RESUMO
SB 9200 is a novel, first-in-class oral modulator of innate immunity that is believed to act via the activation of the RIG-I and NOD2 pathways. SB 9200 has broad-spectrum antiviral activity against RNA viruses including hepatitis C virus (HCV), norovirus, respiratory syncytial virus, and influenza and has demonstrated activity against hepatitis B virus (HBV) in vitro and in vivo. In phase I clinical trials in chronically infected HCV patients, SB 9200 has been shown to reduce HCV RNA by up to 1.9 log10 . Here, we demonstrate the antiviral activity of SB 9200 against a HCV replicon system and patient derived virus. Using the HCV capture-fusion assay, we show that SB 9200 is active against diverse HCV genotypes and is also effective against HCV derived from patients who relapse following direct-acting antiviral treatment, including viruses containing known NS5A resistance-associated sequences. These data confirm the broad antiviral activity of SB 9200 and indicate that it may have clinical utility in HCV patients who have failed to respond to current antiviral regimens.
Assuntos
Antivirais/farmacologia , Hepacivirus/efeitos dos fármacos , Fatores Imunológicos/farmacologia , Genótipo , Hepacivirus/classificação , Hepacivirus/genética , Hepacivirus/isolamento & purificação , Hepatite C Crônica/virologia , Humanos , Testes de Sensibilidade MicrobianaRESUMO
The alkoxycarbonyloxy dinucleotide prodrug R(p), S(p)-2 is an orally bioavailable anti-hepatitis B virus agent. The compound is efficiently metabolized to the active dinucleoside phosphorothioate R(p), S(p)-1 by human liver microsomes and S9 fraction without cytochrome P450-mediated oxidation or conjugation. The conversion of R(p), S(p)-2 to R(p), S(p)-1 appears to be mediated by liver esterases, occurs in a stereospecific manner, and is consistent with our earlier reported studies of serum-mediated hydrolytic conversion of R(p), S(p)-2 to R(p), S(p)-1. However, further metabolism of R(p), S(p)-1 does not occur. The presence of a minor metabolite, the desulfurized product 10 was noted. The prodrug R(p), S(p)-2 was quite stable in simulated gastric fluid, whereas the active R(p), S(p)-1 had a half-life of <15 min. In simulated intestinal fluid, the prodrug 2 was fully converted to 1 in approximately 3 h, whereas 1 remained stable. To ascertain the tissue distribution of the prodrug 2 in rats, the synthesis of (35)S-labeled R(p), S(p)-2 was undertaken. Tissue distribution studies of orally and intravenously administered radiolabeled [(35)S]2 demonstrated that the radioactivity concentrates in the liver, with the highest liver/plasma ratio in the intravenous group at 1 h being 3.89 (females) and in the oral group at 1 h being 2.86 (males). The preferential distribution of the dinucleotide 1 and its prodrug 2 into liver may be attributed to the presence of nucleoside phosphorothioate backbone because phosphorothioate oligonucleotides also reveal a similar tissue distribution profile upon intravenous administration.
Assuntos
Antivirais , Vírus da Hepatite B/efeitos dos fármacos , Oligonucleotídeos Fosforotioatos , Pró-Fármacos , Administração Oral , Animais , Antivirais/química , Antivirais/metabolismo , Antivirais/farmacocinética , Biotransformação , Cromatografia Líquida de Alta Pressão , Desenho de Fármacos , Estabilidade de Medicamentos , Feminino , Suco Gástrico/química , Humanos , Técnicas In Vitro , Injeções Intravenosas , Secreções Intestinais/química , Masculino , Espectrometria de Massas , Microssomos Hepáticos/enzimologia , Microssomos Hepáticos/metabolismo , Modelos Biológicos , Estrutura Molecular , Oligonucleotídeos Fosforotioatos/química , Oligonucleotídeos Fosforotioatos/metabolismo , Oligonucleotídeos Fosforotioatos/farmacocinética , Pró-Fármacos/química , Pró-Fármacos/metabolismo , Pró-Fármacos/farmacocinética , Ratos , Ratos Sprague-Dawley , Estereoisomerismo , Distribuição TecidualRESUMO
SARS-CoV-2 is a novel virus that has rapidly spread, causing a global pandemic. In the majority of infected patients, SARS-CoV-2 leads to mild disease; however, in a significant proportion of infections, individuals develop severe symptoms that can lead to long-lasting lung damage or death. These severe cases are often associated with high levels of pro-inflammatory cytokines and low antiviral responses, which can cause systemic complications. Here, we have evaluated transcriptional and cytokine secretion profiles and detected a distinct upregulation of inflammatory cytokines in infected cell cultures and samples taken from infected patients. Building on these observations, we found a specific activation of NF-κB and a block of IRF3 nuclear translocation in SARS-CoV-2 infected cells. This NF-κB response was mediated by cGAS-STING activation and could be attenuated through several STING-targeting drugs. Our results show that SARS-CoV-2 directs a cGAS-STING mediated, NF-κB-driven inflammatory immune response in human epithelial cells that likely contributes to inflammatory responses seen in patients and could be therapeutically targeted to suppress severe disease symptoms.
Assuntos
COVID-19/metabolismo , Síndrome da Liberação de Citocina , Mediadores da Inflamação/metabolismo , Proteínas de Membrana/metabolismo , NF-kappa B/metabolismo , Nucleotidiltransferases/metabolismo , COVID-19/virologia , Humanos , SARS-CoV-2/isolamento & purificação , Transdução de SinaisRESUMO
The acyloxyalkyl derivatives of a model anti-HBV dinucleotide were synthesized and evaluated as orally bioavailable prodrugs. Our studies have led to the identification of the first orally bioavailable dinucleotide prodrugs for further therapeutic development against the hepatitis B virus (HBV).
Assuntos
Antivirais/química , Vírus da Hepatite B/genética , Hepatite B/tratamento farmacológico , Nucleotídeos/química , Pró-Fármacos/química , Administração Oral , Animais , Antivirais/síntese química , Antivirais/uso terapêutico , Linhagem Celular , Feminino , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Mutação , Nucleotídeos/síntese química , Nucleotídeos/uso terapêutico , Pró-Fármacos/síntese química , Pró-Fármacos/uso terapêutico , Ratos , Ratos Sprague-DawleyRESUMO
Tuberculosis (TB) caused by Mycobacterium tuberculosis (MTB) kills about 1.5 million people each year and the widely used Bacille Calmette-Guérin (BCG) vaccine provides a partial protection against TB in children and adults. Because BCG vaccine evades lysosomal fusion in antigen presenting cells (APCs), leading to an inefficient production of peptides and antigen presentation required to activate CD4 T cells, we sought to boost its efficacy using novel agonists of RIG-I and NOD2 as adjuvants. We recently reported that the dinucleotide SB 9200 (Inarigivir) derived from our small molecule nucleic acid hybrid (SMNH)® platform, activated RIG-I and NOD2 receptors and exhibited a broad-spectrum antiviral activity against hepatitis B and C, Norovirus, RSV, influenza and parainfluenza. Inarigivir increased the ability of BCG-infected mouse APCs to secrete elevated levels of IL-12, TNF-α, and IFN-ß, and Caspase-1 dependent IL-1ß cytokine. Inarigivir also increased the ability of macrophages to kill MTB in a Caspase-1-, and autophagy-dependent manner. Furthermore, Inarigivir led to a Capsase-1 and NOD2- dependent increase in the ability of BCG-infected APCs to present an Ag85B-p25 epitope to CD4 T cells in vitro. Consistent with an increase in immunogenicity of adjuvant treated APCs, the Inarigivir-BCG vaccine combination induced robust protection against tuberculosis in a mouse model of MTB infection, decreasing the lung burden of MTB by 1-log10 more than that afforded by BCG vaccine alone. The Inarigivir-BCG combination was also more efficacious than a muramyl-dipeptide-BCG vaccine combination against tuberculosis in mice, generating better memory T cell responses supporting its novel adjuvant potential for the BCG vaccine.
Assuntos
Adjuvantes Imunológicos , Vacina BCG/imunologia , Mycobacterium tuberculosis/imunologia , Proteína Adaptadora de Sinalização NOD2/metabolismo , Receptores de Superfície Celular/metabolismo , Tuberculose/metabolismo , Tuberculose/prevenção & controle , Adjuvantes Imunológicos/química , Adjuvantes Imunológicos/farmacologia , Animais , Apresentação de Antígeno/imunologia , Antígenos de Bactérias/imunologia , Homólogo 5 da Proteína Cromobox , Citocinas/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Memória Imunológica , Imunomodulação , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Mycobacterium tuberculosis/efeitos dos fármacos , Ligação Proteica , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Tuberculose/genéticaRESUMO
SB 9200, an orally bioavailable dinucleotide, activates the viral sensor proteins, retinoic acid-inducible gene 1 (RIG-I) and nucleotide-binding oligomerization domain-containing protein 2 (NOD2) causing the induction of the interferon (IFN) signaling cascade for antiviral defense. The present study evaluated the overall antiviral response in woodchucks upon induction of immune response, first with SB 9200 followed by Entecavir (ETV) versus reduction of viral burden with ETV followed by SB 9200 immunomodulation. Woodchucks chronically infected with woodchuck hepatitis virus (WHV) were treated orally with SB 9200 (30 mg/kg/day) and ETV (0.5 mg/kg/day). Group 1 received ETV for 4 weeks followed by SB 9200 for 12 weeks. Group 2 received SB 9200 for 12 weeks followed by ETV for 4 weeks. At the end of treatment in Group 2, average reductions of 6.4 log10 in serum WHV DNA and 3.3 log10 in WHV surface antigen were observed whereas in Group 1, average reductions of 4.2 log10 and 1.1 log10 in viremia and antigenemia were noted. Both groups demonstrated marked reductions in hepatic WHV nucleic acid levels which were more pronounced in Group 2. Following treatment cessation and the 8-week follow-up, recrudescence of viral replication was observed in Group 1 while viral relapse in Group 2 was significantly delayed. The antiviral effects observed in both groups were associated with temporally different induction of IFN-α, IFN-ß, and IFN-stimulated genes in blood and liver. These results suggest that the induction of host immune responses by pretreatment with SB 9200 followed by ETV resulted in antiviral efficacy that was superior to that obtained using the strategy of viral reduction with ETV followed by immunomodulation.
Assuntos
Antivirais/uso terapêutico , Guanina/análogos & derivados , Vírus da Hepatite B da Marmota/patogenicidade , Marmota/virologia , Animais , Guanina/uso terapêutico , Vírus da Hepatite B da Marmota/efeitos dos fármacos , Vírus da Hepatite B da Marmota/imunologia , Fígado/virologia , Replicação Viral/efeitos dos fármacosRESUMO
During the past decade, nucleotide analogs have emerged as novel antiviral agents against hepatitis B virus. Adefovir dipivoxil, a prototype phosphonate analog, has been approved for chronic hepatitis B virus therapy, and additional phosphonate analogs and di- and tri-nucleotides are under development. Several innovative prodrug derivatizations have also been reported to improve the oral bioavailability of nucleotide analogs, which usually carry a negative charge.
Assuntos
Antivirais/farmacologia , Vírus da Hepatite B/efeitos dos fármacos , Vírus da Hepatite B/genética , Nucleotídeos/farmacologia , Antivirais/química , Antivirais/metabolismo , Antivirais/uso terapêutico , Humanos , Nucleotídeos/química , Nucleotídeos/metabolismo , Nucleotídeos/uso terapêutico , Pró-Fármacos/química , Pró-Fármacos/metabolismoRESUMO
SB 9200, an oral prodrug of the dinucleotide SB 9000, is being developed for the treatment of chronic hepatitis B virus (HBV) infection and represents a novel class of antivirals. SB 9200 is thought to activate the viral sensor proteins, retinoic acid-inducible gene 1 (RIG-I) and nucleotide-binding oligomerization domain-containing protein 2 (NOD2) resulting in interferon (IFN) mediated antiviral immune responses in virus-infected cells. Additionally, the binding of SB 9200 to these sensor proteins could also sterically block the ability of the viral polymerase to access pre-genomic RNA for nucleic acid synthesis. The immune stimulating and direct antiviral properties of SB 9200 were evaluated in woodchucks chronically infected with woodchuck hepatitis virus (WHV) by daily, oral dosing at 15 and 30 mg/kg for 12 weeks. Prolonged treatment resulted in 2.2 and 3.7 log10 reductions in serum WHV DNA and in 0.5 and 1.6 log10 declines in serum WHV surface antigen from pretreatment level with the lower or higher dose of SB 9200, respectively. SB 9200 treatment also resulted in lower hepatic levels of WHV nucleic acids and antigen and reduced liver inflammation. Following treatment cessation, recrudescence of viral replication was observed but with dose-dependent delays in viral relapse. The antiviral effects were associated with dose-dependent and long-lasting induction of IFN-α, IFN-ß and IFN-stimulated genes in blood and liver, which correlated with the prolonged activation of the RIG-I/NOD2 pathway and hepatic presence of elevated RIG-I protein levels. These results suggest that in addition to a direct antiviral activity, SB 9200 induces antiviral immunity during chronic hepadnaviral infection via activation of the viral sensor pathway.
Assuntos
Antivirais/administração & dosagem , Hepatite B Crônica/tratamento farmacológico , Hepatite B Crônica/imunologia , Imunidade Inata/efeitos dos fármacos , Animais , Modelos Animais de Doenças , Vírus da Hepatite B da Marmota/efeitos dos fármacos , Vírus da Hepatite B da Marmota/patogenicidade , Vírus da Hepatite B/patogenicidade , Hepatite B Crônica/sangue , Hepatite B Crônica/virologia , Humanos , Interferon-alfa/sangue , Interferon beta/sangue , Fígado/metabolismo , Marmota/imunologia , Marmota/virologia , Replicação Viral/efeitos dos fármacosRESUMO
Ultra-fast and efficient functionalization of solid supports such as controlled-pore glass (CPG), amino methyl polystyrene, and Tentagel has been achieved using microwave-assisted procedures. Both amino- and carboxy-terminated supports are easily prepared within minutes, in a reproducible manner, using microwave-assisted methodologies. The resulting functionalized supports are efficiently coupled to nucleosides using dimethylformamide as a solvent in conjunction with a specially designed reactor and workstation called LOTUS. Using these improved protocols, CPG with loadings of 75 to 85 micromol/g can be prepared on a large scale within 3 to 4 days starting from native CPG, as opposed to traditional methods that require 10 to 15 days to achieve the same objective. In addition, the methods described here can potentially be employed for rapid functionalization of other solid matrices such as beads, slides, and pins for applications in microarrays or combinatorial chemistry.
Assuntos
Micro-Ondas , Nucleosídeos/química , Vidro , Reprodutibilidade dos TestesRESUMO
Several pronucleotide analogs of the model anti-HBV dinucleotide 3'-dA-U(2'OMe) have been synthesized and evaluated for stability, bioreversibility and cytotoxicity. These studies have helped identify potential candidates for further evaluation.