Anti-Granulocyte-Macrophage Colony-Stimulating Factor Monoclonal Antibody Gimsilumab for COVID-19 Pneumonia: A Randomized, Double-Blind, Placebo-controlled Trial.

Rationale: GM-CSF (granulocyte-macrophage colony-stimulating factor) has emerged as a promising target against the hyperactive host immune response associated with coronavirus disease (COVID-19). Objectives: We sought to investigate the efficacy and safety of gimsilumab, an anti-GM-CSF monoclonal antibody, for the treatment of hospitalized patients with elevated inflammatory markers and hypoxemia secondary to COVID-19. Methods: We conducted a 24-week randomized, double-blind, placebo-controlled trial, BREATHE (Better Respiratory Education and Treatment Help Empower), at 21 locations in the United States. Patients were randomized 1:1 to receive two doses of intravenous gimsilumab or placebo 1 week apart. The primary endpoint was all-cause mortality rate at Day 43. Key secondary outcomes were ventilator-free survival rate, ventilator-free days, and time to hospital discharge. Enrollment was halted early for futility based on an interim analysis. Measurements and Main Results: Of the planned 270 patients, 225 were randomized and dosed; 44.9% of patients were Hispanic or Latino. The gimsilumab and placebo groups experienced an all-cause mortality rate at Day 43 of 28.3% and 23.2%, respectively (adjusted difference = 5% vs. placebo; 95% confidence interval [-6 to 17]; P = 0.377). Overall mortality rates at 24 weeks were similar across the treatment arms. The key secondary endpoints demonstrated no significant differences between groups. Despite the high background use of corticosteroids and anticoagulants, adverse events were generally balanced between treatment groups. Conclusions: Gimsilumab did not improve mortality or other key clinical outcomes in patients with COVID-19 pneumonia and evidence of systemic inflammation. The utility of anti-GM-CSF therapy for COVID-19 remains unclear. Clinical trial registered with www.clinicaltrials.gov (NCT04351243).

Identification of triazolopyridazinones as potent p38α inhibitors.

Structure-activity relationship (SAR) investigations of a novel class of triazolopyridazinone p38α mitogen activated protein kinase (MAPK) inhibitors are disclosed. From these studies, increased in vitro potency was observed for 2,6-disubstituted phenyl moieties and N-ethyl triazolopyridazinone cores due to key contacts with Leu108, Ala157 and Val38. Further investigation led to the identification of three compounds, 3g, 3j and 3m that are highly potent inhibitors of LPS-induced MAPKAP kinase 2 (MK2) phosphorylation in 50% human whole blood (hWB), and possess desirable in vivo pharmacokinetic and kinase selectivity profiles.

Part 2: Structure-activity relationship (SAR) investigations of fused pyrazoles as potent, selective and orally available inhibitors of p38alpha mitogen-activated protein kinase.

A novel class of pyrazolopyridazine p38alpha mitogen-activated protein kinase (MAPK) inhibitors is disclosed. A structure activity relationship (SAR) investigation was conducted driven by the ability of these compounds to inhibit the p38alpha enzyme, the secretion of TNFalpha in a LPS-challenged THP1 cell line and TNFalpha-induced production of IL-8 in the presence of 50% human whole blood (hWB). This study resulted in the discovery of several inhibitors with IC(50) values in the single-digit nanomolar range in hWB. Further investigation of the pharmacokinetic profiles of these lead compounds led to the identification of three potent and orally bioavailable p38alpha inhibitors 2h, 2m, and 13h. Inhibitor 2m was found to be highly selective for p38alpha/beta over a panel of 402 other kinases in Ambit screening, and was highly efficacious in vivo in the inhibition of TNFalpha production in LPS-stimulated Lewis rats with an ED(50) of ca. 0.08mg/kg.

Part 1: Structure-Activity Relationship (SAR) investigations of fused pyrazoles as potent, selective and orally available inhibitors of p38alpha mitogen-activated protein kinase.

A novel class of fused pyrazole-derived inhibitors of p38alpha mitogen-activated protein kinase (MAPK) is disclosed. These inhibitors were evaluated for their ability to inhibit the p38alpha enzyme, the secretion of TNFalpha in a LPS-challenged THP1 cell line and TNFalpha-induced production of IL-8 in 50% human whole blood. This series was optimized through a SAR investigation to provide inhibitors with IC(50) values in the low single-digit nanomolar range in whole blood. Further investigation of their pharmacokinetic profiles led to the identification of two potent and orally bioavailable p38 inhibitors 10 m and 10 q. Inhibitor 10 m was found to be efficacious in vivo in the inhibition of TNFalpha production in LPS-stimulated Lewis rats with an ED(50) of 0.1mg/kg while 10 q was found to have an ED(50) of 0.05-0.07 mg/kg.

Predictability of peripheral lymphocyte reduction of novel S1P1 agonists by in vitro GPCR signaling profile.

Surrogate readouts of G-protein-coupled receptor signaling pathways using highly engineered systems are often employed in the drug discovery process. However, accumulating data have demonstrated the importance of selecting relevant biological activity rather than technically facile assays to support high-throughout screening and subsequent structure-activity relationship studies. Here we report a case study using sphingosine-1-phosphate receptor 1 (S1P(1)) as the model system to compare compound activity in six different in vitro assays with their ability to predict in vivo efficacy. S1P(1) has long been validated as a therapeutic target for autoimmune diseases. In this article, in vivo and in vitro studies on 19 S1P1 agonists are reported. In vitro activities of these S1P(1) agonists, together with S1P and FTY720p, on Ca(2+) mobilization, adenylyl cyclase inhibition, extracellular signal-related kinase (ERK) phosphorylation, ß-arrestin recruitment, and receptor internalization, were determined. The in vitro potency of these compounds was correlated with their ability to induce peripheral lymphocyte reduction. The results revealed that inhibition of adenylyl cyclase and induction of ß-arrestin recruitment and receptor internalization are good indicators to predict in vivo efficacy, whereas induction of Ca(2+) mobilization through G(qi/5) coupling and ERK phosphorylation is irrelevant. This study demonstrated the importance of identifying an appropriate in vitro assay to predict in vivo activity based on the biological relevance in the drug discovery setting.

Serum haptoglobin concentrations in dairy cattle with lameness due to claw disorders.

In cattle, elevated blood serum concentrations of haptoglobin, an acute phase protein, have been demonstrated in association with several diseases, but not with lameness. Serum haptoglobin was measured in 60 Holstein dairy cattle diagnosed with lameness due to four claw disorders, pododermatitis septica (PS; n=41), pododermatitis circumscripta (PC; n=8), interdigital necrobacillosis (IN; n=7), papillomatous digital dermatitis (PDD; n=4). Haptoglobin was measured on day 1 (0-3 days after lameness was observed but before treatment) and on days 3 and 5. A total of 10 healthy cows served as controls (haptoglobin values <1.0 mg/dL). Each of the claw disorders was associated with elevated haptoglobin on day 1 (PS, PC, IN and PDD: 65.9%, 37.5%, 71.4% and 25.0%, respectively). Trimming and antibiotic treatment led to a reduction in the number of PS and IN cows with increased haptoglobin concentrations, respectively (P<0.05), but trimming did not lead to any reduction in cows with PC. The study showed that lameness due to claw disorders can be associated with a systemic acute phase response and elevated serum haptoglobin in dairy cattle. Based on the course of haptoglobin, treatments seemed effective for all claw disorders except for PC.

Discovery of pyridazinopyridinones as potent and selective p38 mitogen-activated protein kinase inhibitors.

The p38 mitogen-activated protein kinase (MAPK) plays an important role in the production of proinflammatory cytokines, making it an attractive target for the treatment of various inflammatory diseases. A series of pyridazinopyridinone compounds were designed as novel p38 kinase inhibitors. A structure-activity investigation identified several compounds possessing excellent potency in both enzyme and human whole blood assays. Among them, compound 31 exhibited good pharmacokinetic properties and showed excellent selectivity against other related kinases. In addition, 31 demonstrated efficacy in a collagen-induced arthritis disease model in rats.

Discovery and evaluation of 7-alkyl-1,5-bis-aryl-pyrazolopyridinones as highly potent, selective, and orally efficacious inhibitors of p38alpha mitogen-activated protein kinase.

The p38alpha mitogen-activated protein (MAP) kinase is a central signaling molecule in many proinflammatory pathways, regulating the cellular response to a multitude of external stimuli including heat, ultraviolet radiation, osmotic shock, and a variety of cytokines especially interleukin-1beta and tumor necrosis factor alpha. Thus, inhibitors of this enzyme are postulated to have significant therapeutic potential for the treatment of rheumatoid arthritis, inflammatory bowel disease, and Crohn's disease, as well as other diseases where aberrant cytokine signaling is the driver of disease. In this communication, we describe a novel class of 7-alkyl-1,5-bis-aryl-pyrazolopyridinone-based p38alpha inhibitors. In particular, compound 3f is highly potent in the enzyme and cell-based assays, selective in an Ambit kinase screen, and efficacious (ED(50) < or = 0.01 mg/kg) in the rat collagen induced arthritis (CIA) model.

3-amino-7-phthalazinylbenzoisoxazoles as a novel class of potent, selective, and orally available inhibitors of p38alpha mitogen-activated protein kinase.

The p38 mitogen-activated protein kinase (MAPK) is a central signaling molecule in many proinflammatory pathways, regulating the cellular response to a multitude of external stimuli including heat, ultraviolet radiation, osmotic shock, and a variety of cytokines especially interleukin-1beta and tumor necrosis factor alpha. Thus, inhibitors of this enzyme are postulated to have significant therapeutic potential for the treatment of rheumatoid arthritis, inflammatory bowel disease, osteoporosis, and many other diseases where aberrant cytokine signaling is the driver of disease. Herein, we describe a novel class of 3-amino-7-phthalazinylbenzoisoxazole-based inhibitors. With relatively low molecular weight, these compounds are highly potent in enzyme and cell-based assays, with minimal protein shift in 50% human whole blood. Compound 3c was efficacious (ED 50 = 0.05 mg/kg) in the rat collagen induced arthritis (CIA) model.

Discovery of highly selective and potent p38 inhibitors based on a phthalazine scaffold.

Investigations into the structure-activity relationships (SAR) of a series of phthalazine-based inhibitors of p38 are described. These efforts originated from quinazoline 1 and through rational design led to the development of a series of orally bioavailable, potent, and selective inhibitors. Kinase selectivity was achieved by exploiting a collection of interactions with p38alpha including close contact to Ala157, occupation of the hydrophobic gatekeeper pocket, and a residue flip with Gly110. Substitutions on the phthalazine influenced the pharmacokinetic properties, of which compound 16 displayed the most desirable profile. Oral dosing (0.03 mg/kg) of 16 in rats 1 h prior to LPS challenge gave a >50% decrease in TNFalpha production.