**1. Introduction**

Although it was long thought that sphingosine, largely generated from hydrolysis of ceramides within lysosomes [1,2], served primarily as a component of the structural sphingolipid family of molecules, it is now recognized for its importance as a component of sphingosine-1-phosphate (S1P), a crucial messenger molecule. S1P is generated by sphingosine kinase 1 or 2 (sk1, sk2), primarily in red blood cells, platelets and endothelial cells [3,4] (Figure 1). Their G-protein-coupled receptors are widely distributed in many organs and tissues, on cell surface plasma membranes, and on the endoplasmic reticulum and cell nuclei, but it is cytoplasmic membrane-bound receptor sites that have attracted the greatest attention [4]. Five S1P receptor (S1PR-1-5) subtypes have been identified [5,6] (Figure 2a). The translational intracellular pathways activated by S1P-receptor (S1PR) interaction are highly varied because of the multiplicity of receptor subtypes, the varied G-proteins to which they are coupled, the multiple downstream pathways linked to G proteins, and the wide variety cells which express S1PRs (Figure 2b; Table 1). Investigations into the physiological impact of S1P and its receptors, as well as its potential contribution to disease pathogenesis, have revealed important opportunities for therapeutic interventions. This is particularly the case in the treatment of multiple sclerosis (MS). In the

past 10 years, three disease modifying therapies (DMT)s which modulate S1PRs, fingolimod (FGM), siponimod (SPM), and ozanimod (OZM), have been approved for treatment of MS, and a fourth agent, ponesimod (PNM) is under regulatory review.

The first part of this article describes the results of preclinical research, which has expanded the knowledge of the physiological roles of S1P and its receptors, and a discussion of the specific mechanisms of action of the S1PR modulators. This work did not only reveal important insights into S1PR-based DMT mechanisms of action, but also widened the perspective on future therapeutic opportunities for this class of molecules. The second part of this chapter summarizes the results of the pivotal clinical trials of FGM, SPM, OZM, and the phase II and phase III trials of PNM upon clinical efficacy, as well as the reported adverse events (AE) of these S1PR modulators.

Fingolimod (FGM) was the first S1PR modulator to be approved for the treatment of relapsing forms of MS (RMS). Preclinical and clinical studies demonstrated that its use results in the sequestration of lymphocytes, particularly central memory and naïve T and B cell lymphocytes in peripheral lymphoid organs [7–11]. Pathological studies also demonstrated reduced pro-inflammatory cells within the CNS following treatment with this class of agents, and it had been widely believed that the sequestration of these potentially pro-inflammatory cells within lymphoid organs accounted for their therapeutic e fficacy in MS [7–10]. However, preclinical investigations, further detailed below, strongly sugges<sup>t</sup> that these agents may also act directly within the CNS, where there is an abundance of S1PR expression, to ameliorate the impact of inflammatory disease.

**Figure 1.** Metabolic Pathway to Sphingosine-1-phosphate Synthesis and Migration from the Intracellular to Extracellular Space via Sphingosine-1-phosphate Transmembrane Transporters.

**Figure 2.** (**A**) Five G protein-coupled Sphingosine-1-phosphate receptors have been identified. (**B**) Each of the S1PRs is coupled to one or more G proteins, resulting in multiple different downstream messaging targets (See Table 1). S-1-P = Sphingosne-1-phosphate (S-1-P), S1PR = Sphingosine-1-phosphate Receptor.

**Table 1.** G protein-coupled Sphingosine-1-phosphate receptor subset modulation of downstream signaling pathways.


S1PR = Sphingosine-1-phosphate Receptor, Akt = serine/threonine kinase (protein kinase B), Ras = small GTPase, MAPK = mitogen-activated protein kinase, Rac = member of Rho family of small GTPases, DAG = Diacyl Glycerol, PKC = protein kinase C, Rho = ras homolog gene family, ROCK = Rho kinase.

### **2. Preclinical Studies**

As noted above, S1P is a major intracellular signaling molecule via its G-protein-coupled membrane bound receptors [12–15]. Because the S1PR subsets are coupled to different G-proteins, they can modulate multiple downstream intracellular pathways (Table 1).

Although S1P is critically essential for normal CNS development and maturation [3,4,16,17] and may regulate synaptic function [18], it may also be cytotoxic at elevated concentrations, such as when there is a genetic deficiency in its degradative enzymes, producing neuronal apoptosis [19–24]. S1P also regulates calcium signaling [25], and may promote presynaptic calcium overload and cell death [26].

It is now known that S1P, through its interaction with S1PR-1, expressed on the surface of CCR7+ naïve, central memory B, and T cell lymphocytes, regulates the trafficking of these cells from peripheral lymphoid organs [27–30]. The S1PR-1-expressing lymphocytes egress in response to the S1P gradient (Figure 3). This effect of S1P makes its metabolic and translational pathways attractive potential therapeutic targets for the treatment of cell-mediated immunologic disorders such as MS.

**Figure 3.** The effect of Sphingosine-1-phosphate on lymphocyte mobilization from peripheral lymphoid organs. Sphingosine-1-phosphate interaction with S1PR1-expressing lymphocytes over-rides CCR7-induced retention signaling, which results in mobilization and egress of naïve and central memory lymphocytes into circulation in response to the S-1-P gradient. Non-CCR7-expressing lymphocytes, such as effector memory cells, are not S-1-P dependent for their entry into the peripheral circulation. B Cells = B cell lymphocytes, T Cells = T cell lymphocyte, NK cells = natural killer cells, pDC Cells = plasmacytoid dendritic cell, S-1-P = Sphingosine-1-phosphate, S1PR1 = Sphingosine-1-Phosphate Receptor, CCR7 = Chemokine Receptor 7.

### *2.1. S1P Receptors*

Fingolimod, approved for the treatment of relapsing forms of MS, is a non-selective S1PR modulator, which once phosphorylated (FGM-P) by sk2 [31], has affinity for S1PRs 1, 3, 4, and 5 [7,31]. The interaction of FGM-P with the S1PR-1 on the surface of CCR7+ lymphocytes results in the internalization and degradation of S1PR-1 [32,33] (Figure 4), promoting the sequestration of naïve, central memory T, and central memory B cell lymphocytes in peripheral lymphoid organs [34]. The circulation of central effector B and T cells, which do not express CCR7, are unaffected by FGM-P. When used in patients with relapsing forms of MS, FGM results in robust clinical and MRI evidence of efficacy, in parallel with marked reduction in circulating B and T cell numbers.

**Figure 4.** The effect of fingolimod-phosphate upon lymphocyte Sphinogosine-1-phosphate receptor-1. Although initially acting as an agonist, sustained exposure of sphingosine-1-phosphate receptor to fingolimod-phosphate results in receptor internalization and intracellular degradation. As a consequence of sphingosine-phosphate receptor-1 internalization, CCR7-expressing lymphocytes are no longer capable of responding to the sphingosine-1-phhosphate gradient. This results in naïve and central memory cell retention in peripheral lymphoid organs. S1PR1 = Sphingosine-1-phosphate receptor-1.

It has been well-established that the CNS also expresses S1PRs, on neurons, astrocytes, microglia, and oligodendroglia (OLG) during development, maturation, and adult cell states [35–37]. Furthermore, FGM-P, and the newer S1PR modulators SPM, OZM, and PNM, readily cross the blood brain barrier (BBB), and are selectively accumulated within the CNS. Unlike FGM, these latter 3 agents do not require prior phosphorylation to be pharmacologically active. It is possible that some of the therapeutic benefits of S1PR modulators result directly from their effects upon CNS receptors, and in the discussion that follows, we present evidence that supports such a role for these agents. Furthermore, there is evidence to sugges<sup>t</sup> that the therapeutic effects of these agents depends, at least in part, upon their interaction with CNS S1PRs [38].

The use of whole animal inflammatory CNS models, such as experimental allergic encephalitis (EAE), non-inflammatory models of CNS demyelination using in situ cuprizone or lyolecithin, in whole animal, brain tissue slice, and CNS cell culture models [39–42] have all provided important insights into fundamental S1P and S1PR physiology. They have also expanded knowledge of the mechanisms by which S1PR modulation regulates CNS development [4,17], and may protect the CNS during inflammatory insult [38]. What follows is a description of preclinical research which has provided important insights into the role of selective S1PR subtypes, the e ffects of their modulation/inhibition, and also the use of selective S1PR agonists and antagonists to further clarify the physiological roles of these receptors, and their impact on inflammatory CNS insults.

Although multiple S1PRs are expressed in the mammalian CNS, S1PR-1, 2 and 5 have attracted the most interest because of the pivotal roles they play in the development and myelinating function of OLGs, the regulation of astrocytes and microglia, and in maintaining the integrity of the BBB.
