Activating the stimulator of interferon genes (STING) pathway with STING agonists is an attractive immune oncology concept to treat patients with tumors that are refractory to single-agent anti-PD-1 therapy. For best clinical translatability and broad application to cancer patients, STING agonists with potent cellular activation of all STING variants are desired. Novel cyclic dinucleotide (CDN)-based selective STING agonists were designed and synthesized comprising noncanonical nucleobase, ribose, and phosphorothioate moieties. This strategy led to the discovery of 2',3'-CDN 13 (BI 7446), which features unprecedented potency and activates all five STING variants in cellular assays. ADME profiling revealed that CDN 13 has attractive drug-like properties for development as an intratumoral agent. Injection of low doses of CDN 13 into tumors in mice induced long-lasting, tumor-specific immune-mediated tumor rejection. Based on its compelling preclinical profile, BI 7446 has been advanced to clinical trials (monotherapy and in combination with anti-PD-1 antibody).

Introduction: Stimulator of interferon genes (STING) is activated by cyclic dinucleotides that are generated by cyclic GMP-AMP synthase in the presence of cytosolic DNA, resulting in a type 1 interferon response and the secretion of pro-inflammatory cytokines. BI-STING mimics the natural ligand of STING and hijacks the STING signaling pathway to trigger a potent anti-tumor immune response. Here we report the pre-clinical characterization of BI-STING in human cells lines in vitro, in human ex vivo systems and in vivo mouse models. Methods: Activity and specificity of BI-STING were tested in vitro in the human monocytic leukemia cell line THP1. Target engagement was confirmed using ex vivo studies in human whole blood and in human precision cut colorectal cancer slices. For in vivo studies, BI-STING was administered intratumorally (i.tu.) in a range of subcutaneous, syngeneic murine tumor models. The modulation of cytokine secretion over time was recorded and the induction of a tumor-specific immune response demonstrated. Results: Using THP1 cells, activation of down-stream signaling events by BI-STING was seen specifically in STING wild type, but not STING knock out cells. Ex vivo treatment of human whole blood and human precision cut colorectal cancer slices with BI-STING resulted in the significant induction of cytokine secretion (including IFNβ) and in an array of transcriptional changes associated with the activation of STING signaling. Treatment of tumor-bearing mice with a single dose of BI-STING i.tu. led to a transient increase of cytokine levels in tumor and plasma. In all tested models, i.tu. administration of BI-STING resulted in dose-dependent local tumor control. Importantly, animals whose primary tumor was cured by BI-STING treatment did not develop tumors when re-challenged with the same tumor cell line and an abscopal delay in tumor growth was seen for non-injected (or anenestic) lesions in mice carrying bilateral tumors. Abscopal tumor control was further improved when combined with anti-PD-1. Finally, ELISpot analysis resulted in a significantly increased number of immunospots in splenocytes from BI-STING treated animals as compared to vehicle control mice, confirming the induction of a tumor-specific immune response. Conclusions: BI-STING is a potent and specific inducer of the STING signaling pathway. In addition to local tumor control, i.tu. treatment with BI-STING results in a systemic anti-tumor immune response in mice, which was enhanced upon anti-PD-1 combination. A clinical trial to evaluate i.tu. administration of BI-STING alone and in combination with anti-PD-1 in patients with advanced solid tumors is ongoing. Citation Format: Gabriela Gremel, Maria A. Impagnatiello, Sebastian Carotta, Otmar Schaaf, Paolo M. Chetta, Thorsten Oost, Thomas Zichner, Marco Hofmann, Sophia Blake, Tom Bretschneider, Martin Fleck, Achim Grube, Herbert Nar, Georg Rast, Esther Schmidt, Ute Klinkhardt, Kirsten Arndt-Schmitz, Thorsten Laux, Vittoria Zinzalla, Jonathon Sedgwick, Norbert Kraut. Potent induction of a tumor-specific immune response by a cyclic dinucleotide STING agonist [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4522.

Background: Excess activity of neutrophil elastase (NE) in the lung of COPD patients results in the degradation of extracellular matrix (ECM) proteins and an increased activation of other proteases, leading to the destruction of alveolar septa and, ultimately, emphysema development. Direct inhibition of NE is expected to reduce lung parenchyma destruction, emphysema formation and progression in patients with COPD. Aim: We investigated the in vitro and in vivo profile of the novel NE inhibitor BI 1323495. Methods: Inhibition of human NE and the related proteases cathepsin G and proteinase 3 was determined in enzymatic assays using purified enzymes. In addition, inhibition of NE in plasma from zymosan-stimulated human whole blood was measured. An acute lung injury model in mice was used to monitor the in vivo inhibition of NE in the lung by BI 1323495. Lung injury was induced by intratracheal application of 25 µg human NE and haemoglobin concentration as a read-out for lung injury, and neutrophil counts as a read-out for inflammation were measured in bronchioalveolar lavage fluid after 4 hours. Results: BI 1323495 blocked NE in vitro with an IC50 of 0.4 nM and demonstrated a \u003e 4000-fold selectivity versus related proteases cathepsin G and proteinase 3. BI 1323495 fully inhibited the activity of NE released by zymosan-stimulated human neutrophils with a mean IC50 of 1 nM. Treatment of mice with an oral formulation of BI 1323495 attenuated lung damage and inflammation induced by intratracheal instillation of human recombinant NE with an ED50 of 1.9 mg/kg. Conclusions: BI 1323495 is a very potent and highly selective inhibitor of human NE with in vivo efficacy.

Herein we report the discovery of a novel oxindole-based series of vasopressin 1b (V1b) receptor antagonists. Introducing a substituted piperazine moiety and optimizing the southern and the northern aromatic rings resulted in potent, selective and brain penetrant V1b receptor antagonists. Compound 9c was found to be efficacious in a rat model of anti-depressant activity (3 mg/kg, ip). Interestingly, both moderate terminal half-life and moderate bioavailability could be achieved despite sub-optimal microsomal stability.

Although overweight and obesity are highly prevalent conditions, options to treat them are still very limited. As part of our search for safe and effective MCH-R1 antagonists for the treatment of obesity, two series of pyridones and pyridazinones were evaluated. Optimization was aimed at improving DMPK properties by increasing metabolic stability and improving the safety profile by reducing inhibition of the hERG channel and reducing the potential to induce phospholipidosis. Steric shielding of a labile keto moiety with an ortho-methyl group and fine-tuning of the polarity in several parts of the molecule resulted in BI 186908 (11 g), a potent and selective MCH-R1 antagonist with favorable DMPK and CMC properties. Chronic administration of BI 186908 resulted in significant body weight reduction comparable to sibutramine in a 4 week diet-induced obesity model in rats. Based on its favorable safety profile, BI 186908 was advanced to pre-clinical development.

Despite recent success there remains a high therapeutic need for the development of drugs targeting diseases associated with the metabolic syndrome. As part of our search for safe and effective MCH-R1 antagonists for the treatment of obesity, a series of 3,6-disubstituted pyridazines was evaluated. During optimization several issues of the initial lead structures had to be resolved, such as selectivity over related GPCRs, inhibition of the hERG channel as well as the potential to induce phospholipidosis. Utilizing property-based design, we could demonstrate that all parameters can significantly be improved by consequently increasing the polarity of the compounds. By this strategy, we succeeded in identifying potent and orally available MCH-R1 antagonists with good selectivity over M1 and 5-HT2A and an improved safety profile with respect to hERG inhibition and phospholipidosis.

Despite recent approvals of anti-obesity drugs there is still a high therapeutic need for alternative options with higher efficacy in humans. As part of our MCH-R1 antagonist program for the treatment of obesity, a series of biphenylacetamide HTS hits was evaluated. Several issues of the initial lead structures had to be resolved, such as potency, selectivity over related GPCRs and P-gp efflux limiting brain exposure in this series. We could demonstrate that all parameters can be significantly improved by structural modifications resulting in BI 414 as a potent and orally available MCH-R1 antagonist tool compound with acceptable in vivo efficacy in an animal model of obesity.