derivados , Pró-Fármacos/farmacologia , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Antivirais/uso terapêutico , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Feminino , Guanina/uso terapêutico , Humanos , Masculino , Inibidores da Síntese de Ácido Nucleico , Fenilalanina/farmacologia , Fenilalanina/uso terapêutico , Pró-Fármacos/uso terapêutico , Coelhos , Neoplasias do Colo do Útero/tratamento farmacológicoRESUMO
PURPOSE: GS-9219, a novel prodrug of the nucleotide analogue 9-(2-phosphonylmethoxyethyl)guanine (PMEG), was designed as a cytotoxic agent that preferentially targets lymphoid cells. Our objective was to characterize the antiproliferative activity, pharmacokinetics, pharmacodynamics, and safety of GS-9219. EXPERIMENTAL DESIGN: GS-9219 was selected through screening in proliferation assays and through pharmacokinetic screening. The activation pathway of GS-9219 was characterized in lymphocytes, and its cytotoxic activity was evaluated against a panel of hematopoietic and nonhematopoietic cell types. To test whether the prodrug moieties present in GS-9219 confer an advantage over PMEG in vivo, the pharmacokinetics, pharmacodynamics (lymph node germinal center depletion), and toxicity of equimolar doses of GS-9219 and PMEG were evaluated after i.v. administration to normal beagle dogs. Finally, proof of concept of the antitumor efficacy of GS-9219 was evaluated in five pet dogs with spontaneous, advanced-stage non-Hodgkin's lymphoma (NHL) following a single i.v. administration of GS-9219 as monotherapy. RESULTS: In lymphocytes, GS-9219 is converted to its active metabolite, PMEG diphosphate, via enzymatic hydrolysis, deamination, and phosphorylation. GS-9219 has substantial antiproliferative activity against activated lymphocytes and hematopoietic tumor cell lines. In contrast, resting lymphocytes and solid tumor lines were less sensitive to GS-9219. GS-9219, but not PMEG, depleted the germinal centers in lymphoid tissues of normal beagle dogs at doses that were tolerated. In addition, GS-9219 displayed significant in vivo efficacy in five dogs with spontaneous NHL after a single administration, with either no or low-grade adverse events. CONCLUSION: GS-9219 may have utility for the treatment of NHL.
Assuntos
Alanina/análogos & derivados , Antineoplásicos/uso terapêutico , Tecido Linfoide/metabolismo , Linfoma não Hodgkin/tratamento farmacológico , Pró-Fármacos/uso terapêutico , Purinas/uso terapêutico , Alanina/administração & dosagem , Alanina/efeitos adversos , Alanina/farmacocinética , Alanina/uso terapêutico , Animais , Antineoplásicos/efeitos adversos , Antineoplásicos/farmacocinética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cães , Guanina/análogos & derivados , Guanina/uso terapêutico , Tecido Linfoide/citologia , Tecido Linfoide/efeitos dos fármacos , Compostos Organofosforados/uso terapêutico , Pró-Fármacos/efeitos adversos , Pró-Fármacos/química , Pró-Fármacos/farmacocinética , Purinas/administração & dosagem , Purinas/efeitos adversos , Purinas/farmacocinética , Distribuição TecidualRESUMO
A phase I/II trial of a candidate vaccine to prevent HIV infection was carried out in Bangkok, Thailand, testing AIDSVAX B/E (VaxGen, Inc., Brisbane, CA), a bivalent subunit vaccine prepared by combining recombinant gp120 from a subtype B virus (HIV-1MN) with gp120 from a subtype E virus (HIV-1A244) in alum adjuvant. The studies provide human data on the immunogenicity of various dose combination of non-subtype B vaccine antigens. The results suggest that AIDSVAX B/E is safe and immunogenic in humans. The optimal dose for humans in developing countries was 300 microg of each antigen (B and E). Clade E responses were measurably increased by immunizing with gp120 B/E over B alone. Using the B/E combination did not interfere with the response to either clade. Antibodies to AIDSVAX B/E were able to bind to oligomeric gp120 on the surface of cells infected with primary isolates of HIV-1.
Assuntos
Vacinas contra a AIDS/efeitos adversos , Vacinas contra a AIDS/imunologia , Proteína gp120 do Envelope de HIV/imunologia , Infecções por HIV/prevenção & controle , HIV-1/classificação , HIV-1/imunologia , Vacinas contra a AIDS/administração & dosagem , Adjuvantes Imunológicos/administração & dosagem , Adjuvantes Imunológicos/efeitos adversos , Adolescente , Adulto , Compostos de Alúmen , Feminino , Anticorpos Anti-HIV/sangue , Proteína gp120 do Envelope de HIV/genética , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/genética , Humanos , Esquemas de Imunização , Masculino , Pessoa de Meia-Idade , Proteínas Recombinantes/imunologia , Abuso de Substâncias por Via Intravenosa/complicações , Tailândia , VacinaçãoRESUMO
A chemokine receptor from the seven-transmembrane-domain G-protein-coupled receptor superfamily is an essential coreceptor for the cellular entry of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) strains. To investigate nonhuman primate CC-chemokine receptor 5 (CCR5) homologue structure and function, we amplified CCR5 DNA sequences from peripheral blood cells obtained from 24 representative species and subspecies of the primate suborders Prosimii (family Lemuridae) and Anthropoidea (families Cebidae, Callitrichidae, Cercopithecidae, Hylobatidae, and Pongidae) by PCR with primers flanking the coding region of the gene. Full-length CCR5 was inserted into pCDNA3.1, and multiple clones were sequenced to permit discrimination of both alleles. Compared to the human CCR5 sequence, the CCR5 sequences of the Lemuridae, Cebidae, and Cercopithecidae shared 87, 91 to 92, and 96 to 99% amino acid sequence homology, respectively. Amino acid substitutions tended to cluster in the amino and carboxy termini, the first transmembrane domain, and the second extracellular loop, with a pattern of species-specific changes that characterized CCR5 homologues from primates within a given family. At variance with humans, all primate species examined from the suborder Anthropoidea had amino acid substitutions at positions 13 (N to D) and 129 (V to I); the former change is critical for CD4-independent binding of SIV to CCR5. Within the Cebidae, Cercopithecidae, and Pongidae (including humans), CCR5 nucleotide similarities were 95.2 to 97.4, 98.0 to 99.5, and 98.3 to 99.3%, respectively. Despite this low genetic diversity, the phylogeny of the selected primate CCR5 homologue sequences agrees with present primate systematics, apart from some intermingling of species of the Cebidae and Cercopithecidae. Constructed HOS.CD4 cell lines expressing the entire CCR5 homologue protein from each of the Anthropoidea species and subspecies were tested for their ability to support HIV-1 and SIV entry and membrane fusion. Other than that of Cercopithecus pygerythrus, all CCR5 homologues tested were able to support both SIV and HIV-1 entry. Our results suggest that the shared structure and function of primate CCR5 homologue proteins would not impede the movement of primate immunodeficiency viruses between species.
Assuntos
HIV-1/fisiologia , Haplorrinos/virologia , Receptores CCR5/química , Vírus da Imunodeficiência Símia/fisiologia , Strepsirhini/virologia , Sequência de Aminoácidos , Animais , Haplorrinos/classificação , Humanos , Fusão de Membrana , Dados de Sequência Molecular , Filogenia , Receptores CCR5/fisiologia , Homologia de Sequência , Strepsirhini/classificaçãoRESUMO
An effective prophylactic vaccine should reduce frequency of new HIV infections in the target population and delay onset of immunodeficiency among those who become infected after vaccination. A variety of vaccine candidates have been developed, which induce neutralizing antibodies and/or cytotoxic T-lymphocytes. While many of those vaccine candidates exhibited some efficacy in primate model systems, their efficacy against natural HIV-1 infection can only be determined in large-scale phase III clinical trials. In this article, difficulties in HIV vaccine development will be discussed from scientific, technical, and business point of views.
Assuntos
Vacinas contra a AIDS , Síndrome da Imunodeficiência Adquirida/prevenção & controle , Animais , Ensaios Clínicos como Assunto , Anticorpos Anti-HIV , Proteína gp120 do Envelope de HIV/imunologia , HIV-1/imunologia , Humanos , Linfócitos T Citotóxicos/imunologiaRESUMO
DNA shuffling facilitated the evolution of a human immunodeficiency virus type 1 (HIV-1) variant with enhanced replication in pig-tailed macaque peripheral blood mononuclear cells (pt mPBMC). This variant consists exclusively of HIV-1-derived sequences with the exception of simian immunodeficiency virus (SIV) nef. Sequences spanning the gag-protease-reverse transcriptase (gag-pro-RT) region from several HIV-1 isolates were shuffled and cloned into a parental HIV-1 backbone containing SIV nef. Neither this full-length parent nor any of the unshuffled HIV-1 isolates replicated appreciably or sustainably in pt mPBMC. Upon selection of the shuffled viral libraries by serial passaging in pt mPBMC, a species emerged which replicated at substantially higher levels (50 to 100 ng/ml p24) than any of the HIV-1 parents and most importantly, could be continuously passaged in pt mPBMC. The parental HIV-1 isolates, when selected similarly, became extinct. Analyses of full-length improved proviral clones indicate that multiple recombination events in the shuffled region and adaptive changes in the rest of the genome contributed synergistically to the improved phenotype. This improved variant may prove useful in establishing a pig-tailed macaque model of HIV-1 infection.
Assuntos
DNA Recombinante/genética , Variação Genética , HIV-1/genética , Leucócitos Mononucleares/virologia , Macaca nemestrina , Replicação Viral , Animais , Células Cultivadas , Evolução Molecular , Biblioteca Genômica , Infecções por HIV/virologia , HIV-1/classificação , HIV-1/fisiologia , Humanos , Dados de Sequência Molecular , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/genética , Vírus da Imunodeficiência Símia/fisiologia , Proteínas Virais/genética , Proteínas Virais/metabolismo , Proteínas Virais Reguladoras e Acessórias/genéticaRESUMO
We previously reported that high-titered neutralizing antibodies directed against the human immunodeficiency virus type 1 (HIV-1) envelope can block the establishment of a simian immunodeficiency virus (SIV)/HIV chimeric virus (SHIV) infection in two monkeys following passive transfer (R. Shibata et al., Nat. Med. 5:204-210, 1999). In the present study, increasing amounts of neutralizing immunoglobulin G (IgG) were administered to 15 pig-tailed macaques in order to obtain a statistically valid protective neutralization endpoint titer in plasma. Using an in vitro assay which measures complete neutralization of the challenge SHIV, we correlated the titers of neutralizing antibodies in plasma at the time of virus inoculation (which ranged from 1:3 to 1:123) with the establishment of infection in virus-challenged animals. Ten of 15 monkeys in the present experiment were virus free as a result of neutralizing IgG administration as monitored by DNA PCR (peripheral blood mononuclear cells and lymph node cells), RNA PCR (plasma), virus isolation, and the transfer of lymph node cell suspensions (10(8) cells) plus 8 ml of whole blood from protected animals to naïve macaques. The titer of neutralizing antibodies in the plasma calculated to protect 99% of virus-challenged monkeys was 1:38.