ANTIVIRAL EFFICACY AND HOST IMMUNE RESPONSE INDUCTION WITH SB 9200, AN ORAL PRODRUG OF THE DINUCLEOTIDE SB 9000, IN COMBINATION WITH ENTECAVIR IN THE WOODCHUCK MODEL OF CHRONIC HEPATITIS B Kyle Korolowicz 1 , Maria Balarezo 1 , Radhakrishnan Iyer 2 , Seetharamaiyer Padmanabhan 2 , Dillon Cleary 2 , Rayomand Gimi 2 , Anjaneyulu Sheri 2 , Manasa Suresh 1 , Changsuek Yon 1 , Robin Tucker 3 , Nezam Afdhal 2 , and Stephan Menne 1 1 Microbiology and Immunology, Georgetown University Medical Center, Washington, DC, 2 Spring Bank PharmaceuNcals, Milford, MA, 3 Department of ComparaNve Medicine, Georgetown University Medical Center, Washington, DC, United States pany BACKGROUND SB 9200 is a small orally bioavailable dinucleoNde that acNvates the cellular viral sensors RIGI and NOD2 causing the inducNon of IFN signaling cascade for anNviral defense (1, 2). In preclinical studies, SB 9200 has shown to be a potent agent against hepaNNs B virus (HBV) (1, 3). In woodchucks chronically infected with woodchuck hepaNNs virus (WHV), SB 9200 monotherapy for 12 weeks at two select doses resulted in potent anNviral acNvity (4). OBJECTIVES The aim of this study was to evaluate the overall anNviral response in woodchucks upon inducNon of innate immune response first by treatment with SB 9200 followed by Entecavir (ETV) treatment versus reducNon of viral burden with ETV treatment followed by immune modulaNon with SB 9200. SUMMARY At the end of treatment in Group 2, average reducNons of 6.4 log 10 in serum WHV DNA and 3.3 log 10 in WHsAg were observed whereas in Group 1, average reducNons of 4.2 log 10 and 1.1 log 10 in serum WHV DNA and WHsAg were seen compared to pretreatment levels. Treatment demonstrated significant reducNons of WHV DNA RI, cccDNA, and RNA in Group 2 (76%, 49%, and 51%) and Group 1 (57%, 39%, and 41%). Following cessaNon of treatment and the 8week followup period, recrudescence of WHV replicaNon was observed in Group 1, whereas recrudescence in Group 2 was much delayed with viremia and anNgenemia staying at 1.7 and 0.7 log 10 below pretreatment levels. Overall, both treatment regimens slightly reduced hepaNc WHV anNgen expression and slowed liver disease progression. Seroconversion to anNWHs anNbody was not observed. The anNviral effects were also associated with the inducNon of IFNα, IFNβ, OAS1, CXCL10, ISG15, and IL6 in blood and liver, which were more pronounced in Group 2 compared to Group 1. RESULTS CONCLUSIONS REFERENCES MATERIALS & METHODS Two groups of five woodchucks 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. Both groups were monitored for 8 weeks posttreatment. Endpoints included PD, tolerability and anNviral efficacy. Safety parameters included hematology, clinical chemistry, body weights, body temperatures and daily observaNons. Serum DNA load was determined by slot blot hybridizaNon and samples below the limit of detecNon were further evaluated by PCR. Serum surface anNgen (WHsAg) level and anNbod Nter against WHsAg (anNWHs) were assayed by ELISA. HepaNc levels of WHV RNA, covalentlyclosed circular (ccc) DNA, and DNA replicaNve intermediates (RI) were measured quanNtaNvely by Northern or Southern blot hybridizaNon, respecNvely. InducNon of innate immune response to SB 9200 treatment was tested by measuring RNA levels of IFNα, IFNβ, IL6, CXCL10, OAS1 and ISG15 in whole blood and liver. InducNon of host innate immune response by pretreatment with SB 9200 followed by ETV in woodchucks resulted in significant declines in viral DNA, RNA, and anNgens that was superior to that seen using the strategy of viral reducNon with ETV followed by immune modulaNon. These data support the planned Phase II clinical trial of SB 9200 alone and in combinaNon with a nucleoside in the treatment of chronic HBV. 1. Coughlin JE, et al., Bioorg Med Chem Lek 2010;20:17831786 2. Cunningham ME, et al., Hepatology 2013;58:92A207A, Abstract 473 3. Iyer RP, et al., AnNmicrob Agents Chemother 2004;48:23182320 4. Korolowicz K, et al., J Hepatol 2015; 62:S557 Contact InformaHon Stephan Menne: [email protected]; Radhakrishnan P. Iyer: [email protected] REFERENCES This study was supported by IDIQ contract HHSN272201000011I, task order HHSN27200002 (D06) to Georgetown University Medical Center and by R01 grant AI094469 to Spring Bank PharmaceuNcals, Inc. from NIAID/DMID. Fig. 1. Changes in levels of serum WHV DNA (top panels) and WHsAg (bo\om panels) relaNve to T 0 (pretreatment baseline) in individual woodchucks administered ETV + SB 9200 (a, d) or SB 9200 + ETV (b, e), and mean of each group (c, f). Fig. 2. Changes in mean hepaNc levels of WHV RI DNA (a), WHV cccDNA (b), and WHV RNA (c) relaNve to week 1 (pretreatment baseline) in response to treatment with ETV + SB 9200 or SB 9200 + ETV. SequenHal treatment with SB 9200 followed by ETV results in marked suppression of serum viremia and anHgenemia and in delayed recrudescence of viral replicaHon compared to sequenHal treatment with ETV followed by SB 9200 SequenHal treatment with SB 9200 followed by ETV induces more pronounced reducHon in hepaHc levels of WHV nucleic acids than sequenHal treatment with ETV followed by SB 9200 SequenHal treatment with SB 9200 followed by ETV induces pronounced and someHmes longlasHng expression increases of type I IFNs, cytokine and ISGs in blood Fig. 5. Changes in mean blood transcript levels of IFNα, IFNβ, and IL6 (top panels) and of CXCL10, OAS1 and ISG15 (bo\om panels) in response to treatment with ETV + SB 9200 (a, c) or SB 9200 + ETV (b, d). Delayed recrudescence of viral replicaHon following sequenHal treatment with SB 9200 and ETV is associated with expression increases of type I IFNs, cytokine and ISGs in liver Fig. 6. Changes in mean liver transcript levels of IFNα, IFNβ, and IL6 (top panels) and of CXCL10, OAS1 and ISG15 (bo\om panels) in response to treatment with ETV + SB 9200 (a, c) or SB 9200 + ETV (b, d). Fig. 3. Changes in cytoplasmic WHcAg (top panels) and membranous WHsAg expression (bo\om panels) in response to treatment with ETV + SB 9200 (a, c) or SB 9200 + ETV (b, d). Changes in mean serum WHV DNA relaNve to T 0 (pretreatment baseline) is ploked on the leo yaxis. The mean immunohistochemistry (IHC) scores for anNgen expression in liver are ploked on the right yaxis. SequenHal treatment with SB 9200 followed by ETV results in greater reducHon of hepaHc WHV anHgen expression than sequenHal treatment with ETV and SB 9200 SequenHal treatment with SB 9200 and ETV or ETV and SB 9200 reduces liver steatosis Fig. 4. Changes in liver steatosis in response to treatment with ETV + SB 9200 (a) or SB 9200 + ETV (b). Changes in mean serum WHV DNA relaNve to T 0 (pretreatment baseline) is ploked on the leo yaxis. The mean score for hepaNc steatosis is ploked on the right yaxis.