RESUMO
Several novel compounds were found to be potent inhibitors of the HCV (JFH-1 isolate) infection in vitro. Human serum did not significantly reduce antiviral activity of the lead compound, AVR560 (< 4-fold). The immunohistochemistry studies with the Huh7 cell line, infectable with the HCV (JFH-1 strain), demonstrated that AVR560 inhibited the early steps of viral infection and blocked the spread of the HCV infection in tissue culture. The cytotoxicity in Huh7 and Vero-76 cell lines was mild. AVR560 proved to be a specific HCV inhibitor and exhibited no activity against other flaviviruses such as yellow fever (strain 17D), West Nile (strain NY99), and dengue (New Guinea type 2) in in vitro infection experiments. AVR560 also did not inhibit any of the tested human CYP450 isozymes (3A4, 1A2, 2C19 and 2D6). In the pharmacokinetic studies in mice, rats and dogs, favorable pharmacokinetic profiles and good oral bioavailability were observed for AV560. Further pre-clinical studies with this novel HCV inhibitor are in progress.
Assuntos
Antivirais/farmacologia , Hepacivirus/efeitos dos fármacos , Piperazinas/farmacologia , Piperidinas/farmacologia , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Animais , Antivirais/farmacocinética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Chlorocebus aethiops , Sistema Enzimático do Citocromo P-450/metabolismo , Vírus da Dengue/efeitos dos fármacos , Vírus da Dengue/crescimento & desenvolvimento , Cães , Avaliação Pré-Clínica de Medicamentos , Hepacivirus/fisiologia , Hepatite C/tratamento farmacológico , Hepatite C/virologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/virologia , Humanos , Camundongos , Piperazinas/farmacocinética , Piperidinas/farmacocinética , Ratos , Células Vero , Vírus do Nilo Ocidental/efeitos dos fármacos , Vírus do Nilo Ocidental/crescimento & desenvolvimento , Vírus da Febre Amarela/efeitos dos fármacos , Vírus da Febre Amarela/crescimento & desenvolvimentoRESUMO
In vitro immunohistochemical investigations on the human hepatoma cell line (Huh7) infected with hepatitis C virus (HCV) strain JFH-1 showed that AV0012 compound blocks the early stages of viral infection. AV0012 also blocked viral infection spread in tissue culture through the secreted virus and through tight cell-to-cell contact. AV0012 is a specific inhibitor of HCV but not of related pestivirus, flaviviruses and other RNA-containing viruses such as bovine diarrhea (BVDV), Venezuelan equine encephalitis (strain TC-83), dengue type 2 (New Guinea), yellow fever (strain 17D), west Nile fever, parainfluenza (type 3) virus, RSV (strain A2), and Rhinovirus (type 2 strain HGP). It is established that human serum does not significantly affect the antiviral activity of AV0012 in vitro. The drug combination studies with AV0012 and interferon alpha 2a in vitro showed that the two inhibitors act additively, which makes possible the use of this combination in clinical tests. AV0012 is highly soluble and stable in aqueous solutions and murine blood plasma, has limited metabolic stability, low binding to human plasma proteins, high permeability through biological membranes, and only interacts with isoenzymes 2D6 and 3A4 of human cytochrome P450. In animal pharmacokinetic studies, AV0012 was rapidly absorbed into the blood stream upon oral administration, showed sufficiently long half-elimination times, and had high oral bioavailability that reached 92% in monkeys. Further preclinical development of AV0012 is in progress.
Assuntos
Antivirais/farmacologia , Antivirais/farmacocinética , Hepacivirus/metabolismo , Hepatite C/tratamento farmacológico , Hepatite C/metabolismo , Animais , Antivirais/química , Bovinos , Linhagem Celular , Avaliação Pré-Clínica de Medicamentos , Flavivirus/metabolismo , Infecções por Flavivirus/tratamento farmacológico , Infecções por Flavivirus/metabolismo , Haplorrinos , Humanos , Camundongos , RatosRESUMO
Pharmacological safety of a new type of HCV inhibitor, AV0012, was studied including acute, subchronic and chronic toxicity in mice, rats and monkeys. Genotoxicity was assessed using the Ames test and the chromosomal aberrations assay in the bone marrow cells of mice. It is established that AV0012 has low toxicity in SHK line mice, Wistar line rats, and monkey of Rhesus macaques species. Results obtained in the study of genetic toxicity showed that AV0012 exhibits no mutagenic activity. Data on general toxicity and mutagenicity discussed in this paper, together with data on 1 the pharmacological activity, pharmacokinetics, and metabolism published previously, allow us to consider AV0012 as a candidate drug for clinical research phase I.
Assuntos
Antivirais/toxicidade , Hepatite C/tratamento farmacológico , Indóis/toxicidade , Piridinas/toxicidade , Animais , Antivirais/uso terapêutico , Avaliação Pré-Clínica de Medicamentos , Feminino , Indóis/uso terapêutico , Macaca mulatta , Masculino , Dose Máxima Tolerável , Camundongos Endogâmicos , Estrutura Molecular , Testes de Mutagenicidade , Piridinas/uso terapêutico , Ratos Wistar , Testes de Toxicidade Aguda , Testes de Toxicidade Crônica , Testes de Toxicidade SubcrônicaAssuntos
Capsídeo/genética , Genes Virais , Antígenos do Núcleo do Vírus da Hepatite B/genética , Peptídeos/genética , Anticorpos Monoclonais , Capsídeo/imunologia , Clonagem Molecular , Ensaio de Imunoadsorção Enzimática , Escherichia coli/genética , Expressão Gênica , Antígenos do Núcleo do Vírus da Hepatite B/química , Espectroscopia de Ressonância Magnética , Peptídeos/química , Peptídeos/imunologia , Recombinação GenéticaRESUMO
A set of recombinant plasmids with a gene encoding surface antigen of hepatitis B virus (HBsAg) is constructed. The plasmids were transfected by DEAE-dextran method into different lines of cultured cells and transient expression of the HBsAg gene was studied. The results indicate that: transcriptional enhancer of hepatitis B virus situated downstream from HBsAg gene is active in green monkey kidney cells (CVI), promoter of 5 LTR of bovine leukemia virus is trans-activated in the goat or calf cells infected with BLV. The results are discussed in the light of hypothesis on the role of transcriptional enhancers in determination of tissue-specificity of hepatitis B virus.
Assuntos
Regulação da Expressão Gênica , Genes Virais , Código Genético , Antígenos de Superfície da Hepatite B/genética , Vírus da Hepatite B/genética , Transformação Genética , Animais , Células Cultivadas , Humanos , Plasmídeos , Regiões Promotoras Genéticas , Recombinação Genética , Transcrição GênicaRESUMO
The nucleotide sequence of the 3'-terminal region of the cloned bovine leukaemia virus cDNA (1474 bp) was elucidated using both Sanger and Maxam-Gilbert techniques. This DNA region contains U3 and R parts of the BLV LTR and an upstream sequence with four open reading frames (ORF) of unknown function. The comparison of the nucleotide substitutions in these ORF with the two variants of proviral BLV DNA suggests that the only pX1 ORF possesses a coding function. The role of the pX1 protein is discussed.
Assuntos
DNA Viral/análise , DNA/análise , Vírus da Leucemia Bovina/genética , Retroviridae/genética , Animais , Sequência de Bases , Bovinos , Clonagem Molecular , DNA Viral/genéticaAssuntos
Vírus da Leucemia Bovina/genética , Leucemia Experimental/genética , Regiões Promotoras Genéticas , Sequências Repetitivas de Ácido Nucleico , Retroviridae/genética , Ativação Viral , Animais , Linhagem Celular , Células Cultivadas , DNA Viral/genética , Genes Virais , Antígenos de Superfície da Hepatite B/análise , Plasmídeos , Ovinos , Transfecção , Proteínas Virais/análiseAssuntos
Aminoácidos/biossíntese , Escherichia coli/metabolismo , Antígenos de Superfície da Hepatite B , Sequência de Aminoácidos , Aminoácidos/genética , Sequência de Bases , Quimera , Escherichia coli/genética , Escherichia coli/imunologia , Regulação da Expressão Gênica , Genes Virais , Antígenos de Superfície da Hepatite B/genética , PlasmídeosAssuntos
Antígenos de Superfície , Antígenos Virais , Escherichia coli/imunologia , Vírus da Hepatite B/imunologia , Sequência de Aminoácidos , Antígenos de Superfície/genética , Antígenos Virais/genética , Sequência de Bases , Clonagem Molecular , Escherichia coli/genética , Regulação da Expressão Gênica , Genes Bacterianos , Genes Virais , Vírus da Hepatite B/genéticaRESUMO
The entire hepatitis B virus (HBV) genome and its fragments have been cloned into the BamHI site of the plasmid pBR322 vector. The identity of physical maps of cloned and authentic virion DNAs was demonstrated by restriction enzyme analysis. The location of restriction sites is suggestive of a certain similarity between the studied HBV DNA and HBV DNA, subtype ayw (Galibert et al., 1979). From the restriction enzyme analysis of virion DNA repaired and 32P-labeled by the endogenous DNA-polymerase reaction, the new information concerning the location and maximal length (approximately 1500 nucleotides) of the single-stranded region of HBV DNA has been established.
Assuntos
Clonagem Molecular , Genes Virais , Vírus da Hepatite B/genética , Vírion/genética , Sequência de Bases , Enzimas de Restrição do DNA , DNA Viral/genética , PlasmídeosRESUMO
A series of plasmids with tetracycline resistance genes (Tcr-operon) subjected to transcription from chloramphenicol acetyl transferase promoter (Cmr-promoter) have been constructed on the basis of plasmid pBR325, AprCmrTcr. For this purpose, a 0.8 Md fragment in pBR325 DNA bordered by unique EcoRI and HindIII restriction sites was cut out and structural genes of Tcr-operon were fused to the cat gene nucleotides corresponding to Cmr-promoter and first 72 amino acids of cat (alton, Vapnek, 1979; Marcoli et al., 1980). These plasmids with molecular weight amounting to 3 Md confer AprTcr phenotype to host cells. Tetracycline resistance can be eliminated completely by the deletion of a) Cmr-promoter; b) part of the first Tcr-operon gene.