In vitro and in vivo characterization of the selective toll-like receptor 8 agonist GS-9688

POSTER PRESENTATIONS SAT-168 In vitro and in vivo characterization of the selective toll-like receptor 8 agonist GS-9688 S. Daffis1, D. Ramakrishnan1, C. Niu1, J. Zheng1, R. Santos1, M. Mish1, G. Chin1, V. Aktoudianakis1, S. Metobo1, H.-J. Pyun1, H. Yu1, G. Lee1, A. Palazzo1, C. Frey1, S. Pflanz1, W.E. Delaney, IV1, S.P. Fletcher1, R. Mackman1. 1Gilead Sciences, Foster City, CA, United States E-mail: [email protected] Background and Aims: GS-9688 is a small molecule agonist of tolllike receptor 8 (TLR8) in clinical development. The in vitro selectivity of GS-9688 for TLR8 was determined and the cytokine profile of GS9688 in human peripheral blood mononuclear cells (PBMC) from healthy donors (HD) and chronic hepatitis B (CHB) patients was characterized. The pharmacokinetic and pharmacodynamic response of cynomolgus monkeys to a single oral dose of GS-9688 was also evaluated. Methods: Selectivity of GS-9688 for TLR8 was evaluated in HEK293 cells stably expressing individual human innate immune receptors together with a reporter gene under the control of an NF-κB or IRF3 promoter. PBMCs obtained from HDs (n = 10) and age-matched CHB patients (n = 10) were treated for 24 hours with GS-9688 and cytokines analyzed by Luminex® assay. Intracellular cytokines in myeloid dendritic cells (mDCs), monocytes and plasmacytoid dendritic cells ( pDCs) from HD PBMCs (n = 6) were evaluated by flow cytometry. Cynomolgus monkeys (n = 3/group) were dosed orally with 0.03–300 mg/kg GS-9688 and serum cytokines were analyzed by Luminex® assay 2–48 hours post-dose. GS-9688 plasma concentrations were determined by an LC-MS/MS method. Results: GS-9688 displayed >100-fold selectivity for human TLR8 over TLR7 in HEK293 cells. GS-9688 did not activate other human TLRs or innate immune receptors in vitro. In human PBMCs, GS-9688 induced the cellular immune mediators IL-12 and IL-18, and the antiviral cytokines TNF-α and IFN-γ (EC50 from 217 to 326 nM). In contrast, GS-9688 had minimal effects on the levels of IFN-α, a TLR7induced cytokine. GS-9688 strongly induced intracellular TNF-α in mDCs and monocytes but not IFN-α production in pDCs, consistent with GS-9688 being TLR8-selective. The potency and cytokine profile were comparable in PBMCs from HDs and CHB patients. Oral GS-9688 demonstrated high first-pass hepatic clearance and minimal systemic exposure in cynomolgus monkeys. In these animals, GS-9688 induced detectable serum IL-12 and various other cytokines, but not IFN-α or TNF-α. Conclusions: GS-9688 is a potent and selective agonist of human TLR8 that induces IL-12 as well as other cellular immune mediators and antiviral cytokines in human PBMCs from HD and CHB patients. GS-9688 has low oral bioavailability in cynomolgus monkeys but induces serum IL-12 as well as various other cytokines presystemically. Together, these data support investigation of oral GS-9688 for the treatment of CHB. SAT-169 Extracellular vesicles including microparticles from hepatitis B infected patients function as replication bodies and transmit HBV infection S. Sukriti1, M.C. Choudhary2, S. Sharma1, J.S. Maras1, A. Singh1, M. Islam1, S. Sharma1, N. Trehanpati1, E. Gupta2, S.K. Sarin1,3. 1 Molecular and Cellular Medicine; 2Virology; 3Hepatology, Institute of Liver and Biliary Sciences, New Delhi, India E-mail: [email protected] Background and Aims: Extracellular vesicles (EVs), including microparticles (MPs), serve as vehicles for intercellular communication and can mediate transfer of proteins, mRNAs, and microRNAs. While it has been shown that EVs containing hepatitis B virus (HBV) can cause NK-cell dysfunction, role of MPs in the transmission of HBV infection remains unknown. We investigated whether MPs serve as reservoirs of HBV infection, carry HBV DNA after its clearance from

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plasma post-treatment, mediate viral replication and can transmit infection. Methods: MPs were isolated using differential ultracentrifugation method from chronic hepatitis B (CHB) patients (Group1: qHBsAg+ve with undetectable HBV DNA post antiviral therapy [n = 9]; Group2: CHB with detectable HBV DNA > 103 {Median: 4.3 × 104; Range: 1.2 × 103−9.3 × 108}[n = 12]). HBV DNA was quantified (Cobas Taqman) in plasma and MPs from both the groups. Hepatocyte origin was confirmed by ASGPR+ MP levels (mp/ul) by flow-cytometry and mir122 expression in MPs by qRT-PCR and compared with n = 20 healthy controls (HC). Role of MPs in mediating HBV replication and infection was assessed in Huh7 cells. Results: In Gr1, HBV DNA was detected in MPs in n = 5 CHB patients (60%) which was undetected in plasma, whereas in Gr2, HBV DNA was seen in n = 12 (100%) but found lower than the values in plasma in all the patients {MPs HBV DNA, Median: 2.23 × 103; Range: 2.56 × 103– 3.3 × 105}. The probability of detecting HBV DNA in MPs in 60% of Gr1 patients was found significant ( p = 0.08) with relative risk ratio 2.25 (95% of CI 1.08–4.67; p = 0.02) and odds ratio 28.1(95% of CI −1.27 to 619.9; p = 0.03) respectively. ASGPR+ MPs were significantly higher in CHB patients than HC (342.5 MP/uL vs 60.3 MP/uL; p > 0.00) and hepatocyte origin was also confirmed by mir122 expression in CHB MP’s and was 1.5-fold higher than HC. Interestingly, we found MPs from Gr2 transmitted HBV DNA to Huh7 cells and mediated replication (HBV viral load-1.93 × 103 IU/mL at 24 h increased to 3.32 × 104 IU/mL at 72 h), whereas MPs from Gr1 failed to do so. Also, MPs isolated from infected Huh7 cells were unable to cause any infection further in fresh Huh7 cells. Conclusions: Hepatic MPs from CHB patients can act as reservoir of HBV infection and persistence was seen in MPs after clearance of HBV DNA from serum. Also, they are capable of mediating replication in Huh7 cells. MPs from infected Huh7 cultured cells are unable to transmit productive HBV infection in vitro. This novel data may help develop new therapeutic strategies. SAT-170 Hepatic steatosis via controlled attenuation parameter measurements and diabetes contribute to persistent liver fibrosis in the era of long-term nucleoside analogue therapy for chronic hepatitis B W.-K. Seto1, R. Hui1, L.L.Y. Mak1, K.S.H. Liu1, K.-S. Cheung1, J. Fung1, D.K.-H. Wong1, C.-L. Lai1, M.-F. Yuen1. 1Medicine, The University of Hong Kong, Hong Kong, Hong Kong, China E-mail: [email protected] Background and Aims: Natural history studies have shown significant liver fibrosis to be present in >30% of treatment-naïve chronic hepatitis B (CHB). Fibrosis regression is possible during long-term nucleoside analogue (NA) therapy for CHB. The prevalence of significant fibrosis in the era of NA-treated CHB has not been investigated. Methods: From December 2014 onwards, we performed transient elastography (Fibroscan, Echosens, Paris) for liver stiffness (LS) and controlled parameter attenuation (CAP) measurements and comprehensive virologic and metabolic assessment in CHB patients being followed up at the Department of Medicine, Queen Mary Hospital, Hong Kong. Severe fibrosis was defined using the EASL-ALEH criteria (LS >9 kPa in normal alanine aminotransferase). Hepatic steatosis was defined as CAP ≥222 dB/M. Parameters associated with significant fibrosis were determined. Results: 1,581 CHB patients (mean age 52.2 ± 11.3 years, 52.3% male, 89.9% HBeAg-negative) were recruited. 885 patients (55.9%) were treated with NA therapy for a median duration of 6.3 years (interquartile range/IQR 3.2–8.5 years). Median LS and CAP levels were 5.0 kPa (IQR 4.1–7.0 kPa) and 234 dB/m (IQR 198– 275 dB/m) respectively. The prevalence of steatosis, severe fibrosis, cirrhosis were 47.9% (n = 758), 13.9% (n = 220) and 7.0% (n = 111) respectively. Proportion of patients with severe fibrosis increased

Journal of Hepatology 2017 vol. 66 | S543–S750