Effective treatments for neurodegenerative diseases remain elusive and are critically needed since the burden of these diseases increases across an aging global population. Nitric oxide (NO) is a gasotransmitter that binds to soluble guanylate cyclase (sGC) to produce cyclic guanosine monophosphate (cGMP). Impairment of this pathway has been demonstrated in neurodegenerative diseases. Normalizing deficient NO-cGMP signaling could address multiple pathophysiological features of neurodegenerative diseases. sGC stimulators are small molecules that synergize with NO, activate sGC, and increase cGMP production. Many systemic sGC stimulators have been characterized and advanced into clinical development for a variety of non-central nervous system (CNS) pathologies. Here, we disclose the discovery of CY6463, the first brain-penetrant sGC stimulator in clinical development for the treatment of neurodegenerative diseases, and demonstrate its ability to improve neuronal activity, mediate neuroprotection, and increase cognitive performance in preclinical models. In several cellular assays, CY6463 was demonstrated to be a potent stimulator of sGC. In agreement with the known effects of sGC stimulation in the vasculature, CY6463 elicits decreases in blood pressure in both rats and mice. Relative to a non-CNS penetrant sGC stimulator, rodents treated with CY6463 had higher cGMP levels in cerebrospinal fluid (CSF), functional-magnetic-resonance-imaging-blood-oxygen-level-dependent (fMRI-BOLD) signals, and cortical electroencephalographic (EEG) gamma-band oscillatory power. Additionally, CY6463 improved cognitive performance in a model of cognitive disruption induced by the administration of a noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist. In models of neurodegeneration, CY6463 treatment increased long-term potentiation (LTP) in hippocampal slices from a Huntington’s disease mouse model and decreased the loss of dendritic spines in aged and Alzheimer’s disease mouse models. In a model of diet-induced obesity, CY6463 reduced markers of inflammation in the plasma. Furthermore, CY6463 elicited an additive increase in cortical gamma-band oscillatory power when co-administered with donepezil: the standard of care in Alzheimer’s disease. Together, these data support the clinical development of CY6463 as a novel treatment for neurodegenerative disorders.

Background Inflammation in the central nervous system (CNS) is observed in many neurological disorders. Nitric oxide-soluble guanylate cyclase-cyclic guanosine monophosphate (NO–sGC–cGMP) signaling plays an essential role in modulating neuroinflammation. CYR119 is a CNS-penetrant sGC stimulator that amplifies endogenous NO–sGC–cGMP signaling. We evaluated target engagement and the effects of CYR119 on markers of neuroinflammation in vitro in mouse microglial cells and in vivo in quinolinic acid (QA)-induced and high-fat diet-induced rodent neuroinflammation models. Methods Target engagement was verified in human embryonic kidney (HEK) cells, rat primary neurons, mouse SIM-A9 cells, and in rats by measuring changes in cGMP and downstream targets of sGC signaling [phosphorylated vasodilator-stimulated phosphoprotein (pVASP), phosphorylated cAMP-response element binding (pCREB)]. In SIM-A9 cells stimulated with lipopolysaccharides (LPS), markers of inflammation were measured when cells were treated with or without CYR119. In rats, microinjections of QA and vehicle were administered into the right and left hemispheres of striatum, respectively, and then rats were dosed daily with either CYR119 (10 mg/kg) or vehicle for 7 days. The activation of microglia [ionized calcium binding adaptor molecule 1 (Iba1)] and astrocytes [glial fibrillary acidic protein (GFAP)] was measured by immunohistochemistry. Diet-induced obese (DIO) mice were treated daily with CYR119 (10 mg/kg) for 6 weeks, after which inflammatory genetic markers were analyzed in the prefrontal cortex. Results In vitro, CYR119 synergized with exogenous NO to increase the production of cGMP in HEK cells and in primary rat neuronal cell cultures. In primary neurons, CYR119 stimulated sGC, resulting in accumulation of cGMP and phosphorylation of CREB, likely through the activation of protein kinase G (PKG). CYR119 attenuated LPS-induced elevation of interleukin 6 (IL-6) and tumor necrosis factor (TNF) in mouse microglial cells. Following oral dosing in rats, CYR119 crossed the blood–brain barrier (BBB) and stimulated an increase in cGMP levels in the cerebral spinal fluid (CSF). In addition, levels of proinflammatory markers associated with QA administration or high-fat diet feeding were lower in rodents treated with CYR119 than in those treated with vehicle. Conclusions These data suggest that sGC stimulation could provide neuroprotective effects by attenuating inflammatory responses in nonclinical models of neuroinflammation.

Background: Praliciguat (IW-1973) is a soluble guanylate cyclase (sGC) stimulator in clinical development. In animal models, praliciguat has broad tissue distribution and enhances nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) signaling, which has been shown to elicit hemodynamic, anti-inflammatory, and anti-fibrotic effects. This study assessed the metabolic effects of praliciguat in a mouse DIO model. Methods: Male C57BL/6J mice (12 weeks old at the start of treatment) were either maintained on regular chow (lean mice) or switched to a 60% high fat diet (HFD) starting at age 6 weeks (obese mice). At the start of treatment, obese mice received either praliciguat (6 mg/kg) or vehicle in the HFD. The lean mice remained on regular chow. On day 28, an oral glucose tolerance test (OGTT) was performed after a 3-h fast. On day 29, organs and terminal blood were collected for analysis after a 3-h fast. Results: Praliciguat treatment lowered OGTT glucose area under the curve (-22%, P<0.05), fasting plasma insulin (-53%, P<0.01) and HOMA-IR, an index of insulin resistance (-53%, P<0.01) vs. vehicle treatment. Praliciguat-treated mice also had lower liver (-36%, P<0.05) and fasting circulating triglycerides (-30%, P<0.01) vs. vehicle-treated mice. Furthermore, several fasting biomarkers did not change in treated vs. vehicle including beta-hydroxybutyrate, free fatty acids, plasma glucose, cholesterol, leptin, and adiponectin. Conclusions: Praliciguat improved insulin sensitivity, glucose tolerance, and yielded lower circulating and liver triglycerides in a mouse diet-induced obesity model. Disclosure C.D. Schwartzkopf: None. J. Hadcock: None. J.E. Jones: None. M. Currie: Employee; Self; Ironwood Pharmaceuticals, Inc. G.T. Milne: Employee; Self; Ironwood Pharmaceuticals, Inc.. Stock/Shareholder; Self; Ironwood Pharmaceuticals, Inc.. Employee; Spouse/Partner; Catabasis Pharmaceuticals. Stock/Shareholder; Spouse/Partner; Catabasis Pharmaceuticals. J. Masferrer: Employee; Self; Ironwood Pharmaceuticals, Inc..

Pharmacologic antagonism of cannabinoid 1 receptors (CB1 receptors) in the central nervous system (CNS) suppresses food intake, promotes weight loss, and improves the metabolic profile. Since the CB1 receptor is expressed both in the CNS and in peripheral tissues, therapeutic value may be gained with CB1 receptor inverse agonists acting on receptors in both domains. The present report examines the metabolic and CNS actions of a novel CB1 receptor inverse agonist, compound 64, a 1,5,6-trisubstituted pyrazolopyrimidinone. Compound 64 showed similar or superior binding affinity, in vitro potency, and pharmacokinetic profile compared to rimonabant. Both compounds improved the metabolic profile in diet-induced obese (DIO) rats and obese cynomolgus monkeys. Weight loss tended to be greater in compound 64-treated DIO rats compared to pair-fed counterparts, suggesting that compound 64 may have metabolic effects beyond those elicited by weight loss alone. In the CNS, reversal of agonist-induced hypothermia and hypolocomotion indicated that compound 64 possessed an antagonist activity in vivo. Dosed alone, compound 64 suppressed extinction of conditioned freezing (10 mg/kg) and rapid eye movement (REM) sleep (30 mg/kg), consistent with previous reports for rimonabant, although for REM sleep, compound 64 was greater than threefold less potent than for metabolic effects. Together, these data suggested that (1) impairment of extinction learning and REM sleep suppression are classic, centrally mediated responses to CB1 receptor inverse agonists, and (2) some separation may be achievable between central and peripheral effects with brain-penetrating CB1 receptor inverse agonists while maintaining metabolic efficacy. Furthermore, chronic treatment with compound 64 contributes to evidence that peripheral CB1 receptor blockade may yield beneficial outcomes that exceed those elicited by weight loss alone.

Prolonged high fat diet (HFD) accelerates the cardiovascular, renal and metabolic dysfunction in hypertensive rats with altered renal development (ARDev). Soluble guanylate cyclase (sGC) stimulation or sodium-glucose cotransporter 2 (SGLT2) inhibition may improve cardiovascular, renal, and metabolic function in settings of hypertension and obesity. This study examined whether six weeks treatment with an SGLT2 inhibitor (EMPAGLIFLOZIN, 7 mg/kg/day) enhances the cardiovascular, renal and metabolic effects of a sGC stimulator (PRALICIGUAT, 10 mg/kg/day) in hypertensive rats with ARDev and prolonged exposure to HFD. Arterial pressure (AP), renal vascular resistance (RVR), fat abdominal volume (FAV), insulin resistance, leptin and triglycerides levels and intrarenal infiltration of inflammatory cells were higher, but cardiac output and creatinine clearance were lower in hypertensive rats (n=15) than in normotensive rats (n=7). PRALICIGUAT administration (n=10) to hypertensive rats reduced (P<0.05) AP, FAV, plasma concentrations of leptin and triglycerides, and increased (P<0.05) cardiac output and creatinine clearance. EMPAGLIFLOZIN administration (n=8) only increased (P<0.05) glucosuria and creatinine clearance, and decreased (P<0.05) plasma leptin and triglycerides concentrations in hypertensive rats. Simultaneous administration of PRALICIGUAT and EMPAGLIFLOZIN (n=10) accelerated the decrease in AP, improved glucose tolerance, reduced (P<0.05) incremental body weight gain, and decreased (P<0.05) insulin resistance index, RVR and the infiltration of T-CD3 lymphocytes in renal cortex and renal medulla. In summary, the combined administration of PRALICIGUAT and EMPAGLIFLOZIN leads to a greater improvement of the cardiovascular, renal and metabolic dysfunction secondary to prolonged exposure to HFD in hypertensive rats with ARDev than the treatment with either PRALICIGUAT or EMPAGLIFLOZIN alone.