Antiviral Efficacy and Host Innate Immunity Associated with SB 9200

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http://journals.plos.org/plosone/article?id=10.1371%2Fjournal.pone.0161313

Antiviral Efficacy and Host Innate Immunity Associated with SB 9200 Treatment in the Woodchuck Model of Chronic Hepatitis B

Kyle E. Korolowicz, Radhakrishnan P. Iyer, Stefanie Czerwinski, Manasa Suresh, Junming Yang, Seetharamaiyer Padmanabhan, [...view 6 more...], Stephan Menne


Abstract

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-β 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.

Citation: Korolowicz KE, Iyer RP, Czerwinski S, Suresh M, Yang J, Padmanabhan S, et al. (2016) Antiviral Efficacy and Host Innate Immunity Associated with SB 9200 Treatment in the Woodchuck Model of Chronic Hepatitis B. PLoS ONE 11(8): e0161313. doi:10.1371/journal.pone.0161313

Editor: Haitao Guo, Indiana University, UNITED STATES

Received: March 12, 2016; Accepted: August 3, 2016; Published: August 23, 2016

Copyright: © 2016 Korolowicz et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the paper and its Supporting Information files.

Funding: This study was funded by IDIQ Contract HHSN272201000011I, Task Order HHSN27200002 (D06) to the Department of Microbiology & Immunology, Georgetown University Medical Center (SM) from the Division of Microbiology and Infectious Diseases (DMID) of the National Institute of Allergy and Infectious Diseases (NIAID). Support by R01 grant AI094469 to Spring Bank Pharmaceuticals, Inc. (RPI) from NIAID is gratefully acknowledged. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: RPI, SP, and AS are employees of Spring Bank Pharmaceuticals, Inc. RKP is a former employee of Spring Bank Pharmaceuticals, Inc. JKM and JS were consultants to Spring Bank Pharmaceuticals, Inc. All other authors, including KEK, SC, MS, JY, BVK, RDT, and SM have declared that no competing interests exist. This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials.

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Materials and Methods

Investigational drug

SB 9200, an oral prodrug of SB 9000, was manufactured by Spring Bank Pharmaceuticals, Inc. (Milford, MA) and the structure and antiviral characteristics of SB 9200/SB 9000 have previously been described [7, 10]. Doses of SB 9200 were dry mixed with woodchuck diet powder (Dyets, Bethlehem, PA), the blended drug material suspended in ultrapure water, and orally administered to woodchucks within ½ hour after preparation.

Repeat dose study in woodchucks

The animal protocol and all procedures involving woodchucks were approved by the IACUC of Georgetown University and adhered to the national guidelines of the Animal Welfare Act, the Guide for the Care and Use of Laboratory Animals, and the American Veterinary Medical Association. Woodchuck were anesthetized by intramuscular injection of ketamine (50 mg/kg) and xylazine (5 mg/kg). Prior to euthanasia, woodchucks were anesthetized as described above and euthanized by an overdose of Beuthanasia-D solution (80–100 mg/kg) administered by intracardiac injection, followed by bilateral intercostal thoracotomy. Woodchucks used in this study were born in captivity and infected at 3 days of age with WHV. Chronically infected animals were confirmed positive for serum WHV DNA and WHV surface antigen (WHsAg) and had undetectable antibodies against WHsAg (anti-WHs) at approximately one year post-infection. Absence of liver tumors in woodchucks with low gamma-glutamyl transferase (GGT) was confirmed by ultrasonography. Chronic WHV carrier woodchucks were assigned and stratified by gender, body weight, and by pretreatment serum markers (WHV DNA and WHsAg loads and serum GGT and sorbitol-dehydrogenase (SDH) levels) into two groups (n = 5 each). Woodchucks were treated once daily, orally with either a low dose (15 mg/kg) or a high dose (30 mg/kg) of SB 9200 for 12 weeks. As inclusion of a placebo-treated control group was not feasible due to the paucity of WHV-infected woodchucks, the treatment effects mediated by SB 9200 at two separate doses were evaluated by changes in viral and host parameters from pretreatment level which served as the control for comparison. For select assays, blood and liver samples from five age-matched, treatment-naïve chronic WHV carrier woodchucks were included for comparison of basal expression levels of immune response genes in untreated animals with pretreatment levels in SB 9200 treated animals.

Pharmacokinetic analysis of SB 9000

Plasma levels of SB 9000, the active moiety of the prodrug SB 9200, in samples collected at pretreatment and then bi-weekly throughout the study at a single time-point of approximately 2 hours post-dose were evaluated as follows: A 100 μL aliquot of plasma was extracted by solid phase method (Phenomenex Strata X-AW 33 μm, 10 mg bed) and SB 9000 quantified by liquid chromatography using a Waters XBridge C18, 50 x 2.1 mm, 3.5 μm particle size column with a mixture of water/formic (1000:1, v/v; mobile phase A) and methanol/acetonitrile/formic acid (500:500:1, v/v/v; mobile phase B) under gradient conditions (650 μL/minute flow rate). Detection of the analyte was achieved using an AB Sciex API 5000 triple quadrupole LC MS/MS system operating in positive ESI (TurboIonSpray®) mode.

Serum WHV parameters

Depending on the serum concentration, WHV DNA was quantified weekly by either dot blot hybridization or real-time PCR assay as described [15]. Serum levels of WHsAg and anti-WHs were measured weekly by WHV-specific enzyme immunoassays as described [16].

Hepatic WHV parameters

Hepatic levels of WHV nucleic acids were determined in liver biopsy samples collected at pretreatment (week -1), during treatment (week 6), at the end of treatment (week 12), and at the end of the study (week 20). WHV RNA was measured by Northern blot hybridization and WHV DNA replicative intermediates (RI) and WHV covalently-closed circular (ccc) DNA were determined by Southern blot hybridization as described [15]. Liver was imunostained with an antibody against WHV core antigen (WHcAg) using a 1:350 dilution. Liver was further immunostained with a cross-reactive antibody against RIG-1 (Origene Technologies, Rockville, MD) using a 1:125 dilution. The immunohistochemistry (IHC) scores for WHcAg and RIG-I expression in liver were derived from the mean of the stained hepatocyte percentage score combined with the mean of the staining intensity score. A composite IHC score of 0 indicates absence of WHcAg or RIG-I staining in all hepatocytes (0%) whereas 8 indicates presence of strong WHcAg or RIG-I staining in 81–100% of hepatocytes. Specifically, the percentage of WHcAg or RIG-I stained hepatocytes was scored on a 0–5 scale, where 0 = 0%, 1 = 1–20%, 2 = 21–40%, 3 = 41–60%, 4 = 61–80%, and 5 = 81–100% of cells stained. The intensity of WHcAg or RIG-I staining was scored on a 0–3 scale, where 0 = no staining, 1 = weak staining, 2 = moderate staining, and 3 = strong staining. The liver histology score was derived from the mean of the lobular sinusoidal hepatitis score combined with the mean of the portal hepatitis score (n = 1–5 portal tracts examined). The composite histology was scored on a 0–6 scale, where 0 = absent hepatitis, >0–2 = mild hepatitis, >2–4 = moderate hepatitis and >4 = marked to severe hepatitis.

Host immune response parameters

Immune responses associated with treatment were determined by changes in RNA transcript levels of IFN-α, IFN-β, IFN-γ induced protein 10 (IP-10 or CXCL10), interleukin 6 (IL-6), interferon-induced 17 kDa protein (ISG15), and 2'-5'-oligoadenylate synthetase 1 (OAS1) in blood and liver using PCR. Briefly, whole blood was collected into PAXgene blood tubes (Qiagen, Redwood City, CA) at pretreatment (week -1 and T0), during treatment (weeks 6 and 12), and during follow-up (week 18) and stored at -70°C until use. Total RNA was isolated with on-column DNase I digestion using the PAXgene Blood miRNA Kit (Qiagen). Total RNA was further isolated from liver biopsy samples collected as indicated above using the RNeasy Mini Kit (Qiagen) with on-column DNase I digestion using RNase-Free DNase (Qiagen). Following reverse transcription of mRNA with the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) using oligo(dT), complementary (c) DNA samples were amplified on a 7500 Real Time PCR System instrument (Applied Biosystems) using TaqMan Gene Expression Master Mix (Applied Biosystems) and woodchuck-specific primers and probes (Table 1). Woodchuck 18S rRNA expression was used to normalize target gene expression. Transcription levels of target genes were calculated as a fold-change relative to pretreatment level at week -1 (liver) or at T0 (blood) using the formula 2-ΔΔCt. Samples from a subset of woodchucks of the low and high dose groups from which liver biopsies could be collected at all time points during the study were analyzed for expression changes of genes involved in the RIG-I/NOD-2 pathway induced by SB 9200, including RIG-I, NOD2, stimulator of IFN genes (STING; also known as transmembrane protein 173 or TMEM173), and IFN regulatory factor 3 (IRF3).

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Table 1. Oligonucleotides used for analyses of RIG-I/NOD2 pathway and host innate immune responses in blood and liver.
doi:10.1371/journal.pone.0161313.t001

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Drug safety parameters

Various measurements (body weight, body temperature, clinical chemistry, and hematology) were obtained weekly to monthly for monitoring drug safety.

Statistical analyses

All parameters were compared to the values at pretreatment and between both dose groups using unpaired Student’s t-test with equal variance. P values of <0.05 were considered statistically significant.

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Introduction

Infection with hepatitis B virus (HBV) is responsible for approximately 1.2 million deaths per year worldwide due to HBV-associated liver diseases, including hepatic cirrhosis and hepatocellular carcinoma (HCC) [1]. Current antiviral therapies for chronic hepatitis B (CHB) are limited to nucleos(t)ide analogs and (pegylated) interferon-alpha (IFN-α). Prolonged administration of nucleos(t)ide analogs reduces viral load and improves the long-term outcome of CHB, but rarely leads to a cure [2]. Use of these antivirals is further limited due to the emergence of drug-resistant variants during treatment, the risk of relapse upon treatment discontinuation, and unwanted side effects [2]. Long-term IFN-α administration is also associated with treatment-limiting adverse effects and variability in treatment response, but the rate of durable HBV surface antigen loss is higher than with nucleos(t)ide analogs although still only occurs in <10% of patients [2]. There is therefore an urgent need for improvements in therapeutic strategies for inducing functional cure of CHB.

The Eastern woodchuck (Marmota monax) is naturally infected with the woodchuck hepatitis virus (WHV), a hepadnavirus which is genetically closely related to human HBV [3]. Neonatal WHV infection parallels the main route of human (vertical) transmission for chronic HBV infection and displays a disease course similar to that in HBV-infected patients. Thus, chronic WHV infection in woodchucks is a fully immunocompetent model for studying CHB and HBV-induced HCC, and woodchucks have extensively been used to evaluate efficacy and safety of current and new HBV therapeutics [3]. The recent comparison of hepatic transcriptional profiles in woodchucks and humans with acute self-limiting and chronic hepadnaviral infections identified important parallels in the antiviral immune responses induced by WHV and HBV [4]. In addition, these transcription signatures have facilitated the characterization of antiviral immune responses in woodchucks to treatment with GS-9620, a toll-like receptor 7 agonist [5] and IFN-α [6]. As these studies have established the translational value of this animal model for CHB, woodchucks were used to evaluate the antiviral efficacy of SB 9200.

SB 9000 is a small molecule nucleic acid hybrid (a dinucleotide) with antiviral activity against HBV [7, 8], which was further elaborated into an orally bioavailable prodrug compound, SB 9200 [9, 10]. In addition to the direct antiviral property, the host immune stimulating activity of SB 9200 is thought to induce endogenous IFN via the activation of the viral sensor proteins, retinoic acid-inducible gene 1 (RIG-I) and nucleotide-binding oligomerization domain-containing protein 2 (NOD2). Activation is believed to occur by binding of SB 9200/SB 9000 to RIG-I and NOD2 at their nucleotide binding domain. Both cytosolic proteins usually recognize signature patterns of foreign RNA such as the pathogen-associated molecular pattern (PAMP). Once PAMP is recognized, RIG-I and NOD2 become activated resulting in the induction of the IFN signaling pathway and the production of type I and III IFNs, as well as the subsequent induction of interferon-stimulated genes (ISGs), pro-inflammatory cytokines and antiviral immune cells [11, 12]. The direct antiviral activity of SB 9200/SB 9000 is thought to inhibit the synthesis of viral nucleic acids by steric blockage of the viral polymerase, similar to the mechanism recently described for HBV [12]. The blockage, which is independent of the IFN signaling pathway, may be achieved by binding of SB 9200/SB 9000 with RIG-I and NOD2 that associate with viral RNA and that in turn prevents the polymerase from engaging with the pre-genomic (pg) RNA template for viral replication. Although no data currently exist that directly support the assumed mechanism, preliminary in vitro findings indicate that SB 9200/SB 9000 induces rapid translocation of RIG-I on a double-stranded RNA template and that SB 9000 binds to RIG-I in a dose-dependent way (Radhakrishnan P. Iyer; personal communication). Thus, dose-dependent binding of SB 9000/9200 to RIG-I (and NOD2) and consequent rapid shuttling of sensor proteins on a nucleic acid template (i.e., pgRNA) could displace the viral polymerase from accessing the pgRNA for replication. SB 9200 has been intensively tested in vitro and in vivo against HBV [8, 9]. SB 9200 is further antiviral efficacious against hepatitis C virus (HCV) [13], and a Phase I evaluation of SB 9200 in humans for therapy of HCV was recently completed [14]. Here we report a single agent efficacy study in which the host immune stimulating and direct antiviral activities of SB 9200 at two separate doses were evaluated in woodchucks with chronic WHV infection. The results show that oral administration of SB 9200 for 12 weeks at two select doses reduced viral markers in serum and liver that were associated with (or were a result of) the induction of host antiviral immune responses.