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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.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.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.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.

Resistance to DNA-damaging agents is a significant cause of treatment failure and poor outcomes in oncology. To identify unrecognized regulators of cell survival we performed a whole-genome CRISPR-Cas9 screen using treatment with ionizing radiation as a selective pressure, and identified STING (stimulator of interferon genes) as an intrinsic regulator of tumor cell survival. We show that STING regulates a transcriptional program that controls the generation of reactive oxygen species (ROS), and that STING loss alters ROS homeostasis to reduce DNA damage and to cause therapeutic resistance. In agreement with these data, analysis of tumors from head and neck squamous cell carcinoma patient specimens show that low STING expression is associated with worse outcomes. We also demonstrate that pharmacologic activation of STING enhances the effects of ionizing radiation in vivo, providing a rationale for therapeutic combinations of STING agonists and DNA-damaging agents. These results highlight a role for STING that is beyond its canonical function in cyclic dinucleotide and DNA damage sensing, and identify STING as a regulator of cellular ROS homeostasis and tumor cell susceptibility to reactive oxygen dependent, DNA damaging agents., The endoplasmic reticulum-localized adaptor STING regulates the innate immune response through its ability to sense DNA damage. Here the authors reveal that STING functions as a regulator of cellular ROS homeostasis and tumor cell susceptibility to reactive oxygen dependent, DNA damaging agents.

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-β, and Caspase-1 dependent IL-1β 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.

Background Immunotherapy has emerged as a transformative approach for the treatment of cancer. However, a significant percentage of patients are nonresponsive to these immunotherapies or experience disease relapse which highlights the need for new therapies. Recent work has highlighted a major role for Stimulator of Interferon Genes (STING) agonists in immunotherapy. Conceptually, the activation of the STING pathway in immune cells in the tumor microenvironment (TME) and tumor cells could result in the induction of innate and adaptive immunity and subsequent activation of cytotoxic T cells and NK cells for durable anti-tumor responses. SB 11285 is a novel agonist of STING pathway leading to the activation of tumor-resident APCs and priming of tumor antigen specific CD8+ T cells. In our preclinical studies using multiple tumor-derived cell lines, SB 11285 has been observed to cause the induction of cytokines, such as INF-b, INF-a, TNFa and others consistent with engagement of the STING target, as well as tumor cell death by STING-mediated apoptosis. SB 11285 reduced tumor volumes in multiple rodent tumor models when administered intravenously, intraperitoneally and intratumorally. Systemic administration could additionally facilitate trafficking of newly activated CD8+T cells from periphery into the tumor site. In addition, preclinical models indicate that survival and local tumor shrinkage were significantly enhanced when SB11285 was administered with anti-CTLA-4 or anti-PD-1 antibody, suggesting that SB 11285 can be administered with anti-PD-1 and anti-CTLA-4 antibody for synergistic activity. A multiple ascending dose, phase 1a/1b trial of SB11285 in multiple tumor types has been initiated and the objectives of this trial include determining a safe and efficacious dose of intravenous SB 11285 and a preliminary assessment of antitumor activity/efficacy as either monotherapy or in combination with nivolumab. Materials and Methods This open-label, multicenter phase 1a/1b clinical trial (NCT04096638) aims to enroll approximately 110 patients in the dose escalation (Part 1) and expansion cohorts (Part 2). Part 1 of the trial is a dose escalation study with IV SB11285 monotherapy followed by combination with the checkpoint inhibitor nivolumab. Part 1 Dose Escalation of the study will evaluate ascending doses of intravenously administered SB 11285 with respect to dose-limiting toxicities (DLTs), maximum tolerated dose (MTD), recommended phase 2 dose (RP2D) and the pharmacokinetic (PK)/pharmacodynamic profile as monotherapy and in combination with nivolumab. SB 11285, with a starting dose of 0.3 μg/kg, will be administered as monotherapy weekly on Days 1, 8, 15, and 22 of repeated 28-day cycles in escalating doses and in combination with nivolumab administered on Q4W schedule. Part 2 Expansion Cohorts of the study will explore initial signs of efficacy in pre-specified tumor types (such as Melanoma, HNSCC) using the recommended phase 2 dose (RP2D) of SB 11285 in combination with nivolumab. In addition, the biological effects of SB 11285 will be evaluated by changes in immune cell types and activation state, serum cytokines, and gene expression patterns indicative of activation of the immune compartment. The trial is being conducted at multiple sites in the U.S. Disclosure Information A. Abbas: A. Employment (full or part-time); Modest; Spring Bank Pharmaceutical Inc. J. Strauss: E. Ownership Interest (stock, stock options, patent or other intellectual property); Modest; Abbvie, Abbott Laboratories, Bristol-Myers Squibb, Intuitive Surgical, Johnson & Johnson, Merck. F. Consultant/Advisory Board; Modest; Tempus. Other; Modest; Dialectic Therapeutics. F. Janku: None. R. Karim: None. A. Olszanski: F. Consultant/Advisory Board; Modest; Bristol Myers Squibb. J.J. Luke: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Modest; All to institution for clinical trials unless noted) Abbvie, Bristol Myers Squibb, Medimmune, Necktar, Novartis, Merck, Leap, Incyte, Immunocore, Compugen, Corvus, Evil, Five Prime, Genentech, Immatic. F. Consultant/Advisory Board; Modest; Consultant:Akrevia, Algios, Array, Astellas,AstraZeneca, Bayer, Bristol Myers Squibb/Advisory Board:7 Hills, Actym, Alphamab Oncology, Mavu (now part of Abbvie), Pyxis, Spring Bank Pharma, Tempest. Other; Modest; Travel: Akrevia, Bayer, Bristol Myers Squibb, Reflexion, EMD Serono, Incyte, Janssen, Merck, Mersana, Novartis. K. Leach: A. Employment (full or part-time); Modest; Spring Bank Pharmaceuticals Inc. R. Iyer: A. Employment (full or part-time); Modest; Spring Bank Pharmaceuticals Inc.

TPS3162 Background: Activation of the Stimulator of Interferon Genes (STING) pathway in immune cells in the tumor microenvironment (TME) and tumor cells results in the induction of innate and adaptive immunity and subsequent activation of cytotoxic T cells and NK cells for durable anti-tumor responses. SB 11285 is a novel agonist of the STING pathway leading to the activation of tumor-resident APCs and priming of tumor antigen specific CD8+ T cells. In our preclinical studies using multiple tumor-derived cell lines, SB 11285 has been observed to cause the induction of cytokines, such as INF-b, INF- a, TNFa and others consistent with engagement of the STING target, as well as tumor cell death by STING-mediated apoptosis. SB 11285 reduced tumor volumes in multiple rodent tumor models when administered intravenously, intraperitoneally or intratumorally as monotherapy or in combination with checkpoint inhibitors such as anti-CTLA-4 or anti-PD-1 antibody. Systemic administration could additionally facilitate trafficking of newly activated CD8+T cells from periphery into the tumor site. Methods: This open-label, multicenter phase 1a/1b clinical trial (NCT04096638) aims to enroll approximately 110 patients in the dose escalation (Part 1) and expansion cohorts (Part 2). Part 1 of the trial is a dose escalation study with IV SB 11285 monotherapy followed by combination with the checkpoint inhibitor nivolumab. Part 1 Dose Escalation of the study will evaluate ascending doses of intravenously administered SB 11285 with respect to dose-limiting toxicities (DLTs), maximum tolerated dose (MTD), recommended phase 2 dose (RP2D) and the pharmacokinetic (PK)/pharmacodynamic profile as monotherapy and in combination with nivolumab. SB 11285, with a starting dose of 0.3μg/kg, will be administered as monotherapy weekly on Days 1, 8, 15, and 22 of repeated 28-day cycles in escalating doses and in combination with nivolumab administered on Q4W schedule. Part 2 Expansion Cohorts of the study will explore initial signs of efficacy in pre-specified tumor types (such as Melanoma, Head and Neck squamous cell carcinoma) using the recommended phase 2 dose (RP2D) of SB 11285 in combination with nivolumab. In addition, the biological effects of SB 11285 will be evaluated by changes in immune cell types and activation state, serum cytokines, and gene expression patterns indicative of activation of the immune compartment. The trial is being conducted at multiple sites in the U.S . Clinical trial information: NCT04096638 .

Background: Cancer immunotherapy is a highly effective therapeutic option for cancer patients. However, the overall response rate with checkpoint inhibitors and other related modalities has been modest. Targeting innate immune signaling pathways that induce type I IFN production to reprogram tumor microenvironment and restore antitumor immunity represents a novel immunotherapeutic approach. We have previously reported that SB 11285 is a first-in-class synthetic cyclic dinucleotide STING agonist, which has demonstrated potent antitumor activity, when used alone or combined with other antitumor agents, in several syngeneic mouse and rat tumor models when administered by intratumoral, intravenous or intraperitoneal routes. Presented here are studies that provide additional insights into the mechanism of action of SB 11285 and analogs. Methods: (a) SB 11285 induces Type I IFN production and other cytokines in human PBMCs and PBMC-derived monocytes. PBMCs and monocytes, isolated from fresh PBMCs using pan monocyte isolation kit (Miltenyi Biotec), were stimulated with SB 11285. Type I IFNs and other cytokines were quantified using regular and multiplex ELISA assays. (b) SB 11285 directly binds STING. To address whether SB 11285 directly binds wild-type human STING, surface plasmon resonance assay was performed with a Biacore T200 device and a biotinylated SB 11285 analog (Biot-SB 11285). (c) Activity against STING polymorphs. Evaluation of SB 11285 and analogs was carried out using both SZ14 reporter cells (HEK293-derived) and HEK293T cells that stably or transiently express STING polymorphic variants. (d) Pharmacodynamic studies. Normal BALB/c mice were injected intravenously with SB 11285 or its analogs at 9 mg/kg. Serum, spleen, and liver samples were collected to quantify RANTES and TNF-α using ELISA. The expression of representative ISGs in spleen samples, including IRF7, IFIT2, and OAS1b were quantified using real-time PCR. (e) Determination of cellular uptake of SB 11285 by PBMCs. The STING agonist activity of Biot-SB 11285 in inducing type I IFN response was confirmed using SZ14 reporter cells and THP1-Dual-WT reporter cells. Human PBMCs were incubated with Biot-SB 11285 and the cellular uptake of compound by immune cells was evaluated using flow cytometry. Results and Conclusion: Our studies provide significant mechanistic insights into the STING agonistic activity of SB 11285 and analogs in that they: (a) induce Type I IFNs, other cytokines and chemokines in PBMCs and monocytes, monocyte-derived dendritic cells; (b) directly bind STING with nanomolar affinity; (c) activate multiple human STING polymorphic variants; (d) induce cytokines, chemokines, and ISGs in mice following i.v. injection; and (e) are effectively taken up by monocytes and other immune cells. SB 11285 is being advanced to human clinical trials. Citation Format: Shenghua Zhou, Sreerupa Challa, Vishal Nair, Geeta Meher, Anjaneyulu Sheri, Rayomand Gimi, Seetharamaiyer Padmanabhan, Dillon Cleary, Leena Suppiah, Diane Schmidt, Santosh Khedkar, Radhakrishnan Iyer. Mechanistic insights into the antitumor activity of SB 11285—a novel STING agonist [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B87.

Background: Agonists of the Stimulator of Interferon Genes (STING) pathway have great potential in cancer immunotherapy. Activation of STING in tumor cells and/or antige- presenting cells (APCs) can induce type I Interferon production, leading to the induction of innate and adaptive immune response. We recently reported the discovery of the cyclic dinucleotide SB 11285 as a potent, first-in-class, STING agonist. Herein, we report SB 11 and SB 12 as unique linkage analogs of SB 11285. Methods: (a) Synthesis: Several linkage analogs of SB 11285 were synthesized by standard solution-phase phosphoramidite chemistry and screened for STING agonistic activity using the previously described SZ14 reporter cell lines, and SB 11 and SB 12 were identified as lead compounds., (b) Binding affinity: Differential scanning fluorimetry was used to determine binding affinity of SB 11 and 12 to wtSTING, with 2´,3´-cGAMP being used as a positive control. (c) Induction of IRF3 and NF-KB: wtTHP-1 or R232-THP1 or RAW cells carrying the respective reporter constructs were treated with SB 11 and 12 or controls for 22hrs and induction of IRF3 and NF-KB was determined as % fold-change in luminescence compared to vehicle-treated cells. (d) Induction of cytokines: PBMCs and mBMDCs were treated with SB 11 and 12 at different doses, and cell supernatants were analyzed for IFN-β, TNF-α, and RANTES by ELISA or multiplexing Luminex assays. (e) In vivo studies: Both SB 11 and 12 were tested for antitumor efficacy in CT26 syngeneic mouse tumor model after intravenous (i.v.) administration at 3mg/kg on days 1,5,9,14. Results: (i) SB 11 and 12 demonstrated high binding affinity to human wild-type and mouse STING that is evident by 16° thermal shift. (ii) SB 11 and 12 showed potent induction of (a) STING-dependent IRF3 (EC50: 0.8 and 39.7 nM) and NF-κB (EC50: 9 and 5265 nM) signaling induction in wtTHP-1 cells; (b) IRF3 (EC50: 2.6 and 317 nM) and NF-κB (EC50: 83 and 3783 nM) induction in R232-THP-1 cells; (c) IRF3 (EC50: 67 and 46 nM) in RAW macrophages. (iii) Both SB 11 and SB 12 induced STING dependent secretion of IFN-β (~10 ng/ml) in mBMDCs. (iv) In the CT26 colon carcinoma syngeneic mouse models, both SB 11 and 12 showed potent antitumor activity when administered by i.v. route with 98 and 97% TGI and 91 and 79% TGD, respectively. Conclusion: SB 11 and SB 12 showed excellent safety and antitumor activity in syngeneic mouse model when administered by i.v. route. SB 11 and SB 12 were shown to cause STING-dependent activation of IRF3 and NF-κB signaling, as well as the induction of type I IFN signature and ISGs. Further preclinical studies of systemically administered analogs SB 11 and SB 12 are in progress. Citation Format: Shenghua Zhou, Sreerupa Challa, Diane Shmidt, Leena Suppiah, Vishal Nair, Anjaneyulu Sheri, Geeta Meher, Rayomand Gimi, Seetharamaiyer Padmanabhan, Dillon Cleary, Radhakrishnan Iyer. Development of SB 11 and SB 12, structurally unique linkage analogs of SB 11285 as STING agonists for Immuno-oncology [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B53.

Background: Immunotherapy has emerged as a transformative approach for the treatment of cancer. Recent evidence suggests that the activation of Stimulator of Interferon Genes (STING) pathway in immune calls within the tumor microenvironment (TME) can induce the production of Interferons and cytokines that would lead to the induction of innate and adaptive immunity. We have previously disclosed the discovery and development of SB 11285 as a first-in-class synthetic cyclic dinucleotide STING agonist, which has demonstrated potent antitumor activity, when used alone or combined with other antitumor agents, in several syngeneic mouse and rat tumor models when administered by intratumoral, intravenous, or intraperitoneal routes. Presented here is the development of a controlled-release nanoparticle formulation of SB 11285 for subcutaneous administration. Methods: (a) Nanoparticle formulations of SB 11285. SB 11285-NPs were generated using Polylactic-glycolic acid copolymer (PLGA) using double emulsion and nano-precipitation methods (b) Imaging of nanoparticles: Scanning electron microscopy was performed to visualize and measure size of the nanoparticles. (c) Induction of IRF3 and NF-KB. THP-1 cells and RAW macrophages carrying dual reporter constructs were treated with either SB 11285-NPs or naked SB 11285 for 22hrs and induction of IRF3 and NF-KB was calculated as % fold-change in luminescence compared to vehicle-treated cells. (d) Induction of cytokines: PBMCs were treated with different concentrations of 11285-NPs or naked SB 11285, and cell supernatants were analyzed for IFN-β, TNF-α, and RANTES by ELISA. (e) Dendritic cell maturation assay: PBMCs were differentiated into immature dendritic cells by addition of IL4 and GM-CSF followed by treatment with either SB 11285-NP or naked SB 11285. The maturation of dendritic cells was then evaluated by analyzing expression of CD83 and CD86 by flow cytometry. Results: PLGA formulations of SB 11285 prepared by double-emulsion method with an entrapment efficiency of 30 to 40% were spherical particles with an average diameter of 800nm. The nanoparticles were stable in aqueous media with minimal release of the entrapped SB 11285. In cell-culture studies, SB 11285-NPs showed potent induction of: (a) STING-dependent IRF3 induction in THP-1 cells (EC50: 3nM) as well as RAW macrophages (EC50: 3nM); (b) secretion of IFN-β (35 pg/ml), TNF-α (800 pg/ml), and RANTES (200 pg/ml), when tested at a low concentration of 112nM; and (c) SB 11285-NPs effectively induced DC maturation that was evident by increase in CD83 and CD86 expression. Conclusion: PLGA nanoparticles of SB 11285 were successfully prepared with submicron particle size that demonstrated potent expression of STING-dependent Type I IFN and cytokines in immune cells. The nanoparticles also induced maturation of dendritic cells. In vivo evaluation is in progress. Citation Format: Sreerupa Challa, Leena Suppiah, Dillon Cleary, Anjaneyulu Sheri, Rayomand Gimi, Geeta Meher, Seetharamaiyer Padmanabhan, Sumit Shah, Pranav Bhatt, Jovita Tauro, Radhakrishnan Iyer. Nanoparticle formulation of the STING agonist SB 11285 [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B75.

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. β-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 β-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 β-blockers with improved half-lives and pKi values comparable with marketed β-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.

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.

The 35S-labeled, dinucleoside phosphorothioate 1, an orally available agent against hepatitis B virus, was prepared in eight steps with high specific activity and radiochemical purity. Radiolabeled 3H-benzodithiole-3-one-1,1-dioxide was synthesized in four steps from 35S8 and was used as the sulfurizing reagent. Copyright © 2012 John Wiley & Sons, Ltd.

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

Open-chain bis-Reissert compounds 1 were converted to the corresponding bis-oxazolium intermediates via acid-catalyzed intramolecular cyclization. These oxazolium compounds exist in a variety of tautomeric structures in which the meso-ionic form can be intercepted by reaction with dipolarophiles in a 1,3-dipolar-cycloaddition reaction to produce a variety of highly functionalized bis-pyrrole esters 2. In turn, the bis-pyrrole esters could be converted to the corresponding bis-pyrrole tetrols 3 in high yields. J. Heterocyclic Chem., 2011.

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).

A simple quantitative structure activity relationship (QSAR) approach termed local indices for similarity analysis (LISA) has been developed. In this technique, the global molecular similarity is broken up as local similarity at each grid point surrounding the molecules and is used as a QSAR descriptor. In this way, a view of the molecular sites permitting favorable and rational changes to enhance activity is obtained. The local similarity index, calculated on the basis of Petke's formula, segregates the regions into "equivalent", "favored similar", and "disfavored similar" (alternatively "favored dissimilar") potentials with respect to a reference molecule in the data set. The method has been tested on three large and diverse data sets-thrombin, glycogen phosphorylase b, and thermolysin inhibitors. The QSAR models derived using genetic algorithm incorporated partial least square analysis statistics are found to be comparable to the ones obtained by the standard three-dimensional (3D)-QSAR methods, such as comparative molecular field analysis and comparative molecular similarity indices analysis. The graphical interpretation of the LISA models is straightforward, and the outcome of the models corroborates well with literature data. The LISA models give insight into the binding mechanisms of the ligand with the enzyme and allow fine-tuning of the molecules at the local level to improve their activity.

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.

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.

Rapid and efficient functionalization of solid-support by microwave-assisted procedures has been achieved. The functionalized solid-support can be readily loaded with nucleosides for use in oligonucleotide synthesis. This method can also be extended to rapid functionalization of other solid matrices for application in microarrays and combinatorial chemistry.

e14616 Background: The activation of innate and adaptive immunity via Stimulator of Interferon Genes (STING) signaling is a potentially transformative immuno-therapeutic strategy in cancer. We report here in vivo efficacy and safety studies of SB 11285. Methods: Tumor Growth Inhibition (TGI) and Tumor Growth Delay (TGD) studies in syngeneic mouse models were initiated when mean tumor volume (MTV) reached 100mm3: A20 Lymphoma: (10 animals/Group); (A) Saline; (B) 100µg SB 11285, intratumoral (i.t.), days 3,4,6,8,10; (C) 100mg/kg Cyclophosphamide, intraperitoneal (i.p), days 1,2; and (D) combination of cyclophosphamide+SB 11285. CT26 Colon Carcinoma experimental design is shown in Table. Re-challenge study was initiated in tumor-free animals in A20 lymphoma model on day 73, and monitored for an additional 45 days. Presence of activated immune cells intumor tissues was evaluated by immuno-histochemistry. Cytokine response was evaluated in serum after a single i.p. injection of SB 11285 at 10mg/kg. Maximum Tolerated Dose (MTD) in mice was determined by daily i.p. injection of SB 11285 for 10 days. Results: A20 model: % TGI in the treatment groups were—A, 0; B, 86; C, 98, and D, 93; % TGD, day 70: A, 0; B, 64; C, 156; D, 288. In D, day 73, 90% of animals remained tumor-free. CT26 model: Table 1 shows MTV on day 19 and %TGD on day 43; Re-challenge study. All animals from the SB 11285-treated groups remained completely tumor-free on day 45 compared to control group (MTV,1666 mm3); Immuno-histochemistry of SB 11285-treated groups, revealed the infiltration of CD8+T and NK cells into tumor and surrounding tissues; Cytokine analysis did not show systemic inflammatory response; MTD of SB 11285 was 16 mg/kg/day. Conclusions: SB 11285, a novel STING agonist, showed very potent, and highly durable immune response-mediated anti-tumor activity. SB 11285 was well tolerated, safe, and is being advanced to IND-enabling studies. [Table: see text]

Background: Immunotherapy has recently emerged as a transformative approach for the treatment of cancer; nevertheless, many patients remain unresponsive to treatment. It is being recognized that induction of type I interferons (IFN) and interferon-stimulated genes (ISGs) in tumor cells and within the tumor microenvironment (TME) is essential for modulating the host-immune response and inducing apoptosis of tumor cells. Furthermore, the antigen-presenting cells within TME can cause induction of adaptive immune response, through priming of CD8+ T cells and tumor killing. Importantly, the DNA released from damaged cells and cancer cells can be sensed by cyclic GMP-AMP synthase (cGAS) leading to the synthesis of cyclic-GMP-AMP (2',3'-cGAMP), a second messenger that activates Stimulator of Interferon Genes (STING) pathway resulting in the production of type I IFN and ISGs. The cumulative effects of activation of innate and adaptive immune response can result in potent anti-cancer effects. Therefore, therapeutic agents that activate the cGAS-STING signaling pathway in tumor cells and TME are urgently needed. Herein, we describe the discovery of novel potent, first-in-class small molecules for application in immuno-oncology. Methods: Using structure-guided drug design, in conjunction with published crystal structures of cyclic dinucleotides bound to STING, a focused library of dinucleotide compounds was synthesized using phosphoramidite chemistry and evaluated for: (a) Induction of IFN signaling: The compounds were screened for the induction of Interferon regulatory factor (IRF), ISG54, and NF-κB using reporter assays. We used HEK293 cell line (SZ14) stably expressing ISG54 (ISRE)-promoter-driven firefly luciferase reporter gene for screening and the active compounds were further characterized in THP1 cells and human primary PBMCs. The IRF, ISG54, and NF-κB induction was calculated from % fold-change in luminescence compared to DMSO-treated cells and EC50s of the compounds were ascertained to identify active compounds, (b) Expression of IFN-β and IRF7 in THP1 cells: THP1 cells were treated with active compounds or controls for 22hrs. RNA was extracted and the expression of IFN-β, IRF7, was ascertained using semi-quantitative RT-PCR, (c) Induction of pathogen recognition receptors (PRRs) including RIG-I, MDA5, LGP2, and OAS-1 and ISG54: THP1 cells and PBMCs were treated with active compounds, 2',3'-cGAMP (control), or DMSO and the gene expression of different PRRs, ISGs, was determined by quantitative RT-PCR using ΔΔct method, (d) Induction of cGAS-STING signaling using reporter assays: HEK293 cells stably expressing ISG54 were transfected with plasmids encoding human cGAS (wild-type, or K384A, K400A, or K411A mutants) and treated with active compounds, poly (dA:dT) (positive control), or DMSO for 21 hrs. ISG54 induction was calculated as fold-change in luminescence compared to DMSO-treated controls. (e) Cytotoxicity assays: THP1 cells were treated with active compounds or DMSO control with Lipofectamine and cytotoxicity assessed using the CellTiter-Glo® Luminescent assays. Cytotoxicity was calculated from %-fold change in luminescence compared to DMSO-treated sample. Results: Through in vitro assays in conjunction with Structure Activity Relationship studies, we have identified potent compounds that activate cGAS-STING signaling pathway for induction of IRF, IFN, and NF-κB. These compounds also cause induction of expression of PRRs, including RIG-I, MDA5, LGP2, as well as, ISG54 and OAS-1. Conclusion: We have discovered potent, first-in-class agents that cause induction of IFN, NF-κB, ISGs, and PRRs. Further optimization and preclinical evaluation of the compounds for application in immuno-oncology is underway. Citation Format: Shenghua Zhou, Sreerupa Challa, Seetharamaiyer Padmanabhan, Anjaneyulu Sheri, Samantha Delaney, Geeta Meher, Dillon Cleary, Rayomand Gimi, Santosh Khedkar, Radhakrishnan Iyer. Novel dinucleotides that activate STING signaling for immuno-oncology. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B39.

Immunotherapy has recently emerged as a transformative approach for the treatment of cancer; nevertheless, many patients remain unresponsive to treatment. Recent evidence suggests that the activation of Stimulator of Interferon Genes (STING) pathway in tumor cells and/or antigen presenting cells (APCs) within the tumor microenvironment (TME) can induce type I Interferon production leading to apoptosis of tumor cells, as well as, induction of adaptive immune response (through priming of CD8+ T cells to tumor-associated antigens) thereby providing a powerful anti-cancer strategy. Therefore, therapeutic agents that activate STING signaling pathway in tumor cells and APCs in the TME are urgently needed. Herein, we describe the discovery of highly potent and selective first-in-class STING agonists for application in immuno-oncology. Methods: Using structure-guided drug design, in conjunction with published crystal structures of different cyclic dinucleotides bound to STING, a focused library of nucleotide compounds was prepared using standard phosphoramidite chemistry. The compounds were screened for induction of Interferon regulatory factor (IRF), Interferon-stimulated gene 54 (ISG54), and NF-KB using reporter assays. We used HEK293 cell line stably expressing ISG54 (ISRE)-promoter-driven firefly luciferase reporter gene for initial hit discovery and the actives were further characterized in PBMCs and THP1 cells. The IRF and NF-kB induction was calculated from % fold-change in luminescence compared to DMSO-treated cells and EC50 of the compounds were ascertained using Xlfit. Lead STING agonists were further evaluated for: (a) Binding affinity: Binding assays were conducted by Differential Scanning Fluorimetry (DSF) and Tm was calculated using Thermal Shift software, (b) Induction of pathogen recognition receptors (PRRs), ISGs and Programmed Death Ligands 1 & 2 (PDL1, PDL2) genes: THP1 cells and PBMCs were treated with various concentrations of lead compounds or 2,'3'-cGAMP or DMSO and the gene expression of different PRRs, ISGs, PDL1, and PDL2 was determined by quantitative RT-PCR using ΔΔct method, (c) Apoptosis-inducing activity: PBMCs and THP1 cells were treated with various concentrations of lead compounds, 2',3'-cGAMP, or DMSO control and the apoptotic activity was evaluated using Caspase-Glo® 3/7 Assay (Promega), and (d) In vitro anti-tumor activity: STING-dependent anti-tumor activity of lead compounds in various tumor cell lines was assessed by either high-content imaging or through Cell titer Glo® Cytotoxicity Assay (Promega). Cell survival was calculated based upon % reduction of live cells compared to DMSO control. CC50 of the compounds were generated by curve fit in Xlfit. Results: Through in vitro assays in conjunction with Structure Activity Relationship (SAR) studies, we have identified several highly potent and selective first-in-class STING agonists. A promising lead nucleotide compound SB 11285 caused STING-dependent induction of: (a) IRF with an EC50 of 2 nM that is 1000-fold more potent than the natural STING agonist 2',3'-cGAMP, (b) NF-kB with an EC50 of 200 nM that is >200-fold more potent than 2',3'-cGAMP, (c) selective apoptosis of human monocyte leukemic cell lines (CC50, 500 nM) as compared to normal PBMCs through induction of IFN, and NF-kB signaling, and (d) expression of various PRRs and ISGs including RIG-I, MDA-5, LGP2, ISG54 and OAS-1, as well as, PDL1 and PDL2. Finally, SB 11285 showed potent in vitro anti-tumor activity in multiple tumor cell lines. Conclusion: We have discovered highly potent first-in-class STING agonists that show excellent selectivity in induction of IFN, NF-KB, ISGs, and PRRs, and apoptosis of tumor-derived cell lines. The lead STING agonist SB 11285 has potent immune-modulating, as well as, anti-tumor activities and is being advanced for additional preclinical studies for application in immuno-oncology. Citation Format: Sreerupa Challa, Shenghua Zhou, Anjaneyulu Sheri, Seetharamaiyer Padmanabhan, Samantha Delaney, Geeta Meher, Dillon Cleary, Vishal Nair, Rayomand Gimi, Santosh Khedkar, Radhakrishnan Iyer. Nucleotide analogs as novel STING agonists for immuno-oncology. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B40.

The preparation of a controlled-pore-glass (CPG) support carrying a novel linker is described. This support was compatible with the established phosphoramidite method of solid-phase oligonucleotide synthesis. The use of this linker for the synthesis of a library of 3′-thiophosphorylated dinucleotides is described.

The nucleoside bicylic oxazaphospholidine derived from L-, or D-prolinol is a novel synthon with potential for solid-phase stereoselective synthesis of oligonucleoside phosphorothioates.

Modulation of gene expression using oligonucleotides (oligos) is currently an area of intense activity, both from therapeutic, as well as research perspectives. To develop oligos as therapeutic agents, in addition to demonstrable biological activity, in vivo metabolic stability, tissue disposition and pharmacokinetics are important considerations. Oligodeoxynucleoside phosphorothioates are the first-generation antisense analogs that have been studied extensively, and are in clinical trials against a number of disease indications. In an effort to improve the antisense properties of these compounds, mixed-backbone oligos incorporating different chemical modifications have been synthesized and evaluated for antisense activity. The present review will provide an overview of the pharmacokinetics and toxicology following intravenous, intraperitoneal, subcutaneous and oral administration of mixed-backbone oligos as second-generation antisense therapeutics.

Mixed backbone oligonucleotides (MBOs) (containing ionic and non-ionic internucleotidic linkages) in which the non-ionic segments are either methylphosphotriester (PO-OMe) or primary phosphoramidate (PO-NH2) linkages have been prepared using the recently described N-pent-4-enoyl (PNT) nucleoside phosphoramidates and H-phosphonates. Biophysical properties and stability studies suggest that these MBOs are novel antisense molecules.

The use of NH3 gas under pressure offers an efficient alternative to hot aqueous ammonium hydroxide for the deprotection, and cleavage steps during the large-scale synthesis of oligonucleotides, and their analogs prepared using N-pent-4-enoyl (PNT) nucleoside phosphoramidites and H-phosphonates.

Rapid synthesis of phosphoric diester and phosphorothioate oligonucleotides in 1 micromol to 1 millimol scale has been achieved using PNT nucleoside phosphoramidites. Furthermore, facile synthesis of mixed backbone oligonucleotides (MBOs) as antisense agents can be carried out using PNT nucleoside phosphoramidites in conjunction with PNT nucleoside H -phosphonates and PNT nucleoside phosphonamidites. The versatility of the PNT group is further demonstrated by its use in the preparation of bioreversible oligonucleotide conjugates.

Oligonucleotide analogs which are site-specifically derivatized with O-acyloxyaryl groups were prepared using phosphoramidite chemistry. Upon their incubation with pig liver esterase (PLE) or chymortrypsin, the analogs were shown to revert to the parent compound.

The synthesis of mixed-backbone-oligonucleotides (MBOs) (15 to 25-mers) which contain phosphoric diester (PO) or phosphorothioate (PS) backbones along with segments of internucleotidic O-methylphosphate (POOMe) or O-methylphosphorothioate (PSOMe) linkages have been accomplished using N-pent-4-enoyl (PNT) nucleoside phosphoramidite synthons. The Tms of the MBOs reveal that they form stable duplexes with complementary DNA and RNA. The MBOs also showed increased resistance to degradation by nucleases. Consequently, the MBOs with methylphosphotriester segments represent a novel class of antisense oligonucleotides with potential for therapeutic and diagnostic applications.

Improved design, synthesis, and evaluation of an oligonucleotide prodrug is described.

As part of a therapeutic strategy involving multiple targeting, we have synthesized oligonucleotides which are linked via 3′-3′ phosphoric diester linkages. These 3′-3′ analogs showed increased resistance against nuclease-mediated degradation compared to the 5′-3′-linked oligonucleotides.

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-β, 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.

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.

Dowex® resin (H+ form) is a convenient reagent for detritylation of 5′-O-DMT-oligonucleotides. With Dowex, detritylation is rapid and quantitative compared to that with acetic acid. As the liberated dimethoxytrityl species remain bonded to the resin, further processing of oligonucleotides is simplified.

Synthesis of iodoalkylacylates 3a-f and use of 3 c in the preparation of an oligonucleoside phosphorothiate prodrug analog 5 is described.

Oligodeoxynucleotide phosphorothioates containing segments of 2'-O-methyloligoribonucleotide phosphorothioates at both 3'- and 5'-ends (hybrid oligonucleotide) have been shown to be potent antisense agents. In the present study, in vivo biostability, disposition, and excretion of a 25-mer hybrid oligonucleotide were determined in rats after i.v. bolus administration of the 35S-labeled oligonucleotide at a dose of 30 mg/kg. The plasma disappearance curve for the hybrid oligonucleotide could be described by a two-compartmental model, with half-lives of 0.34 and 52.02 hr, respectively. The majority of the radioactivity in plasma was associated with the intact hybrid oligonucleotide. Urinary excretion represented the major pathway of elimination, with 21.98 +/- 3.21% (mean +/- SD) of the administered dose excreted within 24 hr and 38.13 +/- 2.99% over 240 hr post-dosing. The majority of the radioactivity in urine was associated with the degradative products with lower molecular weights, but the intact form was also detected by HPLC analysis. Fecal excretion was a minor pathway of elimination with 2.34 +/- 0.13% of the administered dose excreted over 24 hr and 6.74 +/- 0.40% over 240 hr post-dosing. A wide tissue distribution of hybrid oligonucleotide was observed based on radioactivity levels, and analysis by HPLC showed that the majority of the radioactivity in tissues was associated with the intact hybrid oligonucleotide. Further analyses of the experimental data provided a comprehensive pharmacokinetic analysis of hybrid oligonucleotide in each tissue. Compared with a previously examined oligodeoxynucleotide phosphorothioate (GEM 91) that has a similar nucleotide sequence, the hybrid oligonucleotide had a shorter distribution half-life and a longer elimination half-life, based on the quantitation of radioactivity in plasma. Although it had a similar tissue distribution pattern compared with other oligonucleotide phosphorothioates such as GEM 91, the hybrid oligonucleotide was more stable in vivo, which may be important in the development of antisense oligonucleotides as therapeutic agents.

By incorporating a “capping step” at the start of an oligonucleotide synthesis (“pre-cap”) and following a “SUP” work-up protocol with ammonium hydroxide, an overall improvement is observed in the quality of oligonucleotides synthesized on a large scale on controlled pore glass support (CPG). Rationalization of these results is provided.

Tritium was incorporated into oligonucleotides at a predetermined site in high specific activity through a combination of redox chemistry on solid support.