Safe and Efficient Sigma1 Ligand: A Potential Drug Candidate for Multiple Sclerosis

Abstract

Multiple Sclerosis (MS) is an autoimmune demyelinating and neurodegenerative disease of the central nervous system (CNS). Current management strategies suppress or modulate immune function, all with consequences and known side effects. They demonstrate a high level of success in limiting new relapses. However, the neurodegenerative process still affects both grey and white matter in the central nervous system. The sigma1 (S1R) ligand-regulated chaperone is implicated in many biological processes in various CNS-targeted diseases, acting on neural plasticity, myelination and neuroinflammation. Among the proteins involved in MS, S1R has therefore emerged as a promising new target. Standard and robust methods have been adopted to analyze the adsorption, distribution, metabolism, excretion (ADME) properties, safety pharmacology and toxicology of a previously synthetized simple benzamide-derived compound with nanomolar affinity for S1R, high selectivity, no cytotoxicity and good metabolic stability. The compound was also characterized as an agonist based on well-validated assays prior to in vivo investigations. Interestingly, we found that the oral administration of this compound resulted in an overall significant reduction in clinical progression in an MS experimental model. This effect is mediated through S1R action. Our results further suggest the potential use of this compound in the treatment of MS.

Keywords: CNS; benzamide; multiple sclerosis; sigma1 receptor.

Figure 1

Immunotoxicity analysis. Human peripheral blood mononuclear cells (hPBMCs) were either non-stimulated (basal) or stimulated during the course of a 48-h period. Compound 7i was tested at 10 µM. Immune cell subpopulations were immunophenotyped among hPBMCs by flow cytometry. Cell percentage was evaluated. Data are presented as mean ± SEM from six individual healthy donors. * p < 0.05 vs. control (basal vehicle) condition. Wilcoxon–Mann–Whitney U test.

Figure 2

In vitro compound 7i agonist activity evaluation. The effect of compound 7i (1 and 10 µM) on S1R–BiP association (30-min incubation) was analyzed through co-immunoprecipitation with an S1R antibody (red). BiP level was measured using ELISA. PRE−084 (blue) and NE−100 (yellow) were used for agonist and antagonist of reference, respectively (10 µM). Agonist activity was validated by blockade by NE−100 (yellow hatching). The absence of antagonist activity was validated with PRE−084 (blue hatching). Data are presented as mean ± SEM of n = 6 mice per condition. *** p < 0.0001 vs. non treated group, ^### p < 0.0001 vs. PRE-084 or 7i 10 μM group. Dunnett’s test.

Figure 3

In vivo compound 7i agonist activity evaluation. The effect of compound 7i (0.5 to 5 mg/kg, i.p., red) was analyzed with dizocilpine-induced learning deficit analysis (V, vehicle) through spontaneous alternation performances (panels (A,C)) and passive avoidance latency (panels (B,D)). Agonist activity was validated by NE-100 blockade (3 mg/kg, i.p., yellow). Data are presented as mean ± SEM of n = 6 mice per condition. * p < 0.05, ** p < 0.01 vs. (vehicle + vehicle)-treated group, # p < 0.05, ## p < 0.01 vs. (Dizocilpine + vehicle)-treated group, oo p < 0.01 vs. (Dizocilpine + 7i)-treated group. Dunn’s test.

Figure 4

Pharmacokinetic modeling using the non-compartmental method. Compound 7i was administrated through two methods, i.v. (panel (A)) and p.o. (panel (B)), at 1 mg/kg.

Figure 5

The curative effect of compound 7i in the multiple sclerosis (MS) experimental model. Mice were immunized at day 0 (D0) and their clinical course was followed for 80 days (panels (A,C)). Compound 7i was administrated daily for 15 days when mice reached a score of 2 (panels (B,D)). Five different groups were analyzed depending on the route of administration (i.p. or p.o.) and concentration (0.5 and 1 mg/kg). The involvement of S1R in the beneficial effect of EAE was analyzed during the first relapse (panel B). S1R binding sites were blocked by i.p administration of BD-1047 (10 mg/kg) 20 min before compound 7i administration. Data are presented as mean ± SEM of n = 5–6 mice per condition. ns, non-significant. **** p < 0.0001, ### p < 0.001 vs. EAE—vehicle group. * or # p < 0.05 vs. EAE—vehicle group; o p < 0.05 vs. EAE—BD-1047 and 7i group. Wilcoxon–Mann–Whitney U test.