ABSTRACT
BACKGROUND/AIMS: Novel agents acting against hepatitis B virus (HBV) are needed to improve HBsAg seroclearance or termed as 'functional cure'. Inarigivir (retinoic acid-inducible gene I agonist) has immunomodulatory and direct antiviral actions against HBV. We aimed to determine the safety and efficacy of Inarigivir for the treatment of HBV infection. PATIENTS/METHODS: 80 treatment-naïve patients were randomized in 4 ascending dose cohorts to receive 12 weeks of Inarigivir 25, 50, 100, 200 mg or placebo in a ratio of 4:1. All patients were then given tenofovir for another 12 weeks. RESULTS: Least squares (LS) mean reductions in HBV DNA from baseline increased with higher doses of Inarigivir (0.6116 in 25 mg and 1.5774 in 200 mg groups vs. 0.0352 in placebo group) (95% CI 0.9518-0.2011 and 1.921-1.1634 respectively). LS mean changes in HBV RNA and HBsAg from baseline ranged from -0.3856 to -0.5794 versus -0.1474 and -0.0956 to -0.1818 versus +0.0026 in Inarigivir-treated versus placebo groups respectively. During the tenofovir-treated period, LS mean reductions in HBsAg in the Inarigivir-treated groups ranged from 0.1709 to 0.3529 versus 0.1984 in the placebo group. Inarigivir-treated groups showed mean reductions in ALT from baseline between 23.3 and 33.8 versus 0.7 U/L in the placebo group. Treatment-emergent adverse events related to Inarigivir and placebo occurred in 4.7% and 6.3% patients respectively. CONCLUSIONS: Twelve-week Inarigivir up to 200 mg dose was associated with a reduction of HBV DNA, HBV RNA and antigen levels. A trend for greater HBsAg reduction was observed in Inarigivir pre-treated patients after switching to tenofovir.
Subject(s)
Hepatitis B, Chronic , Hepatitis B , Humans , Hepatitis B Surface Antigens , DNA, Viral , Tenofovir/therapeutic use , Antiviral Agents/adverse effects , Hepatitis B/drug therapy , Hepatitis B virus/genetics , Hepatitis B e Antigens , RNA , Treatment OutcomeABSTRACT
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.
Subject(s)
Antiviral Agents/pharmacology , Hepacivirus/drug effects , Immunologic Factors/pharmacology , Genotype , Hepacivirus/classification , Hepacivirus/genetics , Hepacivirus/isolation & purification , Hepatitis C, Chronic/virology , Humans , Microbial Sensitivity TestsABSTRACT
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.
Subject(s)
Antiviral Agents , Hepatitis B virus/drug effects , Phosphorothioate Oligonucleotides , Prodrugs , Administration, Oral , Animals , Antiviral Agents/chemistry , Antiviral Agents/metabolism , Antiviral Agents/pharmacokinetics , Biotransformation , Chromatography, High Pressure Liquid , Drug Design , Drug Stability , Female , Gastric Juice/chemistry , Humans , In Vitro Techniques , Injections, Intravenous , Intestinal Secretions/chemistry , Male , Mass Spectrometry , Microsomes, Liver/enzymology , Microsomes, Liver/metabolism , Models, Biological , Molecular Structure , Phosphorothioate Oligonucleotides/chemistry , Phosphorothioate Oligonucleotides/metabolism , Phosphorothioate Oligonucleotides/pharmacokinetics , Prodrugs/chemistry , Prodrugs/metabolism , Prodrugs/pharmacokinetics , Rats , Rats, Sprague-Dawley , Stereoisomerism , Tissue DistributionABSTRACT
Quantitative structure-activity relationship (QSAR) is the most versatile tool in computer-assisted molecular design. One conceptual drawback seen in QSAR approaches is the "one chemical-one structure-one parameter value" dogma where the model development is based on physicochemical description for a single molecular conformation, while ignoring the rest of the conformational space. It is well known that molecules have several low-energy conformations populated at physiological temperature, and each conformer makes a significant impact on associated properties such as biological activity. At the level of molecular interaction, the dynamics around the molecular structure is of prime essence rather than the average structure. As a step toward understanding the role of these discrete microscopic states in biological activity, we have put together a theoretically rigorous and computationally tractable formalism coined as eQSAR. In this approach, the biological activity is modeled as a function of physicochemical description for a selected set of low-energy conformers, rather than that's for a single lowest energy conformation. Eigenvalues derived from the "Physicochemical property integrated distance matrices" (PD-matrices) that encompass both 3D structure and physicochemical properties, have been used as descriptors; is a novel addition. eQSAR is validated on three peptide datasets and explicitly elaborated for bradykinin-potentiating peptides. The conformational ensembles were generated by a simple molecular dynamics and consensus dynamics approaches. The eQSAR models are statistically significant and possess the ability to select the most biologically relevant conformation(s) with the relevant physicochemical attributes that have the greatest meaning for description of the biological activity.
Subject(s)
Drug Design , Models, Theoretical , Molecular Conformation , Peptides/chemistry , Quantitative Structure-Activity Relationship , Bradykinin/agonists , Molecular Dynamics SimulationABSTRACT
With the emergence of high throughput screening of bioactive molecules, there is constant need for the development of new strategies for diversity-oriented synthesis. We describe here a novel one-pot multicomponent reaction for the synthesis of uracil derivatives using easily available starting materials. This new synthetic strategy provides easy access to diverse uracil derivatives in moderate to good yields.
Subject(s)
Uracil/analogs & derivatives , Uracil/chemical synthesis , Acrylamides/chemistry , Amines/chemistry , Formates/chemistry , Nitriles/chemistry , Phosgene/analogs & derivatives , Phosgene/chemistryABSTRACT
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).
Subject(s)
Antiviral Agents/chemistry , Hepatitis B virus/genetics , Hepatitis B/drug therapy , Nucleotides/chemistry , Prodrugs/chemistry , Administration, Oral , Animals , Antiviral Agents/chemical synthesis , Antiviral Agents/therapeutic use , Cell Line , Female , Humans , Male , Mice , Mice, Transgenic , Mutation , Nucleotides/chemical synthesis , Nucleotides/therapeutic use , Prodrugs/chemical synthesis , Prodrugs/therapeutic use , Rats , Rats, Sprague-DawleyABSTRACT
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.
Subject(s)
Adjuvants, Immunologic , BCG Vaccine/immunology , Mycobacterium tuberculosis/immunology , Nod2 Signaling Adaptor Protein/metabolism , Receptors, Cell Surface/metabolism , Tuberculosis/metabolism , Tuberculosis/prevention & control , Adjuvants, Immunologic/chemistry , Adjuvants, Immunologic/pharmacology , Animals , Antigen Presentation/immunology , Antigens, Bacterial/immunology , Chromobox Protein Homolog 5 , Cytokines/metabolism , Dendritic Cells/immunology , Dendritic Cells/metabolism , Immunologic Memory , Immunomodulation , Macrophages/immunology , Macrophages/metabolism , Mice , Mycobacterium tuberculosis/drug effects , Protein Binding , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism , Tuberculosis/geneticsABSTRACT
Oligonucleotides carrying a variety of chemical modifications including conjugates are finding increasing applications in therapeutics, diagnostics, functional genomics, proteomics, and as research tools in chemical and molecular biology. The successful synthesis of oligonucleotides primarily depends on the use of appropriately protected nucleoside building blocks including the exocyclic amino groups of the nucleobases, the hydroxyl groups at the 2'-, 3'-, and 5'-positions of the sugar moieties, and the internucleotide phospho-linkage. This unit is a thoroughly revised update of the previously published version and describes the recent development of various protecting groups that facilitate reliable oligonucleotide synthesis. In addition, various protecting groups for the imide/lactam function of thymine/uracil and guanine, respectively, are described to prevent irreversible nucleobase modifications that may occur in the presence of reagents used in oligonucleotide synthesis. © 2017 by John Wiley & Sons, Inc.
Subject(s)
Deoxyribonucleosides/chemistry , Ribonucleosides/chemistry , Acetylation , PhotochemistryABSTRACT
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.
Subject(s)
Antiviral Agents/therapeutic use , Guanine/analogs & derivatives , Hepatitis B Virus, Woodchuck/pathogenicity , Marmota/virology , Animals , Guanine/therapeutic use , Hepatitis B Virus, Woodchuck/drug effects , Hepatitis B Virus, Woodchuck/immunology , Liver/virology , Virus Replication/drug effectsABSTRACT
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.
Subject(s)
Antiviral Agents/pharmacology , Hepatitis B virus/drug effects , Hepatitis B virus/genetics , Nucleotides/pharmacology , Antiviral Agents/chemistry , Antiviral Agents/metabolism , Antiviral Agents/therapeutic use , Humans , Nucleotides/chemistry , Nucleotides/metabolism , Nucleotides/therapeutic use , Prodrugs/chemistry , Prodrugs/metabolismABSTRACT
The present work exploits the potential of in silico approaches for minimizing attrition of leads in the later stages of drug development. We propose a theoretical approach, wherein 'parallel' information is generated to simultaneously optimize the pharmacokinetics (PK) and pharmacodynamics (PD) of lead candidates. ß-blockers, though in use for many years, have suboptimal PKs; hence are an ideal test series for the 'parallel progression approach'. This approach utilizes molecular modeling tools viz. hologram quantitative structure activity relationships, homology modeling, docking, predictive metabolism, and toxicity models. Validated models have been developed for PK parameters such as volume of distribution (log Vd) and clearance (log Cl), which together influence the half-life (t1/2) of a drug. Simultaneously, models for PD in terms of inhibition constant pKi have been developed. Thus, PK and PD properties of ß-blockers were concurrently analyzed and after iterative cycling, modifications were proposed that lead to compounds with optimized PK and PD. We report some of the resultant re-engineered ß-blockers with improved half-lives and pKi values comparable with marketed ß-blockers. These were further analyzed by the docking studies to evaluate their binding poses. Finally, metabolic and toxicological assessment of these molecules was done through in silico methods. The strategy proposed herein has potential universal applicability, and can be used in any drug discovery scenario; provided that the data used is consistent in terms of experimental conditions, endpoints, and methods employed. Thus the 'parallel progression approach' helps to simultaneously fine-tune various properties of the drug and would be an invaluable tool during the drug development process.
Subject(s)
Adrenergic beta-Antagonists/pharmacokinetics , Computer Simulation , Drug Design , Receptors, Adrenergic, beta/metabolism , Adrenergic beta-Antagonists/chemistry , Adrenergic beta-Antagonists/toxicity , Half-Life , Humans , Molecular Dynamics Simulation , Quantitative Structure-Activity Relationship , Reproducibility of Results , Structural Homology, ProteinABSTRACT
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.
Subject(s)
Antiviral Agents/administration & dosage , Hepatitis B, Chronic/drug therapy , Hepatitis B, Chronic/immunology , Immunity, Innate/drug effects , Animals , Disease Models, Animal , Hepatitis B Virus, Woodchuck/drug effects , Hepatitis B Virus, Woodchuck/pathogenicity , Hepatitis B virus/pathogenicity , Hepatitis B, Chronic/blood , Hepatitis B, Chronic/virology , Humans , Interferon-alpha/blood , Interferon-beta/blood , Liver/metabolism , Marmota/immunology , Marmota/virology , Virus Replication/drug effectsABSTRACT
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.
Subject(s)
Microwaves , Nucleosides/chemistry , Glass , Reproducibility of ResultsABSTRACT
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.
Subject(s)
Adenine/analogs & derivatives , Antiviral Agents , Hepatitis B virus/drug effects , Oligodeoxyribonucleotides , Adenine/pharmacology , Animals , Antiviral Agents/chemical synthesis , Antiviral Agents/pharmacology , Cattle , Drug-Related Side Effects and Adverse Reactions , Hepatitis B virus/genetics , Hepatitis B virus/metabolism , Oligodeoxyribonucleotides/chemical synthesis , Oligodeoxyribonucleotides/pharmacology , Organophosphonates/chemical synthesis , Organophosphonates/pharmacology , Prodrugs , Rabbits , Reverse Transcriptase Inhibitors/chemical synthesis , Reverse Transcriptase Inhibitors/pharmacology , Swine , Virus Replication/drug effectsABSTRACT
Several nucleoside analogs are under clinical development for use against hepatitis B virus (HBV). Lamivudine (3TC), a nucleoside analog, and adefovir dipivoxil (ADV), an acyclonucleotide analog, are clinically approved. However, long-term treatment can induce viral resistance, and following the cessation of therapy, viral rebound is frequently observed. There continues to be a need for new antiviral agents with novel mechanisms of action. A library of more than 600 di- and trinucleotide compounds synthesized by parallel synthesis using a combinatorial strategy was screened for potential inhibitors of HBV replication using the chronically HBV-producing cell line 2.2.15. Through an iterative process of synthesis, lead optimization, and screening, three analogs were identified as potent inhibitors of HBV replication: dinucleotides ORI-7246 (drug concentration at which a 10-fold reduction of HBV DNA was observed [EC(90)], 1.4 microM) and ORI-9020 (EC(90), 1.2 microM) and trinucleotide ORI-7170 (EC(90), 7.2 microM). These analogs inhibited the replication of both strands of HBV DNA. No suppression of HBV protein synthesis or intracellular core particle formation by these analogs was observed. No inhibition of HBV DNA strand elongation by the analogs or their 5'-triphosphate versions was apparent in in vitro polymerase assays. Although the exact mechanism of action is not yet identified, present data are consistent with an inhibition of the HBV reverse transcriptase-directed priming step prior to elongation of the first viral DNA strand. In transient-transfection assays, these analogs inhibited the replication of 3TC-resistant HBV. Synergistic interactions in combination treatments between the analogs and either 3TC or ADV were observed. These compounds represent a novel class of anti-HBV molecules and warrant further investigation as potential therapeutic agents.
Subject(s)
Adenine/analogs & derivatives , Antiviral Agents/chemical synthesis , Antiviral Agents/pharmacology , Hepatitis B virus/drug effects , Oligodeoxyribonucleotides/chemical synthesis , Oligodeoxyribonucleotides/pharmacology , Organophosphonates , Organothiophosphorus Compounds/chemical synthesis , Organothiophosphorus Compounds/pharmacology , Adenine/pharmacology , DNA, Viral/biosynthesis , Hepatitis B virus/genetics , Hepatitis B virus/metabolism , Lamivudine/pharmacology , Mutation/genetics , Nucleic Acid Synthesis Inhibitors , Reverse Transcriptase Inhibitors/chemical synthesis , Reverse Transcriptase Inhibitors/pharmacology , Viral Proteins/biosynthesis , Virus Replication/drug effectsABSTRACT
ORI-9020, a novel dinucleotide, evaluated in transgenic mice expressing hepatitis B virus (HBV), significantly reduced liver HBV DNA (P = 0.001). Levels of HBeAg and HBsAg in serum and of HBcAg in liver were not affected by treatment. A minimal effective dosage was determined to be between 1.6 and 0.5 mg/kg of body weight/day, which was similar to that observed for adefovir dipivoxil.