Discovery Medicine ® www.discoverymedicine.com n ISSN: 1539-6509; eISSN: 1944-7930

A Mechanistically Novel, First Oral Therapy for Multiple Sclerosis: The Development of Fingolimod (FTY720, Gilenya)

Jerold ChuN ANd Volker BrINkmANN

Abstract: Multiple sclerosis (MS) is a chronic Introduction autoimmune disorder affecting the central nervous system (CNS) through demyelination and neurode - Multiple sclerosis (MS) is a neurological disorder char - generation. Until recently, major therapeutic treat - acterized by demyelination and neurodegeneration ments have relied on agents requiring injection within the central nervous system (CNS) with common delivery. In September 2010, fingolimod/FTY720 relapsing forms produced through autoimmune inflam - (Gilenya, Novartis) was approved by the FDA as the matory mechanisms (Compston and Coles, 2002) first oral treatment for relapsing forms of MS. involving autoreactive lymphocytes that penetrate the Fingolimod is a novel compound produced by chem - blood-brain barrier to attack the nervous system (Figure ical modification of a fungal precursor. Its active 1). MS is a major cause of nervous system disability metabolite, formed by in vivo phosphorylation, mod - affecting individuals in the prime of life with a 2-3:1 ulates sphingosine 1-phosphate (S1P) receptors that female to male ratio (Compston and Coles, 2002) and are a subset of a larger family of cell-surface, G pro - an estimated global prevalence of 2.5 million that is tein-coupled receptors (GPCRs) mediating the highest in Northern latitudes particularly amongst effects of bioactive lipids known as lysophospho - Caucasians (Noseworthy et al. , 2000; Rosati, 2001). lipids. Fingolimod’s mechanism of action in MS is The initiating causes of MS remain unknown not completely understood; however, its relevant (Baranzini et al. , 2010). biology indicates a fundamentally different mecha - nism compared to all previously approved MS ther - Several MS forms have been identified, the most com - apies, with evolving research supporting both mon of which shows a waxing and waning of signs and immunological and nervous system activities. This symptoms to produce a “relapsing-remitting” clinical duality may herald a paradigm shift in the treat - presentation. “Relapsing remitting MS” (RRMS) ment of MS and other neurological disorders. affects ~85% of patients (Compston and Coles, 2002). [Discovery Medicine 12(64):n-n, September 2011] Within a decade, approximately a third of these patients will suffer from “secondary progressive MS” (SPMS) (Weinshenker et al. , 1989), a form that shows less inflammation and more neurodegeneration. A distinct form of MS that is not clearly associated with relapses, Jerold Chun , M.D., Ph.D., is at the Department of “primary progressive MS” (PPMS) shows neurodegen - Molecular Biology and Dorris Neuroscience Center, eration with a relative lack of inflammatory change. The Scripps Research Institute, 10550 North Torrey Late stage sequelae from advanced MS include perma - Pines Rd., La Jolla, California 92037, USA. nent disability and death. Volker Brinkmann , Ph.D., is at the Department of Over the last two decades, therapeutic approaches to Autoimmunity, Transplantation & Inflammation, MS have emerged that can reduce the relapse frequen - Novartis Institutes for BioMedical Research, CH-4056 cy and the rate of disability progression. First-line Basel, Switzerland. therapies include interferon- β formulations (Avonex, Corresponding authors: Jerold Chun, M.D., Ph.D. Biogen-Idec and Rebif, Merck-Serono) and glatiramer ([email protected]) and Volker Brinkmann, Ph.D. acetate (Copaxone, Teva) (PRISMS Study Group, ([email protected]).

© Discovery Medicine . All rights reserved. Discovery Medicine , Volume 12, Number 64, Pages n-n, September 2011 n The development of Fingolimod for multiple Sclerosis

1998; Jacobs et al. , 1996; Johnson et al. , 1995; The side effects are a major concern for all currently used IFNB Multiple Sclerosis Study Group, 1993). Second- agents. Interferons are often associated with injection- line therapies include a humanized antibody natalizum - site reactions and flu-like symptoms (Patti, 2010), with ab (Tysabri, Elan/Biogen-Idec) that recognizes the α4 other less commonly reported events including liver subunit of α4β1 integrin, a cell adhesion molecule dysfunction and cytopenias (Rice et al. , 2001). Of expressed on lymphocytes (Polman et al. , 2006; greater concern with the efficacious agent natalizumab Steinman, 2005). All of these therapies target the is the increasing incidence of progressive multifocal immune system and require parenteral delivery through leukoencephalopathy (PML), a rare but serious infec - injection or infusion, which can be associated with both tion associated with immunosuppression (Berger and patient compliance issues as well as deleterious side Major, 1999; Focosi et al. , 2010; Steinman, 2005) that effects. Moreover, it is debatable whether any of the has resulted in multiple fatalities in MS patients receiv - current therapies can alter neurodegenerative endpoints ing this therapy (Weissert, 2011). Another example is of MS, a view underscored by the current absence of the cytostatic agent mitoxantrone that has a cumulative any approved therapies for PPMS. There is a clear med - dose-dependent cardiac toxicity with additional risk of ical need for new therapies, particularly those with new leukemia, both of which limit its long-term use mechanisms of action that might promote direct CNS (Kingwell et al. , 2010). The latter two agents under - activities as part of their disease modifying effects. score immunosuppressive liabilities of these and other current and future MS therapies that primarily inhibit In addition to efficacy issues for current MS therapies, immune function as their primary mechanism of action.

Figure 1 . Model for the role of T cells and astrocytes in the pathology of MS. Recent data suggest a necessary role for Th17 cells and astrocytes in EAE pathology. Activated myelin-specific Th17 cells of the central memory phe - notype infiltrate the CNS and are restimulated to produce IL-17 by local dendritic cells to undergo clonal expan - sion and differentiation into effectors/effector memory T cells. Astrocytes respond via expression of IL-17R to T cell-released IL-17 and produce leukocyte-attracting chemokines in an Act1-dependent manner. Astrocyte-derived chemokines then recruit a second wave of peripheral inflammatory cells, which mediate EAE relapses and pro - gression via Th17 cell-mediated bystander demyelination (adapted from Rodgers et al ., 2010).

Discovery Medicine , Volume 12, Number 64, September 2011 The development of Fingolimod for multiple Sclerosis n

Efforts to develop new MS treatments, as evidenced by Noguchi and Chun, 2011). The development of fin - those in current development, underscore both limita - golimod also epitomizes a convergence of basic and tions and a need for therapies with more convenient, clinical science that points to untapped mechanisms for effective, and safe treatment profiles. the treatment of MS, as well as potentially other CNS disorders. Despite their introduction nearly 20 years ago, interfer - on therapies still represent the most commonly pre - Origins of Fingolimod: A Folk Medicine from Fungi scribed disease modifying agents for MS. This situation epitomizes deficiencies in the current state of therapeu - Fingolimod emerged from the study of the fungal phy - tics using agents that lack ease of delivery, such as those lum Ascomycota (Cavalier-Smith, 1998; Sung et al. , requiring injection, as well as efficacy characteristics, 2007), historically, commonly, and taxonomically particularly for preventing neurodegeneration. Drugs referred to as class Ascomycetes within the kingdom under development for MS include the monoclonal Fungi. Within this group of “sac fungi” is the species antibodies named rituximab, ocrelizumab, and ofatu - Cordyceps of family Cordycipitaceae, which consti - mumab (which target CD20 to deplete B cells); alem - tutes over 400 members characterized prominently by tuzumab/Campath-1H (which targets CD52 to deplete their entomopathogenic activity wherein the fungus T and B cells and some monocyte-derived dendritic infects a range of insect host species at different points cells) (Buttmann, 2010); and small molecules including in their development, parasitize them to grow out of the the oral agents. These oral agents include cladribine (a corpse to form the stalk and fruiting body of the fungus cytotoxic adenosine deaminase-resistant purine nucleo - (Sung et al. , 2007). This biological cycle was at least in side, recently withdrawn from commercial develop - part recognized in Chinese herbal medicine through the ment), dimethylfumarate BG-12 [an activator of the name, “Dong Chong Xia Cao,” meaning “winter worm, nuclear factor-E2-related factor 2 (Nrf2) transcriptional summer grass” (Zhou et al. , 2009) that describes the pathway that alters glutathione levels (Lin et al. , 2011), infected insect larval form followed later by the fungal but is also associated with tumorigenesis (DeNicola et stalk form of most Cordyceps species (Figure 2; shown al. , 2011) and immunosuppression (Lehmann et al. , for Isaria sinclairii ). Folk medicine recognized an 2007; Vandermeeren et al. , 1997)], laquinimod extensive range of health benefits produced by ingest - (unknown cellular target), and teriflunomide (an ing the fungus (Ng and Wang, 2005), and modern ana - inhibitor of dihydroorotate dehydrogenase, which cat - lytical techniques have identified important fungal alyzes the rate-limiting step in the de novo synthesis of metabolites that have human biological activities, pyrimidines). All of these agents target lymphocytes as including Cordycepin (3′-deoxyadenosine) that inhibits well as other immune-related cells (Niino and Sasaki, tumor growth and was derived from C. militaris that 2010). As of this writing, they also all remain subject to parasitizes the silkworm (Ng and Wang, 2005; additional clinical and regulatory evaluation before Paterson, 2008), Cyclosporine, a classical immunosup - possible entry into the therapeutic armamentarium. pressant derived from C. subsessilis that infects the scarab beetle (Borel and Kis, 1991; Illana Esteban, A major step towards achieving the goal of a mechanis - 2007), and an immunosuppressant, myriocin (ISP-1) tically novel, orally bioavailable agent has recently (Fujita et al. , 1994). Myriocin is a metabolite of Isaria been taken through FTY720/fingolimod (commercial sinclairii (Figures 2 and 3) — the anamorph (asexual)- name Gilenya; Novartis) that represents the first oral stage name historically referenced by its presumed telo - MS therapy approved by the United States Food and morph-stage name, Cordyceps sinclairii (Fujita et al. , Drug Administration (FDA), the European Union, and 1994; McNeill and International Association for Plant several other countries. This compound interacts with a Taxonomy, 2006) — that parasitizes Cicada nymphs new, until now clinically unassessed molecular target: (Figure 2) (Chiba et al. , 1996; Miyake et al. , 1995). receptors for the signaling lysophospholipid known as Efforts to produce chemically modified compounds sphingosine 1-phosphate (S1P). Here we review high - derived from myriocin (Adachi et al. , 1995; Suzuki et lights of the discovery of fingolimod, its receptor tar - al. , 1996a) led, in 1994, to the identification of a novel gets, clinical efficacy and safety profiles, and evolving compound, fingolimod (Adachi et al. , 1995; Fujita et understanding of its mechanism of action in MS that al. , 1995), whose original name, FTY720 (Figure 3), has revealed effects in not only the immune system, but reflects the discoverers: Fujita and colleagues at Kyoto direct action in the CNS. Additional detailed informa - University, Taito Company (now Mitsui Seito that pro - tion on fingolimod can be found elsewhere (Brinkmann duces sugar), and Yoshitomi Pharmaceutical Industries et al. , 2010; Chun and Hartung, 2010; Cohen et al. , (now Mitsubishi Tanabe Pharma). The compound was 2010; Cohen and Chun, 2011; Kappos et al. , 2010; of interest based upon preclinical data that supported

Discovery Medicine , Volume 12, Number 64, September 2011 n The development of Fingolimod for multiple Sclerosis activities that might be relevant to improved organ Consistent with an altered activity profile and contrast - transplantation (Chiba et al. , 1996; Mitsusada et al. , ing with myriocin, fingolimod did not inhibit activa - 1997; Suzuki et al. , 1998; Yuzawa et al. , 1998), and this tion, proliferation, or memory formation of T cells; led to interest by the industrial producers of moreover, it did not affect the production of antibodies cyclosporine (Cyclosporin A, Cyclosporine), Sandoz by B cells or cytokines by T cells and, as a conse - (Stahelin, 1996), which merged with Ciba-Geigy in quence, did not impair immune defence against sys - 1996 to form Novartis. Fingolimod showed activity in temic viral infection (Pinschewer et al. , 2000; models of organ transplantation when combined with Brinkmann et al. , 2001; 2000). Thus, fingolimod Cyclosporin A, which led to in-licensing of fingolimod appeared to be acting in a way distinct from classically by Novartis for evaluation as a therapeutic agent in defined immunosuppressants through an unclear renal transplantation. molecular target(s) and mechanism of action. The mechanism of fingolimod seemed to be quite dif - Parallel Discovery of Lysophospholipid S1P ferent from two other fungal agents isolated from Receptors, a Molecular Target for Fingolimod Cordyceps : Cyclosporin A that interacts with a protein phosphatase, calcineurin, and myriocin that inhibits Around the same period of fingolimod’s discovery, a serine-palmitoyl-transferase (SPT) (Chen et al. , 1999; completely independent line of research was investigat - Miyake et al. , 1995). Fingolimod is involved in sphin - ing genes involved in CNS development (Hecht et al. , gosine metabolism (Miyake et al. , 1995). In particular, 1996), which led to the identification of the first mem - chemical modification efforts of the parental myriocin ber for what is now known as the lysophospholipid compound (Figure 3) to produce fingolimod might family of receptors (Choi et al. , 2010; Chun, 2007; have been expected to maintain some of the myriocin Fukushima et al. , 2001; Ishii et al. , 2004; Mutoh et al. , activities; however, fingolimod had no activity against 2011). These G protein-coupled receptors (GPCRs) SPT (Chen et al. , 1999; Miyake et al. , 1995). mediate the actions of at least two major classes of sig -

Figure 2 . Fungal source of fingolimod precursors compounds. Fingolimod is a chemical derivative of myriocin/ISP-1, a fungal metabolite that was isolated from Isaria sinclairii , the anamorph stage of the ascomycete species Cordyceps . Along with ginseng and the young antlers of deer, Cordyceps fungi were considered as one of the three oriental medicines that give ‘eternal youth.’ Cordyceps fungi enter into living insects, feed on the insides of the host, and eventually grow onto the surface of the cadaver in the summer. Drugs derived from Cordyceps are Cordycepin (3′ deoxyadenosine) which inhibits tumor growth, Cyclosporin, the classical immunosuppressant used in transplant medicine, and Myriocin (ISP-1), the immunosuppressant that targets serine palmitoyl transferase (SPT). Fingolimod, despite being a chemi - cal derivative of myriocin, has lost activity on SPT but targets the class of G protein-coupled S1P receptors. Isaria sinclairii , photo courtesy of Mr. Clive Shirely; cicada nymph, photo courtesy of Alastair Robertson and Maria Minor; soil bugs, from An Illustrated Guide to New Zealand Soil Invertebrates, http://soilbugs. massey.ac.nz. © Massey University.

Discovery Medicine , Volume 12, Number 64, September 2011 The development of Fingolimod for multiple Sclerosis n naling lysophospholipids that are known as lysophos - and Chun, 2008; Yung et al. , 2011). These receptors phatidic acid (LPA) and sphingosine 1-phosphate also have prominent expression in the immune system (S1P), the latter of which comprises the major receptor as well as other tissues, and have been implicated in a target for fingolimod. The first family member recog - broad range of biological and pathophysiological nized was for LPA (Fukushima et al. , 1998; Hecht et processes (Gardell et al. , 2006; Herr and Chun, 2007; al. , 1996) and this identity aided deorphanization of Noguchi and Chun, 2011; Ye and Chun, 2010). other LPA receptors, as well as the S1P receptors (An et al. , 1997; Lee et al. , 1998b) that at one point were The structural similarity of fingolimod to sphingosine hypothesized to interact with both LPA and S1P (Lee et led to the key identification of S1P receptors as the tar - al. , 1998a) reflecting their marked amino acid sequence get for phosphorylated fingolimod (Brinkmann et al. , similarities. There are currently 11 receptors, six for 2002; Mandala et al. , 2002). Sphingosine is a metabo - LPA and five for S1P. Lysophospholipid receptors were lite of the cell-membrane-derived sphingolipids such as first identified by exploiting knowledge of their expres - sphingomyelin and ceramide (Brinkmann et al. , 2010). sion and activity in the developing CNS, and their other Phosphorylation is produced by the action of endoge - activities and the cell types on which they express have nous sphingosine kinases (SPHKs) to produce fin - now been identified within the CNS (Choi et al. , 2008; golimod-phosphate (fingolimod-P) (Brinkmann et al. , Gardell et al. , 2006; Noguchi and Chun, 2011; Rivera 2002; Mandala et al. , 2002), primarily involving the

Figure 3 . Fingolimod-relevant chemical structures. Fingolimod and fingolimod-phosphate (fingolimod-P) are struc - tural analogues of sphingosine and sphingosine 1-phosphate (S1P), respectively. S1P is generated via the intracellu - lar ceramide pathway, and ceramide is formed through de novo biosynthesis or degradation of the cell membrane con - stituent sphingomyelin. Ceramide is N-deacetylated to yield sphingosine, and both sphingosine and fingolimod are phosphorylated by sphingosine kinases to yield S1P and ( S)-fingolimod-P, respectively, whereas ( R)-fingolimod-P is not found in vivo . The ( S)-fingolimod-P is the biologically active principle of the drug in animal models of autoim - mune disease (Albert et al. , 2005; Brinkmann et al. , 2002).

Discovery Medicine , Volume 12, Number 64, September 2011 n The development of Fingolimod for multiple Sclerosis enzyme SPHK2 (Figure 3) (Zemann et al. , 2006). 2007; Foster et al. , 2009)]. These animal studies also Receptor studies revealed that fingolimod-P (but not identified a range of possible side-effects, including parent fingolimod) is an agonist at S1P 1, S1P 4, S1P 5 S1P 1-dependent transient bradycardia (Forrest et al. , (EC 50 values of ~ 0.3-0.6 nM), and S1P 3 (EC 50 values 2004; Gergely et al. , 2009; Koyrakh et al. , 2005; Sanna of ~ 3 nM), with essentially no activity at S1P 2 at these et al. , 2004) and S1P 1-dependent vascular permeability concentrations (Brinkmann et al. , 2002; Mandala et al. , (Sanna et al. , 2004; Oo et al. , 2011). The possible safe - 2002). Stereochemical analyses identified ( S)-fin - ty concerns have not been borne-out by serious adverse golimod-P as the biologically active form in vivo events in human clinical trials (discussed next), under - (Brinkmann et al. , 2002), whereas ( R)-enantiomer was scoring species differences and/or the use of much high - not detected (Albert et al. , 2005). Thus, the discoveries er fingolimod concentrations in the animal studies com - of fingolimod and of their targeted lysophospholipid pared to the approved dose in humans. receptors provided a basis for understanding fin - golimod’s actions. However, the therapeutic indications Initial human studies with fingolimod led to two pivotal for fingolimod evolved in unanticipated ways. Phase III clinical trials focused on a possible reduction in the annualized relapse rate (ARR) as the primary end - From Renal Transplantation to Multiple Sclerosis point using fingolimod in relapsing remitting MS (RRMS): a placebo-controlled trial of 1,272 patients Initial studies driving the discovery and development of (the FREEDOMS trial) and an active comparator trial fingolimod focused on its utility in preventing organ involving 1,292 patients (the TRANSFORMS trial). In graft rejection in animal models using high-dose fin - FREEDOMS, fingolimod was investigated in a double golimod (10 mg/kg) in monotherapy (Chiba et al ., blind, 2-year study that involved patients randomized to 1996; Enosawa et al. , 1996; Suzuki et al ., 1996a), and a reduced dose arm of 0.5 mg or 1.25 mg as compared doses of 0.3-3 mg/kg in combination with classical to a placebo (Kappos et al. , 2010). Once again, the ARR immunosuppressive agents (Brinkmann et al. , 2002; was significantly reduced in both experimental arms Chiba et al. , 1996; Mitsusada et al. , 1997; Suzuki et al. , (0.18 and 0.16, respectively) compared to the placebo 1998; 1996b; Yanagawa et al. , 1998; Yuzawa et al. , (0.40), as was risk of disability progression (17.7% and 1998). However, the apparent synergy observed 16.6%, respectively) compared to placebo (24.1%) and between fingolimod and cyclosporine in some animal MRI-detected lesions, which included the previously models was not substantiated in human Phase III clini - assessed gadolinium-enhancing lesions, and interesting - cal trials for renal transplantation, where fingolimod did ly, a reduction in brain-volume loss (atrophy) (-0.7% in not support reduction of the dose of cyclosporine, com - both fingolimod arms compared to -1.0% with placebo). pared to standard-of-care treatment (Mansoor and In TRANSFORMS, a 1-year, double blind, double- Melendez, 2008). This lack of superior efficacy in pre - dummy trial, fingolimod was compared to IFN²-1a venting transplant rejection was likely related to insuf - (Avonex, Biogen Idec), a major, first-line therapy for ficient immunosuppression by fingolimod, but the MS (Cohen et al. , 2010). The same fingolimod doses effects on cell migration/trafficking opened up other were compared to standard administration of IFN β-1 a. possible therapeutic indications including MS and other Both fingolimod arms showed an ARR that was signif - autoimmune disorders. icantly lower (0.16 and 0.20, respectively), compared to the IFN β-1 a arm (0.33). Disability progression was sim - In addition, basic studies on lysophospholipid receptors ilar, albeit difficult to assess during the short, 1-year identified expression patterns and functions that were trial; however, both fingolimod doses showed superior consistent with possible roles in a range of CNS cell MRI endpoints of fewer new or enlarged lesions on T2- types and processes relevant to MS, including receptor weighted images, gadolinium-enhancing lesions, and expression in myelinating cells (Chun et al. , 2000; reduced brain atrophy at 12 months. Weiner and Chun, 1999; Weiner et al. , 1998) that are affected by demyelination in MS, and immunological Serious adverse event rates in the 2-year FREEDOMS effects that included T lymphocytes that can damage the trial (Kappos et al. , 2010) were similar amongst all CNS (Brinkmann et al. , 2002; Chun et al. , 2002; Goetzl groups including placebo (10.1-13.4%), whereas in the and An, 1999; Huang et al. , 2002). Fingolimod efficacy 1-year TRANSFORMS study (Cohen et al. , 2010), the in MS animal models, particularly experimental autoim - incidence of adverse events was higher in the high dose mune encephalomyelitis (EAE), was observed inde - arm (1.25 mg) but comparable in the 0.5 mg fingolimod pendently by multiple investigators (Brinkmann et al. , vs. IFN β-1 a arms. The incidence of adverse events that 2002; Fujino et al. , 2003; Kataoka et al. , 2005; Webb et resulted in drug discontinuation was similar between al. , 2004), and was supported by findings in other ani - the 0.5 mg fingolimod and all control groups, but was mal models [e.g., dark agouti rat model (Balatoni et al. ,

Discovery Medicine , Volume 12, Number 64, September 2011 The development of Fingolimod for multiple Sclerosis n higher in the 1.25 mg fingolimod groups. The combined and abrogation of receptor-mediated S1P signaling. data from both Phase III trials did not suggest an increased incidence of either infections or malignancies The orchestrated role for S1P signaling that allows lym - associated with fingolimod treatment (Cohen et al. , phocytes to egress from the lymph nodes predominates 2010; Kappos et al. , 2010). The two lethal herpes infec - over another signaling system mediated by chemokines tions (one primary varicella zoster infection and one and the receptor known as CCR7, which promotes case of herpes simplex encephalitis) that occurred in retention of lymphocytes in secondary lymphoid organs patients receiving 1.25 mg fingolimod (Cohen et al. , with preferential effects on “naïve” and early “central” 2010) may have involved confounding factors related memory T-cells rather than late, terminally differentiat - to the use of high-dose steroids. In view of the compa - ed “effector” memory T-cells (Henning et al. , 2001; rable efficacy of the lower 0.5-mg fingolimod dose, Pham et al. , 2008; Sallusto et al. , 1999). Thus, S1P 1 sig - along with its improved safety profile, the 0.5-mg fin - naling appears to predominate over CCR7-mediated golimod dose was nominated for regulatory approval in retention by promoting lymphocyte egress from lymph providing the best risk-to-benefit profile, and during nodes, whereas functional antagonism of S1P 1 by fin - 2010-2011, fingolimod (commercial name Gilenya) golimod inhibits egress from lymph nodes. By contrast, received approvals in the United States, the European egress of CCR7-negative effector memory T cells Union, and several other countries. (Sallusto et al. , 2004) appears to occur independently of S1P 1 receptor signaling and these cells are refractory to Fingolimod Immunological Activities the trafficking effects of fingolimod. This latter point is supported by studies in mice (Brinkmann et al. , 2004; In part reflecting its origins in transplantation research, Metzler et al. , 2008; Pham et al. , 2008) and humans fingolimod has been extensively studied for its effects (Brinkmann, 2009; 2004; Mehling et al. , 2008; Metzler on immune system. Early studies raised the possibility et al. , 2008). that fingolimod might interfere with T cell trafficking rather than function, and that its mode of action may Two corollaries with particular relevance to MS involve G αi protein-coupled receptors (Brinkmann et through this differential effect of fingolimod on CCR7- al. , 2000). It was then found that conversion of fin - positive vs. CCR7-negative T cells may contribute to golimod to its phosphorylated metabolite, fingolimod- efficacy and safety, respectively. First, fingolimod may P, and the interaction with cognate S1P receptors result produce efficacy by sequestering the CCR7-positive in trafficking effects as demonstrated by the sequestra - cells, which include naïve and central memory T cells, tion of lymphocytes in secondary lymphoid organs the latter of which have a key role in immunological (Brinkmann et al. , 2002; Mandala et al. , 2002). memory. Following antigen exposure, central memory T lymphocytes can undergo clonal expansion and dif - These studies provided a link to the biological effects of ferentiation to generate effectors/effector memory T fingolimod, shown in transfected cell lines, to produce, cells which provide adaptive immunity against recog - paradoxically, S1P receptor internalization, removing nized antigens (Iezzi et al. , 2001; Sallusto et al. , 2004). them from the cell surface (Graler and Goetzl, 2004) to Central memory T cell retention by fingolimod could inhibit S1P signaling, despite the initial characterization function as a therapy in MS since more than 90% of T of fingolimod as a receptor agonist (after phosphoryla - cells that are found in the cerebrospinal fluid (CSF) tion) (Brinkmann et al. , 2002). Immunological studies appear to be of the central memory subset (Kivisakk et of knockout mice for S1P 1 — whereby this receptor al. , 2004). The contained autoreactive, pathological T was removed from lymphocytes — resulted in lympho - cells could therefore be prevented from entering the cyte trafficking defects that were similar to the effects CNS by fingolimod sequestration, thereby abrogating of fingolimod exposure, suggesting that fingolimod was their differentiation into pathological effectors and acting as a “functional antagonist” to inhibit S1P recep - effector memory T cells upon interaction with CNS-res - tors (Brinkmann et al. , 2004; Matloubian et al. , 2004). ident antigen-presenting dendritic cells. In animal mod - Interestingly, mutations in the S1P 1 receptor that abro - els, fingolimod prevented accumulation of pathological gated internalization, but not signaling of the receptor, Th17 cells in the nervous system (Zhang et al. , 2008; were sufficient to blunt lymphocyte trafficking effects 2009), supporting Th17 cell- or Th17 cell precursor of fingolimod, confirming that receptor internalization sequestration as an efficacy mechanism. Accordingly, rather than signaling was required, presumably to pre - phenotypic Th17 cells were reduced in the circulation vent S1P-directed migration of cells (Oo et al. , 2011). in fingolimod-treated MS patients (Mehling et al. , In other words, although fingolimod-P initially acts as 2010). In addition to efficacy by CCR7-positive cell an S1P receptor agonist, chronic exposure to it results in sequestration of pathological T cells, fingolimod could S1P receptor loss from the surface of the cell (Figure 4) provide safety through maintained immunosurveil -

Discovery Medicine , Volume 12, Number 64, September 2011 n The development of Fingolimod for multiple Sclerosis lance. Such functionality would be produced by prefer - of the sinus, most T cells retract and migrate back into entially not affecting CCR7-negative effector memory the parenchyma in fingolimod-treated animals, due to a T cells of any functional phenotypes (Mehling et al. , failure of the cells to establish adhesion on the sinus, 2008), which may leave lymph nodes, independent of whereas G αi2-deficient T cells adhere firmly on the S1P 1 signaling (Pham et al. , 2008). sinus, which prevents their retraction, facilitating their transmigration of the lymphatic endothelial barrier. In support of the above, another study proposed that Interestingly, G αi2-deficient T lymphocytes are Gαi2 null T cells egress independent of S1P-mediated hyper ‐responsive for T cell receptor signaling and chemotaxis (Zhi et al. , 2011), and these cells were also cytokine production, with a relaxed costimulatory not retained by fingolimod. Intravital imaging of lymph requirement (Huang et al. , 2003) — a phenotype nodes revealed that T cells approach and engage corti - matching effector memory T cells — again supporting cal sinusoids in lymph nodes similarly in the presence sparing of this subset by fingolimod. or absence of fingolimod. However, after engagement

Figure 4 . Modulation of S1P 1 receptors by S1P and fingolimod. To ensure that extracellular stimuli are translated into intracellular signals of appropriate magnitude and specificity, most signalling cascades are tightly regulated. One of the major mechanisms involved in the regulation of G protein-coupled receptors (GPCRs), including S1P receptors, involves their endocytic trafficking. GPCR endocytic trafficking entails the targeting of receptors to discrete endocytic sites at the plasma membrane, followed by receptor internalization and intracellular sorting to terminate the signal. Shown is the fate of S1P- and fingolimod-P-signalling at S1P 1 receptors. S1P produces a signal that is terminated by internalization of the S1P-S1P 1 complex. After dissociation of S1P from S1P 1, the receptor is recycled to the cell mem - brane. The higher affinity of fingolimod (compared to S1P) to S1P 1 leads to tight binding and less recycling of S1P 1 and, as a consequence, to proteasomal degradation of the drug-receptor complex. This results in termination of an ini - tial agonistic signal, and a “functional antagonism” of S1P 1 receptors that, in models of MS, terminates T cell inflam - mation and astrogliosis.

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Collectively, the data show that activation and prolifer - between clinical scores and peripheral lymphocyte lev - ation of naïve and central memory T-cells, as well as els seen in some EAE animal models (Webb et al. , differentiation and trafficking of effector memory T- 2004). Receptor-mediated S1P signaling has been doc - cells, may not be significantly affected by fingolimod, umented in CNS cell lineages that have relevance to thereby preserving this arm of the adaptive immune MS, consistent with broad expression of lysophospho - system that can reduce the risk of infection and cancers lipid receptors in general within the brain (Chun, 1999; common with immunosuppressive agents. Consistent Mutoh and Chun, 2008; Noguchi and Chun, 2011). with this mechanism, the combined data from both Astrocytes in particular express S1P 1 and S1P 3 (Choi et aforementioned Phase III trials did not suggest an al. , 2011; Mullershausen et al. , 2007; Osinde et al. , increased incidence of either infections or malignancies 2007; Pebay et al. , 2001; Rao et al. , 2003; Sorensen et associated with fingolimod treatment (Cohen et al. , al. , 2003; Wu et al. , 2008), and these two receptors 2010; Kappos et al. , 2010). have been reported to be up-regulated in MS astrocytes (Van Doorn et al. , 2010). Oligodendrocytes and their In addition to the immunomodulatory effects, fin - precursor cells also express S1P receptors (Dev et al. , golimod also simultaneously accesses a completely dif - 2008; Jaillard et al. , 2005; Miron et al. , 2008; Mutoh et ferent biology: direct CNS actions on cells relevant to al. , 2011; Terai et al. , 2003) particularly S1P 5 in mature MS, which if present, would be an efficacy mechanism oligodendrocytes. Neural progenitor cells, and likely independent of immunomodulation. While the exact some neurons, can also express S1P 1, along with other role that fingolimod’s CNS actions might have in MS S1P receptor subtypes (Choi et al. , 2011; Kajimoto et are not known, a growing body of basic and clinical lit - al. , 2007; Kimura et al. , 2007; McGiffert et al. , 2002; erature supports direct CNS influences that are dis - Mizugishi et al. , 2005). In addition, resident non-neural cussed next. cells like microglia can also express S1P receptors (Durafourt et al. , 2011; Schilling et al. , 2002; Tham et Fingolimod CNS Activities al. , 2003). The diversity of both cell types and S1P receptor subtypes underscore potential effector activi - Based upon its relationship to sphingosine, and its inter - ties of fingolimod within the CNS in MS. The receptor actions with lysophospholipid S1P receptors, fin - mechanisms could involve some degree of transient, golimod was likely to have direct CNS effects. As an initial agonism; however, continuous exposure to fin - analog of the lipid sphingosine, fingolimod could have golimod would be expected to produce functional a range of possible roles within the CNS, since that is antagonism — a net loss of S1P receptor signaling — at where sphingosine and related sphingolipids (e.g., least for S1P that has been best characterized in non- phospholipids that contain sphingosine, like sphin - 1 neural cells. Pharmacological S1P loss through func - gomyelin) were first identified from early studies of the 1 tional antagonism can be rigorously modeled by use of brain during the 1800s (Thudichum, 1884; Vauquelin, genetics via the production of null mutation knockouts. 1812). The functions of sphingosine were then as enig - matic as the Sphinx, from which its name was coined This knockout strategy was used to address functional (Merrill et al. , 1997; Thudichum, 1884). Consistent consequences of removing S1P 1 from specific cell line - with CNS actions, fingolimod and fingolimod-P local - ages in the CNS while leaving the immune system ize to the CNS as revealed by radio-labeling studies intact, combined with challenge by EAE (Choi et al. , (Foster et al. , 2007). Independent support for CNS 2011). The resulting mutants were then assessed for 1) activities came from studies of lysophospholipid recep - effects on fingolimod activity, and 2) effects on clinical tors that were first identified from the brain (Hecht et disease, independent of fingolimod exposure, combined al. , 1996), with most, including S1P receptors, with other analyses (Choi et al. , 2011). S1P 1 was con - expressed in CNS lineages where they have a rich neu - ditionally deleted (using loxP technologies) from vari - robiology (Brinkmann, 2009; Choi et al. , 2010; ous CNS cell lineages while still maintaining immuno - Fukushima et al. , 2001; Ishii et al. , 2004; Kingsbury et logical competence as evidenced by normal responses al. , 2003; Mizugishi et al. , 2005; Mullershausen et al. , of peripheral blood lymphocytes to fingolimod expo - 2007; Trimbuch et al. , 2009; Weiner and Chun, 1999; sure, and an ability of mutant lymphocytes to produce Yung et al. , 2011). disease following adoptive transfer from mutant into normal animals. Of the lineages assessed, S1P deletion Data supporting possible direct CNS effects included 1 from astrocytes but not neurons produced a dramatic the aforementioned fingolimod localization within the effect, eliminating fingolimod activity in EAE, com - CNS (Foster et al. , 2007), the rapid onset of therapeutic pared to vehicle controls, and also attenuating MS-like effects with a “rescue” therapy started 40 days after dis - disease. Consistent with these clinical assessments of ease onset (Foster et al. , 2009), and a discordance

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disease, S1P 1 deletion on its own protected against his - al. , 2007). However, risk of serious neoplastic and/or tologically detected damage as compared to control ani - infectious adverse events limits their use. This issue has mals challenged by EAE, including a marked reduction been underscored by the rare occurrence of progressive in astrogliosis — a reactive state of astrocytes that multifocal leukoencephalopathy (PML) associated with increases their number and alters their morphology — the use of natalizumab or rituximab (Buttmann, 2010). along with preservation of axons and myelin that would T cell immunosuppression may be involved in both usually be damaged by EAE. Fingolimod exposure dur - cases; in addition to its effects on T cell trafficking, ing EAE produced a similar histological picture when natalizumab may interfere with the VLA4-VCAM1 assayed in normal (non-mutant) animals, and competi - costimulatory pathway that is critical to human CD4 T tive receptor binding assays using membrane prepara - cell proliferation (Weitz-Schmidt et al. , 2001), and ther - tions from brains of these animals confirmed down- apeutic B cell depletion by rituximab was shown to modulation of S1P receptors by the drug, supporting the impair B cell antigen-presentation and, as a conse - functional antagonism model of S1P 1 loss that had been quence, CD4 T cell activation and clonal expansion in previously observed in the immune system, a receptor response to pathogen challenge (Bouaziz et al. , 2007). mechanism that was further shown to occur in astro - Therefore, the sparing of effector memory T cells in cytes as well. both CD4 and CD8 populations by fingolimod could be critical to immunosurveillance; in the meninges of Overall, these data identify S1P 1 signaling in astrocytes mice, fingolimod preferentially reduced naive and cen - as a major influence on models of MS, as well as a nec - tral memory T cells, whereas anti-VLA4 treatment pri - essary component of fingolimod efficacy (Figure 1). marily depleted the effector memory population The combined effects of the drug on lymphocyte traf - (Derecki et al. , 2010) (see also above for the key role of ficking and astrogliosis may reduce neurodegeneration circulating central memory T cells in pathology of MS). and favor remyelination after damage, as observed in models of EAE and cuprizone-induced demyelination Compared to human CD8 T cells, circulating CD4 T (Balatoni et al. , 2007; Foster et al. , 2009; Kim et al. , cells contain larger numbers of CCR7-positive naive 2011). Other S1P receptor subtypes and/or involved cell and central memory T cells, and this could explain the lineages may also have related influences (Soliven et more profound retention of CD4 T cells in lymph nodes al. , 2011; Wu et al. , 2008), and these remain to be by fingolimod (Brinkmann et al. , 2010). Importantly, addressed in MS models. One or more of these process - infection-relevant effector memory T cells could still be es might explain the reduction in brain atrophy generated in lymph nodes and would recirculate inde - observed with fingolimod treatment in MS Phase III tri - pendent of S1P1 (Pham et al. , 2008); thus, the reduced als (Cohen et al. , 2010; Kappos et al. , 2010), contrast - total CD4 T cell count in blood may not prove useful as ing with distinct and at times increasing atrophy sig - an indicator of immunosuppression in fingolimod-treat - nals, observed with immunologically targeted therapeu - ed patients. tics like natalizumab (Miller et al. , 2007). The above data support the notion that fingolimod at its Immunomodulatory Approaches to MS Therapy approved dose may not act as a potent immunosuppres - sant: 1) CNS effects are unrelated to immunosuppres - A notable corollary of the dual immunological and CNS sion; 2) suboptimal prevention of graft rejection was fingolimod mechanisms is that fingolimod does not fit achieved in renal transplantation studies in combination the profile of an immunosuppressive agent like those in with cyclosporine, despite being at 10X the approved common use in the transplantation field - e.g., “classi - MS dose; 3) immunological constituents are maintained cal” immunosuppressive agents like calcineurin (cellular and humoral), with reversible effects on cell inhibitors [Cyclosporine, Tacrolimus (Borel and Kis, location of some (but not all) lymphocyte subsets — 1991; Juhasz et al. , 2009; Stahelin, 1996)], high dose without inhibition of proliferation, differentiation, and corticosteroids [e.g., Prednisone (Goetzl, 2008)], and cytotoxicity; 4) immunological surveillance is main - cytotoxic and/or antimitotic agents [azathioprine, tained through relatively unaffected effector memory T- mycophenolate, or cladribine (Goetzl, 2008; Neuhaus cells; and 5) clinically, the overall incidence of infec - et al. , 2007)] or biologicals, including a growing num - tions as well as of serious and severe infections was not ber of humanized antibodies raised against immune cell increased over placebo control in the FREEDOMS trial targets [CD3, IL-2 receptor, integrins, CD52 in phase III studies. Overall, the emerging picture iden - (Buttmann, 2010; Nitta et al. , 1992; Steinman, 2005; tifies S1P receptor pathways in MS that can provide Wolff et al. , 2004)]. Early approaches to the treatment efficacy through mechanisms different from classical of MS utilized classical immunosuppressive strategies, immunosuppressants. some of which continue to be used today (Neuhaus et

Discovery Medicine , Volume 12, Number 64, September 2011 The development of Fingolimod for multiple Sclerosis n

Future Prospects helpful discussions, and Mr. Clive Shirely, Dr. Alastair Robertson, and Dr. Maria Minor for use of their photo - Fingolimod is the first compound targeting lysophos - graphs. This review was supported in part by the NIH pholipid receptors to receive regulatory approval as a (J.C.) and Novartis (V.B., J.C.). human medicine. Its direct effects on both the immune and nervous systems via a defined class of molecular Disclosure targets, lysophospholipid S1P receptors, make it unique amongst approved MS therapies. Fingolimod also pro - J.C. has been a consultant, received honoraria or vides human validation for the efficacy and safety of research support relevant to lysophospholipids from: S1P receptor modulation in MS. Evidence for direct Amira Pharmaceuticals, Biogen-Idec, Celgene, Eli Lilly CNS activity raises the possibility that fingolimod and Co., GlaxoSmithKline, Kyorin Pharmaceutical could access mechanisms relevant to non-relapsing Corp., Mitsubishi Tanabe Pharma, Merck & Co., forms of MS, particularly PPMS or SPMS. There are Novartis, Ono Pharmaceutical Co., and Pfizer. V.B. is an currently no approved therapies for PPMS, and a Phase employee of the Novartis Institutes for BioMedical III trial has been started to assess the ability of fin - Research. golimod to improve disability progression over a 3-5 year period towards assessing its efficacy in this form References of MS. Adachi K, Kohara T, Nakao N, Arita M, Chiba K, Mishina T, Sasaki S, Fjujita T. Design, synthesis, and structure-activity relationships of Beyond MS, fingolimod and other S1P receptor modu - 2-substituted-2-amino-1,3-propanediols: discovery of a novel lating compounds could have relevance to both immunosuppressant, FTY720. Bioorg Med Chem Lett 5(8):853-856, 1995. immunological diseases as well as those of the CNS. Autoimmune disorders that may be susceptible to S1P Albert R, Hinterding K, Brinkmann V, Guerini D, Muller-Hartwieg receptor modulators include lupus, psoriasis, arthritis, C, Knecht H, Simeon C, Streiff M, Wagner T, Welzenbach K, Zecri F, Zollinger M, Cooke N, Francotte E. Novel immunomodulator and diabetes (Brinkmann, 2007; Brinkmann and Lynch, FTY720 is phosphorylated in rats and humans to form a single 2002). In particular, the effects of fingolimod on IL-17 stereoisomer. Identification, chemical proof, and biological charac - cytokine-secreting Th17 lymphocytes that reduce IL-17 terization of the biologically active species and its enantiomer. J mediated inflammatory sequelae (Mehling et al. , 2010; Med Chem 48(16):5373-5377, 2005. Zhang et al. , 2008; Zhang et al ., 2009) may be of spe - An S, Bleu T, Huang W, Hallmark OG, Coughlin SR, Goetzl EJ. cial relevance. Additionally, fingolimod’s direct action Identification of cDNAs encoding two G protein-coupled receptors on neural cells, especially astrocytes (Choi et al. , 2011), for lysosphingolipids. FEBS Lett 417(3):279-282, 1997. portend the use of fingolimod or related lysophospho - Balatoni B, Storch MK, Swoboda EM, Schonborn V, Koziel A, lipid receptor-modulatory compounds in a range of Lambrou GN, Hiestand PC, Weissert R, Foster CA. FTY720 sus - other neurological diseases. This in part reflects the tains and restores neuronal function in the DA rat model of MOG- induced experimental autoimmune encephalomyelitis. Brain Res important roles for astrocytes in most major neurologi - Bull 74(5):307-316, 2007. cal disorders that include stroke and neurodegenerative disease (Alzheimer’s and Parkinson’s disease), which Baranzini SE, Mudge J, Van Velkinburgh JC, Khankhanian P, Khrebtukova I, Miller NA, Zhang L, Farmer AD, Bell CJ, Kim RW, gives rise to the possibility of therapeutically treating May GD, Woodward JE, Caillier SJ, Mcelroy JP, Gomez R, Pando disease through the pharmacological modulation of S1P MJ, Clendenen LE, Ganusova EE, Schilkey FD, Ramaraj T, et al. receptors. More broadly, the documented effects of Genome, epigenome and RNA sequences of monozygotic twins dis - lysophospholipid signaling in the CNS, which include cordant for multiple sclerosis. Nature 464(7293):1351-1356, 2010. cell survival for myelinating (Contos et al. , 2000; Berger JR, Major EO. Progressive multifocal leukoencephalopathy. Weiner and Chun, 1999) and neural progenitor cells Semin Neurol 19(2):193-200, 1999. (Hecht et al. , 1996; Herr et al. , 2011; Kingsbury et al. , Borel JF, Kis ZL. The discovery and development of cyclosporine 2003; Yung et al. , 2011) as well as modulation of (Sandimmune). Transplant Proc 23(2):1867-1874, 1991. synaptic activity (Trimbuch et al. , 2009), suggest new approaches to treat major human diseases through Bouaziz JD, Yanaba K, Venturi GM, Wang Y, Tisch RM, Poe JC, Tedder TF. Therapeutic B cell depletion impairs adaptive and lysophospholipid receptor modulation: a first step along autoreactive CD4+ T cell activation in mice. Proc Natl Acad Sci U this path has now been taken with the introduction of S A 104(52):20878-20883, 2007. fingolimod into clinical practice for MS. Brinkmann V. Sphingosine 1-phosphate receptors in health and dis - ease: mechanistic insights from gene deletion studies and reverse Acknowledgments pharmacology. Pharmacol Ther 115(1):84-105, 2007.

The authors thank Dr. Kathie T. Hodge, Dr. Takao Brinkmann V. FTY720 (fingolimod) in Multiple Sclerosis: thera - Shimizu, Dr. Pascale Burtin, and Dr. Gordon Francis for peutic effects in the immune and the central nervous system. Br J Pharmacol 158(5):1173-1182, 2009.

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