Sustained efficacy and seroconversion with the Toll-like receptor 7 agonist GS-9620 in the Woodchuck model of chronic hepatitis B.

BACKGROUND & AIMS: New therapies for chronic hepatitis B (CHB) are urgently needed since current treatments rarely lead to cure. We evaluated whether the oral small molecule toll-like receptor (TLR7) agonist GS-9620 could induce durable antiviral efficacy in woodchucks chronically infected with woodchuck hepatitis virus (WHV), a hepadnavirus closely related to human hepatitis B virus (HBV). METHODS: After evaluating the pharmacokinetics, pharmacodynamics and tolerability of oral GS-9620 in uninfected woodchucks, adult woodchucks chronically infected with WHV (n = 7 per group) were dosed with GS-9620 or placebo for 4 or 8 weeks with different treatment schedules. RESULTS: GS-9620 treatment induced rapid, marked and sustained reduction in serum viral DNA (mean maximal 6.2log10 reduction), and hepatic WHV DNA replicative intermediates, WHV cccDNA and WHV RNA, as well as loss of detectable serum WHV surface antigen (WHsAg). GS-9620 treatment also induced a sustained antibody response against WHsAg in a subset of animals. Strikingly, treatment reduced the incidence of hepatocellular carcinoma (HCC) from 71% in the placebo group to 8% in GS-9620-treated woodchucks with sustained viral load reduction. GS-9620 treatment was associated with reversible increases in serum liver enzymes and thrombocytopenia, and induced intrahepatic CD8(+) T cell, NK cell, B cell and interferon response transcriptional signatures. CONCLUSIONS: The data demonstrate that short duration, finite treatment with the oral TLR7 agonist GS-9620 can induce a sustained antiviral response in the woodchuck model of CHB, and support investigation of this compound as a therapeutic approach to attain a functional cure in CHB patients.

GS-9620, an oral agonist of Toll-like receptor-7, induces prolonged suppression of hepatitis B virus in chronically infected chimpanzees.

BACKGROUND & AIMS: Direct-acting antiviral agents suppress hepatitis B virus (HBV) load, but they require life-long use. Stimulation of the innate immune system could increase its ability to control the virus and have long-lasting effects after a finite regimen. We investigated the effects of immune activation with GS-9620--a potent and selective orally active small molecule agonist of Toll-like receptor 7--in chimpanzees with chronic HBV infection. METHODS: GS-9620 was administered to chimpanzees every other day (3 times each week) for 4 weeks at 1 mg/kg and, after a 1-week rest, for 4 weeks at 2 mg/kg. We measured viral load in plasma and liver samples, the pharmacokinetics of GS-9620, and the following pharmacodynamics parameters: interferon-stimulated gene expression, cytokine and chemokine levels, lymphocyte and natural killer cell activation, and viral antigen expression. Clinical pathology parameters were monitored to determine the safety and tolerability of GS-9620. RESULTS: Short-term oral administration of GS-9620 provided long-term suppression of serum and liver HBV DNA. The mean maximum reduction of viral DNA was 2.2 logs, which occurred within 1 week of the end of GS-9620 administration; reductions of >1 log persisted for months. Serum levels of HBV surface antigen and HBV e antigen, and numbers of HBV antigen-positive hepatocytes, were reduced as hepatocyte apoptosis increased. GS-9620 administration induced production of interferon-α and other cytokines and chemokines, and activated interferon-stimulated genes, natural killer cells, and lymphocyte subsets. CONCLUSIONS: The small molecule GS-9620 activates Toll-like receptor 7 signaling in immune cells of chimpanzees to induce clearance of HBV-infected cells. This reagent might be developed for treatment of patients with chronic HBV infection.

Pharmacokinetic and pharmacodynamic properties of GS-9620, a novel Toll-like receptor 7 agonist, demonstrate interferon-stimulated gene induction without detectable serum interferon at low oral doses.

GS-9620 [8-(3-(pyrrolidin-1-ylmethyl)benzyl)-4-amino-2-butoxy-7,8-dihydropteridin-6(5H)-one] is a potent, orally bioavailable small-molecule agonist of Toll-like receptor 7 (TLR7) developed for finite treatment of chronic hepatitis B viral (HBV) infection, with the goal of inducing a liver-targeted antiviral effect without inducing the adverse effects associated with current systemic interferon-α (IFN-α) therapies. We characterized the pharmacodynamic response of GS-9620 in CD-1 mice and cynomolgus monkeys following intravenous or oral administration and showed that GS-9620 induces the production of select chemokines and cytokines, including IFN-α and interferon-stimulated genes (ISGs). It is noteworthy that we also demonstrated that, in animals and healthy human volunteers, oral administration of GS-9620 can induce a type I interferon-dependent antiviral innate immune response, as measured by whole-blood mRNA of the ISGs 2'5'-oligoadenylate synthetase 1 (OAS1) and myxovirus resistance 1 (MX1), without the induction of detectable systemic IFN-α, i.e., a presystemic response. Additionally, presystemic induction of hepatic OAS1 and MX1 mRNA was observed in CD-1 mice in the absence of detectable systemic IFN-α. We propose that the mechanism of this presystemic response is likely its high intestinal absorption, which facilitates localized activation of TLR7, probably in plasmacytoid dendritic cells at the level of gut-associated lymphoid tissue and/or the liver. This localized response is further supported by data that indicate only minimal contributions of systemic immune stimulation to the overall pharmacodynamic response to orally administered GS-9620. These data demonstrate that GS-9620 can induce an antiviral innate immune response without inducing a systemic IFN-α response and thus suggest the therapeutic potential of this approach in the treatment of chronic HBV infection.

Assessment of GS-9219 in a pet dog model of non-Hodgkin's lymphoma.

PURPOSE: To assess, in dogs with naturally occurring non-Hodgkin's lymphoma, pharmacokinetics, safety, and activity of GS-9219, a prodrug of the nucleotide analogue 9-(2-phosphonylmethoxyethyl) guanine (PMEG), which delivers PMEG and its phosphorylated metabolites to lymphoid cells with preferential cytotoxicity in cells with a high proliferation index such as lymphoid malignancies. EXPERIMENTAL DESIGN: To generate proof-of-concept, a phase I/II trial was conducted in pet dogs (n = 38) with naturally occurring non-Hodgkin's lymphoma using different dose schedules of GS-9219. A subset of dogs was further evaluated with 3'-deoxy-3'-(18)F-fluorothymidine positron emission tomography/computed tomography imaging before and after treatment. RESULTS: The prodrug had a short plasma half-life but yielded high and prolonged intracellular levels of the cytotoxic metabolite PMEG diphosphate in peripheral blood mononuclear cells in the absence of detectable plasma PMEG. Dose-limiting toxicities were generally manageable and reversible and included dermatopathy, neutropenia, and gastrointestinal signs. Antitumor responses were observed in 79% of dogs and occurred in previously untreated dogs and dogs with chemotherapy-refractory non-Hodgkin's lymphoma. The median remission durations observed compare favorably with other monotherapies in dogs with non-Hodgkin's lymphoma. High 3'-deoxy-3'-(18)F-fluorothymidine uptake noted in lymphoid tissues before treatment decreased significantly after treatment (P = 0.016). CONCLUSIONS: GS-9219 was generally well tolerated and showed significant activity against spontaneous non-Hodgkin's lymphoma as modeled in pet dogs and, as such, supports clinical evaluation in humans.

GS-9191 is a novel topical prodrug of the nucleotide analog 9-(2-phosphonylmethoxyethyl)guanine with antiproliferative activity and possible utility in the treatment of human papillomavirus lesions.

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.

GS-9219--a novel acyclic nucleotide analogue with potent antineoplastic activity in dogs with spontaneous non-Hodgkin's lymphoma.

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.

Antifibrotic Effects of the Dual CCR2/CCR5 Antagonist Cenicriviroc in Animal Models of Liver and Kidney Fibrosis.

BACKGROUND & AIMS: Interactions between C-C chemokine receptor types 2 (CCR2) and 5 (CCR5) and their ligands, including CCL2 and CCL5, mediate fibrogenesis by promoting monocyte/macrophage recruitment and tissue infiltration, as well as hepatic stellate cell activation. Cenicriviroc (CVC) is an oral, dual CCR2/CCR5 antagonist with nanomolar potency against both receptors. CVC's anti-inflammatory and antifibrotic effects were evaluated in a range of preclinical models of inflammation and fibrosis. METHODS: Monocyte/macrophage recruitment was assessed in vivo in a mouse model of thioglycollate-induced peritonitis. CCL2-induced chemotaxis was evaluated ex vivo on mouse monocytes. CVC's antifibrotic effects were evaluated in a thioacetamide-induced rat model of liver fibrosis and mouse models of diet-induced non-alcoholic steatohepatitis (NASH) and renal fibrosis. Study assessments included body and liver/kidney weight, liver function test, liver/kidney morphology and collagen deposition, fibrogenic gene and protein expression, and pharmacokinetic analyses. RESULTS: CVC significantly reduced monocyte/macrophage recruitment in vivo at doses ≥20 mg/kg/day (p < 0.05). At these doses, CVC showed antifibrotic effects, with significant reductions in collagen deposition (p < 0.05), and collagen type 1 protein and mRNA expression across the three animal models of fibrosis. In the NASH model, CVC significantly reduced the non-alcoholic fatty liver disease activity score (p < 0.05 vs. controls). CVC treatment had no notable effect on body or liver/kidney weight. CONCLUSIONS: CVC displayed potent anti-inflammatory and antifibrotic activity in a range of animal fibrosis models, supporting human testing for fibrotic diseases. Further experimental studies are needed to clarify the underlying mechanisms of CVC's antifibrotic effects. A Phase 2b study in adults with NASH and liver fibrosis is fully enrolled (CENTAUR Study 652-2-203; NCT02217475).

Web resources for drug toxicity.

Information on drug toxicity is used primarily by three segments of the health care industry: the consumer or patient, healthcare practitioners, and research and development (R&D) scientists in the pharmaceutical industry. The major focus of both consumers and health care practitioners is the provision of suitable information for the safe use of drugs for both individuals and specific patient populations. Therefore, accurate information on potential side effects and drug-drug and food-drug interactions is critical. For pharmaceutical scientists, the use of toxicity information is more complex and has become an essential part of the R&D process. The high rate of failures in drug development has precipitated the utilization of all available resources to gather relevant information that may improve the process of drug development. The web is a growing source of information and services for the pharmaceutical industry. Web-based resources for non-clinical drug development include tools for virtual discovery such as silico ADME/Toxicity programs, access to information regarding in vitro and in vivo testing, new data management options, and the latest regulatory guidelines and industry news. These resources are reviewed from the perspective of a toxicologist.

Safety, pharmacokinetics and pharmacodynamics of GS-9620, an oral Toll-like receptor 7 agonist.

BACKGROUND: GS-9620 is a novel oral agonist of Toll-like receptor 7 (TLR7) in development for the treatment of chronic viral hepatitis. TLR7 is a highly conserved innate immune receptor expressed primarily on plasmacytoid dendritic cells and B lymphocytes. The aim of this double-blind, placebo-controlled, single ascending-dose study was to investigate the safety, tolerability, pharmacokinetics and pharmacodynamics of GS-9620 in healthy volunteers. METHODS: In total, 75 healthy volunteers (8 subjects in each of the 10 cohorts; 5 subjects participated in two cohorts) were randomized (6:2) to receive a single dose of GS-9620 (0.3, 1, 2, 4, 6, 8 or 12 mg) or placebo. RESULTS: GS-9620 was well-absorbed and well-tolerated in oral doses up to 12 mg. Minimal treatment-related adverse events were seen at doses up to 8 mg. Serum interferon (IFN)-α was only detected in subjects who received 8 or 12 mg doses, and the adverse event profile at 8 and 12 mg doses was generally consistent with that associated with IFN-α exposure (flu-like symptoms), consistent with the mechanism of TLR7 agonism. All adverse events resolved within 72 h. Induction of chemokines/cytokines and IFN-stimulated genes were seen at GS-9620 doses ≥ 2 mg, well below doses that induced serum IFN-α or led to clinical adverse events. CONCLUSIONS: GS-9620 demonstrates safety and pharmacodynamic activity at doses up to 12 mg. Pharmacodynamic activity is seen before adverse events, suggesting the potential for induction of an antiviral response without systemic adverse events in subjects with chronic viral hepatitis.