Background and PurposeWe conducted preclinical and clinical studies to examine the pharmacological, particularly cardiac, effects of amiselimod (MT‐1303), a second‐generation sphingosine 1‐phosphate (S1P) receptor modulator, designed to reduce the bradycardia associated with fingolimod and other S1P receptor modulators.Experimental ApproachThe selectivity of the active metabolite amiselimod phosphate (amiselimod‐P) for human S1P receptors and activation of G‐protein‐coupled inwardly rectifying K+ (GIRK) channels in human atrial myocytes were assessed. Its cardiac distribution was determined in rats, and cardiovascular telemetry was assessed in monkeys. We also examined the pharmacokinetics, pharmacodynamics and safety of amiselimod in healthy humans.Key ResultsAmiselimod‐P showed potent selectivity for S1P1 and high selectivity for S1P5 receptors, with minimal agonist activity for S1P4 and no distinct agonist activity for S1P2 or S1P3 receptors and approximately five‐fold weaker GIRK activation than fingolimod‐P. After oral administration of amiselimod or fingolimod at 1 mg·kg−1, the concentration of amiselimod‐P in rat heart tissue was lower than that of fingolimod‐P, potentially contributing to the minimal cardiac effects of amiselimod. A telemetry study in monkeys confirmed that amiselimod did not affect heart rate or ECG parameters. In healthy human subjects, peripheral blood lymphocyte counts gradually reduced over the 21 day dosing period, with similar lymphocyte count profiles with the highest doses by day 21, and no clinically significant bradycardia observed on day 1 or during the study.Conclusions and ImplicationsAmiselimod exhibited potent therapeutic efficacy with minimal cardiac effects at the anticipated clinical dose and is unlikely to require dose titration.

No abstract

No abstract

Cytotoxic necrotizing factor types 1 and 2 (CNF1 and -2) produced by pathogenic Escherichia coli strains have 90% conserved residues over 1,014-amino-acid sequences. Both CNFs are able to provoke a remarkable increase in F-actin structures in cultured cells and covalently modify the RhoA small GTPases. In this study, we demonstrated that CNF2 reduced RhoA GTPase activity in the presence and absence of P122RhoGAP. Subsequently, peptide mapping and amino acid sequencing of CNF2-modified FLAG-RhoA produced in E. coli revealed that CNF2 deamidates Q63 of RhoA-like CNF1. In vitro incubation of the C-terminal domain of CNF2 with FLAG-RhoA resulted also in deamidation of the FLAG-RhoA, suggesting that this region contains the enzymatic domain of CNF2. An oligopeptide antibody (anti-E63) which specifically recognized the altered G-3 domain of the Rho family reacted with glutathione S-transferase (GST)-RhoA and GST-Rac1 but not with GST-Cdc42 when coexpressed with CNF2. In addition, CNF2 selectively induced accumulation of GTP form of FLAG-RhoA and FLAG-Rac1 but not of FLAG-Cdc42 in Cos-7 cells. Taken together, these results indicate that CNF2 preferentially deamidates RhoA Q63 and Rac1 Q61 and constitutively activates these small GTPases in cultured cells. In contrast, anti-E63 reacted with GST-RhoA and GST-Cdc42 but not with GST-Rac1 when coexpressed with CNF1. These results indicate that CNF2 and CNF1 share the same catalytic activity but have distinct substrate specificities, which may reflect their differences in toxic activity in vivo.

No abstract