The CNS-penetrant soluble guanylate cyclase stimulator CYR119 attenuates markers of inflammation in the central nervous system

Abstract

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.

Keywords: CREB; High-fat diet; Microglia; Neuroinflammation; Nitric oxide; Quinolinic acid; Soluble guanylate cyclase; cGMP; sGC.

Fig. 1

In vitro activity of CYR119. A GloSensor^® luciferase assay in HEK293 cells in the presence of 0 to 30 µM DETA-NONOate. B, C Effect of CYR119 on cGMP and CREB phosphorylation in rat primary neurons. Representative concentration response of CYR119 as measured by cellular phosphorylation of CREB in the presence of 10 µM DETA-NONOate in rat primary neurons. Data are normalized to vehicle-control wells. EC₅₀s are noted in each figure

Fig. 2

Measurement of LPS-induced cytokines in SIM-A9 following incubation with CYR119. A Phosphorylation of VASP (pVASP/total VASP) in SIM-A9 cells after incubation with CYR119 (1 or 10 µM) with or without DETA (30 µM). Data analyzed with a one-way ANOVA followed by a Dunnett’s multiple comparison test vs. vehicle-control wells. B, C Effect of CYR119 ± 30 µM DETA on IL-6 and TNF protein production, respectively, in SIM-A9 cells after LPS stimulation. Data analyzed with a one-way ANOVA followed by a Bonferroni’s multiple comparison test vs. LPS-treated control wells. Data are expressed as mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, non-significant (ns) effects are not highlighted

Fig. 3

Concentration of CYR119 and cGMP in rat CSF following oral delivery. A Hour post-dose compound concentration (nM) in the CSF of rats administered 10 mg/kg CYR119. B One hour after dosing, concentrations of cGMP in the CSF were higher in rats dosed orally with 10 mg/kg CYR119 vs. vehicle-treated rats (*p < 0.05; two-tailed t-test). Data are expressed as mean ± SEM

Fig. 4

Effects of CYR119 on markers of inflammation following QA administration. Rats were administered 10 mg/kg CYR119 or vehicle for 7 days following QA administration into the dorsal striatum. Normalized gene expression was analyzed for A TNF and B CD40 or immunohistochemistry staining of C glial fibrillary acidic protein (GFAP), and D IBA1, represented as percent (%) area of Iba1 staining, and E pCREB, represented as intensity as a cumulative distribution and average intensity in the dorsal striatum. A representative image from each group is shown in F GFAP, G IBA1, and H pCREB. Data were analyzed by one-way ANOVA followed by Sidak’s multiple comparison test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). Data are expressed as mean ± SEM

Fig. 5

Effects of CYR119 on inflammatory genes in the prefrontal cortex of DIO mice. CYR119 (10 mg/kg) was orally administered to DIO mice for 6 weeks and prefrontal cortex analyzed for gene expression of A intercellular adhesion molecule 1 (ICAM1), B NADPH oxidase 2 (Cybb), C glial fibrillary acidic protein (GFAP), and D glucose transporter 1 (GLUT1). Data were analyzed with a one-way ANOVA followed by a Dunnett’s multiple comparison test vs. DIO vehicle-control mice. Data are expressed as mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001