The discovery of fingolimod (FTY720/Gilenya; Novartis), an orally active immunomodulatory drug, has opened up new approaches to the treatment of multiple sclerosis, the most common inflammatory disorder of the central nervous system. Elucidation of the effects of fingolimod--mediated by the modulation of sphingosine 1-phosphate (S1P) receptors--has indicated that its therapeutic activity could be due to regulation of the migration of selected lymphocyte subsets into the central nervous system and direct effects on neural cells, particularly astrocytes. An improved understanding of the biology of S1P receptors has also been gained. This article describes the discovery and development of fingolimod, which was approved by the US Food and Drug Administration in September 2010 as a first-line treatment for relapsing forms of multiple sclerosis, thereby becoming the first oral disease-modifying therapy to be approved for multiple sclerosis in the United States.

The immunomodulatory drug FTY720 is phosphorylated in vivo, and the resulting FTY720 phosphate as a ligand for sphingosine-1-phosphate receptors is responsible for the unique biological effects of the compound. So far, phosphorylation of FTY720 by murine sphingosine kinase (SPHK) 1a had been documented. We found that, while FTY720 is also phosphorylated by human SPHK1, the human type 2 isoform phosphorylates the drug 30-fold more efficiently, because of a lower K m of FTY720 for SPHK2. Similarly, murine SPHK2 was more efficient than SPHK1a. Among splice variants of the human SPHKs, an N-terminally extended SPHK2 isoform was even more active than SPHK2 itself. Further SPHK superfamily members, namely ceramide kinase and a "SPHK-like" protein, failed to phosphorylate sphingosine and FTY720. Thus, only SPHK1 and 2 appear to be capable of phosphorylating FTY720. Using selective assay conditions, SPHK1 and 2 activities in murine tissues were measured. While activity of SPHK2 toward sphingosine was generally lower than of SPHK1, FTY720 phosphorylation was higher under conditions favoring SPHK2. In human endothelial cells, while activity of SPHK1 toward sphingosine was 2-fold higher than of SPHK2, FTY720 phosphorylation was 7-fold faster under SPHK2 assay conditions. Finally, FTY720 was poorly phosphorylated in human blood as compared with rodent blood, in line with the low activity of SPHK1 and in particular of SPHK2 in human blood. To conclude, both SPHK1 and 2 are capable of phosphorylating FTY720, but SPHK2 is quantitatively more important than SPHK1.FTY720 is an immunomodulatory drug, which is highly efficacious in models of transplantation and of autoimmune diseases (1). It was recently found to be effective in kidney transplantation in humans (2). FTY720 elicits a lymphopenia resulting from the reversible redistribution of lymphocytes from the circulation to secondary lymphoid organs, without leading to general immunosuppression (3, 4). Conversion of FTY720 to its monophosphate appears to be essential for the effects of the drug on lymphocyte homing, since FTY720 phosphate acts as an agonist at four of the five G-protein-coupled receptors for sphingosine-1-phosphate (S1P) 1 (5, 6); it is assumed that at least one S1P receptor is critical to the lymphopenic response induced by FTY720 treatment (2). More recently, FTY720 was found to stimulate multidrug transporterdependent T-cell chemotaxis to lymph nodes (7); in this instance, FTY720 phosphate as the active metabolite is hypothesized to be responsible for stimulation of efflux activity of the lipid transporter Abcb1. FTY720 has been reported to be phosphorylated ex vivo by rodent lymphoid tissues (5) and whole blood of several species (6), and is rapidly phosphorylated in vivo (5, 6). After oral application of FTY720 to rats, the blood levels of the monophosphate exceeded those of the parent compound 2-4 fold (5). FTY720 was shown to be a substrate for recombinant murine sphingosine kinase 1a (muSPHK1a) (5). Studies with chiral analogs of FTY720 (namely the R-an...

Targeting sphingosine-1-phosphate receptors with the oral immunomodulator drug FTY720 (fingolimod) has demonstrated substantial efficacy in the treatment of multiple sclerosis. The drug is phosphorylated in vivo, and most of the clinical effects of FTY720-phosphate (FTY720P) are thought to be mediated via S1P1 receptors on lymphocytes and endothelial cells, leading to sequestration of lymphocytes in secondary lymphoid organs. FTY720P was described to act as a "functional antagonist" by promoting efficient internalization of S1P1 receptors. We demonstrate here that S1P1 receptors activated by FTY720P retain signaling activity for hours in spite of a quantitative internalization. Structural analogs of FTY720P with shorter alkyl side chains retained potency and efficacy in a functional assay but failed to promote long-lasting receptor internalization and signaling. We show that persistent signaling translates into an increased chemokinetic migration of primary human umbilical vein endothelial cells, which suggests persistent agonism as a crucial parameter in the mechanism of action of FTY720.

FTY720[2-amino-2-[2-(4-octylphenyl)ethyl]propane-1,3-diol hydrochloride] is an oral sphingosine-1-phosphate receptor modulator under development for the treatment of multiple sclerosis (MS). The drug is phosphorylated in vivo by sphingosine kinase 2 to its bioactive form, FTY720-P. Although treatment with FTY720 is accompanied by a reduction of the peripheral lymphocyte count, its efficacy in MS and experimental autoimmune encephalomyelitis (EAE) may be due to additional, direct effects in the central nervous system (CNS). We now show that FTY720 localizes to the CNS white matter, preferentially along myelin sheaths. Brain trough levels of FTY720 and FTY720-P in rat EAE are of the same magnitude and dose dependently increase; they are in the range of 40 to 540 ng/g in the brain tissue at efficacious doses and exceed blood concentrations severalfold. In a rat model of chronic EAE, prolonged treatment with 0.03 mg/kg was efficacious, but limiting the dosing period failed to prevent EAE despite a significant decrease in blood lymphocytes. FTY720 effectiveness is likely due to a culmination of mechanisms involving reduction of autoreactive T cells, neuroprotective influence of FTY720-P in the CNS, and inhibition of inflammatory mediators in the brain.FTY720 is an oral sphingosine-1-phosphate (S1P) receptor modulator (Baumruker et al., 2007) under development for the treatment of multiple sclerosis (MS), representing the first of a new class of immunomodulatory agents. Promising results in phase II trials with relapsing MS patients mirror the striking efficacy of FTY720 in MS models of experimental autoimmune encephalomyelitis (EAE), shown by preventive and therapeutic treatment (Brinkmann et al., 2002;Fujino et al., 2003;Webb et al., 2004;Kataoka et al., 2005;Balatoni et al., 2007). FTY720 is converted in vivo to its biologically active phosphate ester metabolite (FTY720-P), which acts as a high-affinity agonist for four of the five known G-protein-coupled S1P receptors, namely S1P 1 and S1P 3-5 (Brinkmann et al., 2002;Mandala et al., 2002). Sphingosine kinase (SPHK) 2 is the primary enzyme required for FTY720-P formation, as we and others subsequently confirmed in SPHK2 knockout mice (Kharel et al., 2005;Zemann et al., 2006). The fact that SPHK1 null mice become lymphopenic after FTY720 administration further supports the view that SPHK2 is sufficient for the functional activation of FTY720 (Allende et al., 2004).Emerging evidence suggests that the effectiveness of FTY720 in the central nervous system (CNS) extends beyond immunomodulation to encompass other aspects of MS pathophysiology, including an influence on the blood-brain barrier and glial repair mechanisms that could ultimately contribute to restoration of nerve function (Baumruker et al., 2007; This work was supported by Novartis Pharma AG. 1

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FTY720, a potent immunomodulatory drug in phase 2/3 clinical trials, induces rapid and reversible sequestration of lymphocytes into secondary lymphoid organs, thereby preventing their migration to sites of inflammation. As prerequisite for its function, phosphorylation of FTY720 to yield a potent agonist of the sphingosine-1-phosphate receptor S1P 1 is required in vivo, catalyzed by an as-yet-unknown kinase. Here, we report on the generation of sphingosine kinase 2 (SPHK2) knockout mice and demonstrate that this enzyme is essential for FTY720 phosphate formation in vivo. Consequently, administration of FTY720 does not induce lymphopenia in SPHK2-deficient mice. After direct dosage of FTY720 phosphate, lymphopenia is only transient in this strain, indicating that SPHK2 is constantly required to maintain FTY720 phosphate levels in vivo. IntroductionNaive T cells regularly circulate between the bloodstream and lymphatic tissue in search for foreign antigen, as well as for tumor and autoantigen. Their activation in secondary lymphoid organs followed by regulated egress back into the circulation to reach sites of inflammation is a prerequisite for any adaptive immune response in the T-cell compartment. Recently, one of the G protein-coupled receptors for sphingosine-1-phosphate (S1P), namely the S1P 1 receptor, was shown to be crucial for the tempo-spatial trafficking of T cells into and out of the secondary peripheral lymphoid organs. 1 The importance of S1P 1 in lymphocyte trafficking became clear through studies with FTY720, an analog of sphingosine. FTY720, after phosphorylation in vivo to FTY720 phosphate (FTY720-P), induces a reversible sequestration of lymphocytes into lymph nodes and Peyer patches. 2,3 FTY720-P thereby acts as a functional antagonist of the S1P 1 receptor, thus inducing aberrant internalization and consequently rendering T cells unresponsive to the obligatory egress signal provided by S1P. 1,[4][5][6] FTY720 has emerged as a potent immunomodulatory agent with usefulness in the control of organ transplant rejection and for treatment of autoimmune diseases. In animals, FTY720 is efficacious in prolonging graft survival, as well as in models of multiple sclerosis, acute lung injury, autoimmune diabetes, atherosclerosis, and renal ischemia-reperfusion injury. 7 Promising results have been obtained from human trials on FTY720 for indications in renal transplantation and multiple sclerosis. 7,8 Since FTY720 prodrug activation is essential for its action on T (and B) cells, understanding how the drug gets phosphorylated in vivo is of high interest, in particular for the design of novel analogs with altered pharmacologic properties.FTY720 is known to be phosphorylated in vitro by the 2 mammalian sphingosine kinases SPHK1 and 2, with SPHK2 being considerably more efficient. [9][10][11] As shown by a recent study in SPHK1-deficient mice, 12 this enzyme appears to be dispensable for the action of FTY720 in vivo, as Sphk1-null mice are still rendered lymphopenic by the drug.In this study, we describe th...

Summary Toll-Like Receptor (TLR) signaling is a key component of innate immunity. Aberrant TLR activation leads to immune disorders via dysregulation of cytokine production, such as IL-12/23. Herein we identify and characterize PIKfyve, a lipid kinase, as a critical player in TLR signaling using apilimod as an affinity tool. Apilimod is a potent small molecular inhibitor of IL-12/23 with an unknown target and has been evaluated in clinical trials for patients with Crohn’s disease or rheumatoid arthritis. Using a chemical genetics approach, we show that it binds to PIKfyve and blocks its phosphotransferase activity, leading to selective inhibition of IL-12/23p40. Pharmacological or genetic inactivation of PIKfyve is necessary and sufficient for suppression of IL-12/23p40 expression. Thus, we have uncovered a novel phosphoinositide-mediated regulatory mechanism that controls TLR signaling.

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