ASP4058, a Novel Agonist for Sphingosine 1-Phosphate Receptors 1 and 5, Ameliorates Rodent Experimental Autoimmune Encephalomyelitis with a Favorable Safety Profile

<div><p>Sphingosine-1-phosphate (S1P) is a biologically active sphingolipid that acts through the members of a family of five G protein-coupled receptors (S1P₁–S1P₅). S1P₁ is a major regulator of lymphocyte trafficking, and fingolimod, whose active metabolite fingolimod phosphate acts as a nonselective S1P receptor agonist, exerts its immunomodulatory effect, at least in part, by regulating the lymphocyte trafficking by inducing down regulation of lymphocyte S1P₁. Here, we detail the pharmacological profile of 5-{5-[3-(trifluoromethyl)-4-{[(2<i>S</i>)-1,1,1-trifluoropropan-2-yl]oxy}phenyl]-1,2,4-oxadiazol-3-yl}-1<i>H</i>-benzimidazole (ASP4058), a novel next-generation S1P receptor agonist selective for S1P₁ and S1P₅. ASP4058 preferentially activates S1P₁ and S1P₅ compared with S1P_{2, 3, 4} in GTPγS binding assays <i>in vitro</i>. Oral administration of ASP4058 reduced the number of peripheral lymphocytes and inhibited the development of experimental autoimmune encephalomyelitis (EAE) in Lewis rats. Further, ASP4058 prevented relapse of disease in a mouse model of relapsing-remitting EAE. Although these immunomodulatory effects were comparable to those of fingolimod, ASP4058 showed a wider safety margin than fingolimod for bradycardia and bronchoconstriction in rodents. These observations suggest that ASP4058 represents a new therapeutic option for treating multiple sclerosis that is safer than nonselective S1P receptor agonists such as fingolimod.</p></div

Chemical structure of ASP4058 (C19H12F6N4O2.HCl).

<p>Chemical structure of ASP4058 (C19H12F6N4O2.HCl).</p

Agonistic effects of ASP4058 and fingolimod phosphate on human S1P receptor subtypes.

<p>Agonistic effects of ASP4058 and fingolimod phosphate on human S1P receptor subtypes.</p

Plasma concentrations of ASP4058 and blood concentration of fingolimod phosphate during and after intravenous infusion of rats.

<p>Plasma concentrations of ASP4058 and blood concentration of fingolimod phosphate during and after intravenous infusion of rats.</p

Effects of ASP4058 or fingolimod phosphate on bronchoconstriction in anesthetized rats.

<p>To assess the effect of compound on base-line airway pressure, ASP4058 (0.3 mg/kg/min) or fingolimod phosphate (fingolimod-P) (0.003, 0.03, 0.3 mg/kg/min) were administered by continuous intravenous infusion, and airway pressure was measured just before the initiation of compound infusion (baseline) and approximately 20 min after the initiation of compound infusion, which is sufficient time to reach a plateau. Results are shown as the percent change from baseline value and represent the mean ± S.E. The number of animals in each group is shown in parentheses. **<i>P</i><0.01 compared with the vehicle-treated group (Dunnett's multiple comparison test).</p

Effects of ASP4058 and fingolimod-P on heart rate and mean blood pressure in conscious rats.

<p>Vehicle, ASP4058 (1 or 3 mg/kg) or fingolimod phosphate (fingolimod-P) (0.01, 0.03, or 0.1 mg/kg) were continuously administered for 10 min using an infusion pump (1 ml/kg/min) through a catheter inserted into the femoral vein, and the effects of ASP4058 and fingolimod-P on heart rate and mean blood pressure were determined (ASP4058, A and C, respectively; fingolimod-P, B and D, respectively). Heart rates and mean blood pressures were recorded for 20 min after the start of infusion. Heart rate was determined at 1-min intervals, and mean blood pressures were determined 0, 2, 4, 6, 8, 10, 15, and 20 min after the start of infusion. All values represent the mean ± S.E. for 5 rats per group, except for the 20-min time points of the groups treated with 1 mg/kg of ASP4058 (mean ± S.E. for 4 rats) or 0.1 mg/kg fingolimod phosphate (mean ± S.E. for 3 rats).</p

Plasma or blood concentrations of ASP4058 and fingolimod-P after repeated dosing.

<p>ASP4058 (0.1 mg/kg) or fingolimod (0.1 mg/kg) was administered once-daily for 14 days in male Lewis rats. Plasma concentration of ASP4058 and blood concentration of fingolimod phosphate (fingolimod-P) in rats were measured just before the last administration, 0.25 (for fingolimod-P) or 0.5 (for ASP4058), 1, 3, 8, and 24 h after the last administration. Data represent the mean ± S.E. (<i>n</i> = 5).</p

Epigenetic regulation of Nedd9 gene during osteoclastogenesis.

<p>(A) Smad2/3 binding and histone modification changes of <i>Nedd9</i> gene during osteoclastogenesis. Histone modification patterns in BMMs changed from K4(+)K27(+) to K4(+)K27(-) patterns by TGF-ß and returned to K4(+)K27(+) patterns after RANKL treatment. (B) <i>Nedd9</i> mRNA expression after TGF-ß or RANKL stimulation. The expression increased by TGF-ß stimulation and was reduced after RANKL treatment. The expression remained at low levels in the presence of SB431542.</p

Identification of Smad2/3 binding sites.

<p>(A) BMMs were treated with 2 ng/ml TGF-ß for 1.5 h and cells were subjected to ChIP-seq analysis using anti-Smad2/3 antibody. Three known TGF-ß target genes (<i>Cdkn1a</i>, <i>Serpine1</i>, <i>and Smad7</i>) and a negative control gene (<i>Hprt1</i>) were analyzed as representative examples. Smad2/3-binding regions (SBRs; peak signal ratio ≥8) and the peak position of each SBR are shown by black bars. (B) Eight positive regions and two negative regions for Smad2/3 binding were selected from ChIP-seq data and validated by realtime PCR. ChIP using mouse IgG was used as control. Values are presented as n-fold enrichment over Hprt1. (C) Average Smad2/3 signal profile around transcriptional start site (TSS) in ChIP-seq analysis. Smad2/3 binding was enriched around TSS.</p

Nedd9 is critical for osteoclast differentiation.

<p>(A) Realtime PCR analysis of effects of retroviral overexpression of <i>Nedd9</i> gene on RANKL-induced osteoclastogenesis, as indicated by expression of <i>Nedd9</i> and <i>Cathepsin K</i> mRNAs. (B) Effects of SB431542 (SB) treatment on osteoclastogenesis, as evaluated by TRAP staining, in <i>Nedd9</i>-overexpressing cells treated with RANKL. Overexpression of <i>Nedd9</i> increased RANKL-induced osteoclastogenesis. SB431542 suppressed osteoclastogenesis, which was partly recovered by <i>Nedd9</i> overexpression. Cultures were stained by TRAP. Bars = 100 μm. (C) The expression of <i>Nedd9</i> gene as determined by realtime PCR. (D) The number of TRAP positive osteoclasts was significantly increased by <i>Nedd9</i> overexpression, and the suppression of osteoclastogenesis by SB431542 was partly suppressed by <i>Nedd9</i> overexpression. *<i>P</i> < 0.05.</p