N-Succinylaspartic-Acid-Conjugated Riluzole Is a Safe and Potent Colon-Targeted Prodrug of Riluzole against DNBS-Induced Rat Colitis

Abstract

In our previous study, riluzole azo-linked to salicylic acid (RAS) was prepared as a colon-targeted prodrug of riluzole (RLZ) to facilitate the repositioning of RLZ as an anticolitic drug. RAS is more effective against rat colitis than RLZ and sulfasalazine, currently used as an anti-inflammatory bowel disease drug. The aim of this study is to further improve colon specificity, anticolitic potency, and safety of RAS. N-succinylaspart-1-ylRLZ (SAR) and N-succinylglutam-1-ylRLZ (SGR) were synthesized and evaluated as a "me-better" colon-targeted prodrug of RLZ against rat colitis. SAR but not SGR was converted to RLZ in the cecal contents, whereas both conjugates remained intact in the small intestine. When comparing the colon specificity of SAR with that of RAS, the distribution coefficient and cell permeability of SAR were lower than those of RAS. In parallel, oral SAR delivered a greater amount of RLZ to the cecum of rats than oral RAS. In a DNBS-induced rat model of colitis, oral SAR mitigated colonic damage and inflammation and was more potent than oral RAS. Moreover, upon oral administration, SAR had a greater ability to limit the systemic absorption of RLZ than RAS, indicating a reduced risk of systemic side effects of SAR. Taken together, SAR may be a "me-better" colon-targeted prodrug of RLZ to improve the safety and anticolitic potency of RAS, an azo-type colon-targeted prodrug of RLZ.

Keywords: N-succinylated acidic amino acids; colitis; colon-targeted drug delivery; prodrug; riluzole.

Figure 1

Synthesis of SAR and SGR and structure of RAS. (A) Synthetic scheme of SAR and SGR. SAR: (N-succinyl-L-aspart-1-yl)riluzole, SGR: (N-succinyl-L-glutam-1-yl)riluzole, AR: N-(L-aspart-1-yl)riluzole, GR: N-(L-glutam-1-yl)riluzole, TEA: triethylamine, CDI: 1,1′-carbonyldiimidazole, ACN: acetonitrile, and DCM: dichloromethane. (B) Structure of RLZ azo-linked salicylic acid (RAS).

Figure 2

SAR is colon specific. (A) RLZ, RAS, SAR, and SGR (500 µM, 2 mL) dissolved in the medium were added to the apical compartment of the Caco-2 cell monolayer and incubated for 24 h. At appropriate time intervals, the concentrations of each drug were determined in the basolateral compartment filled with medium (3 mL) using HPLC. (B) RAS, SAR, and SGR (1 mM) were incubated with suspension of the cecal contents (10.0%). The supernatants obtained using centrifugation were extracted with EA, and the organic layer in a new mircotube was evaporated, and then dissolved in mobile phase A. After filtration, the filtrates (20 μL) were injected to HPLC to determine the concentration of RLZ. (C) Male Sprague-Dawley rats (250–260 g) were starved for 24 h except for water. Rats were treated orally with RLZ (10 mg/kg), RAS (16.4 mg/kg, equivalent to 10 mg/kg of RLZ), or SAR (19.2 mg/kg, equivalent to 10 mg/kg of RLZ) suspended in PBS (pH 7.4). The concentration of RLZ in the cecum was determined using HPLC at 4, 8, and 12 h after the treatment. The data are represented as the mean ± SD (n = 5).

Figure 3

SAR is more effective in reducing systemic absorption of RLZ than RAS. Rats were treated orally with SSZ (30 mg/kg), RAS (16.4 mg/kg, equivalent to 10 mg/kg of RLZ), SAR (L) (9.6 mg/kg, equivalent to 5 mg/kg), and SAR (H) (19.2 mg/kg, equivalent to 10 mg/kg) dissolved in 1.0 mL of PBS (pH 7.4) once per day, and continued for 6 days. The medications began 3 days after induction of colitis using DNBS. Anticolitic effects of the drugs were evaluated 24 h after the last dose. (A) Left panel: Images of the serosal and luminal sides of the distal colons of rats. Right panel: Colonic-damage score (CDS) was assigned for each treatment. (B) The colonic tissue sections of rats were subjected to H & E staining. Upper panel: Representative images of 100× magnification. Lower panel: Representative images of 200× magnification for the dotted boxes in the upper panel. (C) Myeloperoxidase (MPO) activity, (D) CINC−3 level, and (E) iNOS and COX−2 level in the inflamed distal colons (4.0 cm) were assessed. For Western blot analysis, α−Tubulin was used as a loading control to normalize iNOS and COX−2 levels. NM: not measurable. (F) The levels of p−GS and GS in the inflamed distal colon were monitored using Western blot analysis. NM: not measurable. (G) The levels of IL10 in the inflamed distal colon were determined using an ELISA kit. The data are represented as mean ± SD (n = 5). * p < 0.05, vs. DNBS control ^# p < 0.05.

Figure 3

SAR is more effective in reducing systemic absorption of RLZ than RAS. Rats were treated orally with SSZ (30 mg/kg), RAS (16.4 mg/kg, equivalent to 10 mg/kg of RLZ), SAR (L) (9.6 mg/kg, equivalent to 5 mg/kg), and SAR (H) (19.2 mg/kg, equivalent to 10 mg/kg) dissolved in 1.0 mL of PBS (pH 7.4) once per day, and continued for 6 days. The medications began 3 days after induction of colitis using DNBS. Anticolitic effects of the drugs were evaluated 24 h after the last dose. (A) Left panel: Images of the serosal and luminal sides of the distal colons of rats. Right panel: Colonic-damage score (CDS) was assigned for each treatment. (B) The colonic tissue sections of rats were subjected to H & E staining. Upper panel: Representative images of 100× magnification. Lower panel: Representative images of 200× magnification for the dotted boxes in the upper panel. (C) Myeloperoxidase (MPO) activity, (D) CINC−3 level, and (E) iNOS and COX−2 level in the inflamed distal colons (4.0 cm) were assessed. For Western blot analysis, α−Tubulin was used as a loading control to normalize iNOS and COX−2 levels. NM: not measurable. (F) The levels of p−GS and GS in the inflamed distal colon were monitored using Western blot analysis. NM: not measurable. (G) The levels of IL10 in the inflamed distal colon were determined using an ELISA kit. The data are represented as mean ± SD (n = 5). * p < 0.05, vs. DNBS control ^# p < 0.05.

Figure 4

SAR has a greater ability to restrict systemic absorption of RLZ than RAS.