Siponimod is a Functional Agonist for the S1P5 Receptor (P3.404)

Objective: To investigate the effects of siponimod on the internalization of sphingosine-1-phosphate (S1P) receptor subtypes 1 and 5 in a preclinical assay. Background: Siponimod, a potent and highly selective agonist for S1P1 and S1P5, is being developed for the treatment of multiple sclerosis (MS). Its therapeutic mode of action involves the down-modulation (i.e. internalization and degradation) of S1P1 receptors on T lymphocytes which are then sequestered within secondary lymphoid tissues and prevented from recirculating into the central nervous system. In addition, S1P5 receptor-mediated direct effects on oligodendrocytes and reduced demyelination have been suggested, raising the question about a potential link with S1P5 down-modulation. Design/Methods: Flow cytometry assays using Chinese Hamster Ovary cells overexpressing N-terminal (extracellular) tagged human S1P1 (myc tag), or S1P5 (myc or flag tag) receptors were generated to quantify, with the help of an anti-myc/flag-epitope antibody, their level of surface expression after 1–3 hours incubation with siponimod (0.01–1 μM). In such conditions, a reduction in fluorescence would indicate the disappearance of the tagged receptor from the surface. Results were compared to those obtained with fingolimod-phosphate. Results: Incubation of myc-tagged human S1P1 cells with siponimod (0.01 μM) for 1 hour caused approximately 90% internalization of the S1P1 receptors, with no reappearance within 3 hours after washout. An identical behavior was observed with fingolimod-phosphate. In contrast, no internalization of S1P5 receptors was observed in myc/flag-tagged mouse/human-S1P5 cells incubated for 1 hour with siponimod up to 1 μM. Similarly, fingolimod-phosphate treatment failed to show any relevant internalization of S1P5 receptors up to 1 μM. Conclusions: These observations suggest that, in contrast to the S1P1 receptor, the S1P5 receptor is not down-modulated by agonists such as siponimod or fingolimod. Consequently, S1P5 agonist function, and not functional antagonism, should be considered when studying the direct neuroprotective effects of siponimod. Study Supported by: This study was funded by Novartis Pharma AG, Basel, Switzerland Disclosure: Dr. Bigaud has received personal compensation for consulting, serving on a scientific advisory board, speaking, or other activities with Employee of Novartis Pharma AG, Basel, Switzerland. Dr. Nuesslein-Hildesheim has received personal compensation for consulting, serving on a scientific advisory board, speaking, or other activities with employee of Novartis Pharma AG, Basel, Switzerland. Dr. Tran has received personal compensation for consulting, serving on a scientific advisory board, speaking, or other activities with Employee of Novartis Pharma AG, Basel, Switzerland. Dr. Guerini has received personal compensation for consulting, serving on a scientific advisory board, speaking, or other activities with Employee of Novartis Pharma AG, Basel, Switzerland.