Fingolimod the path from a fungal metabolite to fda approved drug-biom255-sp14-chun lecture ucsd pharmacology2014
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This document discusses sphingosine 1-phosphate (S1P) receptor signaling in multiple sclerosis (MS). It describes how S1P receptors signal in both the immune system and central nervous system (CNS), with effects on astrocytes in the CNS. Fingolimod, an FDA-approved drug for MS, acts as an S1P receptor modulator and has immune and CNS effects. The document provides contact information for Dr. Jerold Chun who studies lysophospholipid receptors including S1P receptors and their roles in diseases like MS.
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Fingolimod the path from a fungal metabolite to fda approved drug-biom255-sp14-chun lecture ucsd pharmacology2014
- 1. Jerold Chun, MD, Ph.D., jchun@scripps.edu ! Fingolimod: the path from a fungal metabolite to an FDA- approved drug for multiple sclerosis (MS) Lysophospholipids and Their Receptors Lysophosphatidic Acid (LPA) receptors Sphingosine 1-Phosphate (S1P) receptors S1P & fingolimod in MS Effects in the immune system Effects in the CNS involving astrocytes Molecular and Cellular Neuroscience Department Dorris Neuroscience Center The Scripps Research Institute jchun@scripps.edu Google or Yahoo: Chun Lab
- 2. Jerold Chun, MD, Ph.D., jchun@scripps.edu ! CNS mechanisms of sphingosine 1-phosphate (S1P) receptor signalling multiple sclerosis (MS)! Lysophospholipids and Their Receptors Lysophosphatidic Acid (LPA) receptors Sphingosine 1-Phosphate (S1P) receptors S1P fingolimod in MS Effects in the immune system Effects in the CNS involving astrocytes Department of Molecular Biology Dorris Neuroscience Center The Scripps Research Institute jchun@scripps.edu Google or Yahoo: Chun Lab
- 3. Jerold Chun, MD, Ph.D., jchun@scripps.edu Phospholipids, including! Sphingolipids, are ! the most abundant! brain lipid and were first! discovered in the brain! during the 19th century! ! Over 50% of the brain’s! dry weight is lipid!
- 4. Jerold Chun, MD, Ph.D., jchun@scripps.edu Lipids are best known as structural ! parts of cell membranes: neurons! glia, other cell types! ! Membrane surface area of brain: ! ~4 football fields (25,000 m2)!
- 5. Jerold Chun, MD, Ph.D., jchun@scripps.edu Phospholipids make-up the lipid membrane bilayer!
- 6. Jerold Chun, MD, Ph.D., jchun@scripps.edu O CH2OH OH NH CH3(CH2)n Sphingomyelin Sphingolipids contain Sphingosine Ceramide CH2OH OH NH2 OH P O O(CH2)2N+ (CH3)3 CH2O OH NH O CH3(CH2)n Glycerophospholipids Phosphatidic Acid CH2O OH P OH O R1 O O R2 O O CH2O OH P O O X R1 O O R2 O O
- 7. Jerold Chun, MD, Ph.D., jchun@scripps.edu Trends Mol. Med. 12, 65 (2006)! Lysophospholipids! Lysophosphatidic Acid (LPA) Sphingosine 1-phosphate (S1P)
- 8. Jerold Chun, MD, Ph.D., jchun@scripps.edu 1800s NL Vauquelan: Brain phospholipids JLW Thudichum Gobley: lecithin, cephalin, sphingomyelin from BRAIN. 1914 1960’s 1978 1989 “Signaling” Lysophospholipids Smooth muscle effects (Vogt W, Biochem Pharmacol 1963, 12:415)! Antiihypertensive (Sen S. Am J Physiol, 1968, 214:337). LPA was isolated from soybean; injection into rodents was shown to affect blood pressure (Tokumura et al. Lipids, 13:468 572). Formation of S1P in erythrocytes was reported (Stoffel. Hoppe- Seyler'sZ Physiol Chem, 351:635). 1970 G protein signaling pathways mediated by LPA (van Corven et al. Cell, 59:45). Lysophospholipids: some history Through mid-1990s Neurite retraction and electrophysiological responses to LPA or S1P (Tigyi Miledi JBC 267:21360. Jalink, et al., Cell Gr. Diff. 4:247. Postma et al., Embo J. 15:2388). 1990 Orphan GPCR Edg-1 identified (Hla and Maciag, JBC; 265:9308). S1P mobilizes Ca++ (Zhang et al. 1991, JCB 114:155).Phosphatidic Acid from Cabbage leaves (Chibnall A.C., Biochem J 1927, 21, 233). 1927
- 9. Jerold Chun, MD, Ph.D., jchun@scripps.edu Possible LP mechanisms Calcium chelator Ionophore Membrane disruptor Second messenger Intracellular receptor Extracellular receptor
- 10. Jerold Chun, MD, Ph.D., jchun@scripps.edu Identification of a first LP receptor from the brain Degenerate PCR! For GPCRS! Followed by ! In situ hybridization! Present in serum Heat stable Morphogenic Proliferative LPA1 J Cell Bio 135:1071 (1996) Cell lines! Neurite retraction! Ventricular zone cells! Ventricular zone expression! “VZG-1”! Homologous to CB1! ! !
- 11. Jerold Chun, MD, Ph.D., jchun@scripps.edu Lysophospholipid (LPA and S1P) G protein-coupled receptors (GPCRs)
- 12. Jerold Chun, MD, Ph.D., jchun@scripps.edu 1996 1998 2003 2006 LPA4 (p2y9/GPR23) a dissimilar LPA receptor (Noguchi et al. J Biol Chem, 278:22600). LPA5 (GPR92) another receptor (Kotarsky K et al. J Pharm Exp Ther, 318:619). (Lee CW et al, J Biol Chem, 281:23589) Lysophospholipids: Receptor Discoveries Other homologous receptors: S1P2,3 (An et al. FEBS Lett, 417:279). 1997 S1P1 (Edg-1) as a S1P receptor (Lee M et al. Science, 279:1552 Zondag GCM et al. Biochem J, 330:605) First LP receptor, LPA1 (VZG1) (Hecht et al J Cell Bio 135:1071). Heterologous expression using B103 and RH7777 lines (Fukushima et al., PNAS 95:6151) First knockouts: LPA1 and S1P1 (Contos et al., PNAS 97:13384; Liu et al., JCI 106:951) 2000 FTY720-P as a S1P receptor agonist (Mandala et al. Science 293:346; Brinkmann et al., JBC 277:21453). 2002 LPA6 (P2Y5) (Pasternack et al., (2008) Nat. Genet. 40: 329–34.) 2008
- 13. Jerold Chun, MD, Ph.D., jchun@scripps.edu Lysophospholipid (LP) Receptors 30% 40% 50% 60% Approximate amino acid identity Agonist LPA LPA LPA S1PSPC S1PSPC S1PSPC S1PSPC S1PSPC LPA G proteins Gi/o, G12/13, Gq Gi/o, G12/13, Gq Gi/o, Gq Gi/o Gi/o, G12/13, Gq, Gs Gi/o, G12/13, Gq, Gs Gi/o, G12/13 Gi/o, G12/13, Gs LPA1 (LPAR1) LPA2 (LPAR2) LPA3 (LPAR2) S1P1 (S1PR1) S1P3 (S1PR3) S1P2 (S1PR2) S1P5 (S1PR5) S1P4 (S1PR4) LPA4 (LPAR4) LPA5 (LPAR5) Receptor (gene*) Gene structure LPA G12/13, Gq, Gs? G12/13, Gq /i?, Gs Ann. Rev. Biochem. 73, 321 (2004) JBC 279, 20555 (2004) Ann. Rev. Pharm. Tox. 50,157 (2010) Pharmacol. Rev. 62, 579 (2010) * HUGO MGI! gene names! LPA6 ! (LPAR6) LPA Gs?
- 14. Jerold Chun, MD, Ph.D., jchun@scripps.edu Choi et al., Ann. Rev. Pharm. Toxicol. 2010! ! ! Mutoh et al., Brit. J. Pharmacol. (2012)!
- 15. Jerold Chun, MD, Ph.D., jchun@scripps.edu
- 16. Jerold Chun, MD, Ph.D., jchun@scripps.edu What are physiological and ! pathophysiological roles for ! lysophospholipid signaling?! ! “What’s it good for?”! !
- 17. Jerold Chun, MD, Ph.D., jchun@scripps.edu Lysophospholipid receptor-‐null knockout mice Mutants (yellow color = produced in-‐house white other groups) LPA1 (Contos et al., Proc. Natl. Acad. Sci. USA (2000); (GSK) LPA1 conditional (unpublished) LPA2 LPA3 (Ye et al., Nature (2005)) LPA1/A2 (Contos et al., Mol. Cell Biol., 22, 6921 (2002)) LPA1/A2/A3 (Ye et al., Biol. Reprod. 79, 328 (2008)) LPA4 (Lee et al., Mol Biol Cell 19, (2008); Sumida et al., Blood (2010) LPA5 (Lin et al., JBC (in press) Autotaxin (constitutive and conditional (W. Moolenaar; J. Aoki)) Other combinations of LPA/S1P receptor KOs S1P1 conditional (Liu et al., JCI 2000 Choi et al., PNAS 2011) S1P2 (MacLennan et al., EJN 2001 unpublished) S1P3 (Ishii et al., JBC 2001) S1P2/S1P3 (Ishii et al., JBC 2002; Herr et al., J. Neurosci. 2007) S1P1,2,3 (Kono et al., JBC 2004) and unpublished) S1P4 (Several groups, unpublished and in progress) S1P5 (Wyeth; Jaillard et al., JN, 2005; GSK, Jenne et al., JEM 2009) Sphingosine kinases (Allende et al., JBC 2004; Mizugushi et al., MCB 2005; Pappu et al., Science 2007)
- 18. Jerold Chun, MD, Ph.D., jchun@scripps.edu Inflammation fibrosis! S1P1/ S1P3 S1P2/ S1P3 S1P 1-5 S1P2 LPA2 LPA1 Survival Migration MS (EAEmodel) Lymphocyte trafficking Survival Proliferation Migration LPA1 LPA3 LPA1 LPA2 Neural development Sandhoff‘s Disease Neuropathic pain Hydrocephalus MS (EAEmodel) Schwann cells Oligodendrocytes Hearing, balance S1P1 migration of VSMCs and VECs bradycardia (S1P3) HDL vasorelaxation proliferation of VSMCs/ECs Barrier maintentance Myocyte survival and adherens junction assembly in VECs Cardiovascular system Nervous system Immune System Reproductive System Litter size Female: implantation Male: Spermatogenesis CANCER S1P1/2/3/ Respiratory System (LPA/S1P) LPA1-3 Inflamma:on fibrosis Lung, GI, Liver, Kidney (Modified from Anliker and Chun, JBC 2004)! S1P1
- 19. Jerold Chun, MD, Ph.D., jchun@scripps.edu ! CNS mechanisms of sphingosine 1-phosphate (S1P) receptor signalling multiple sclerosis (MS)! Lysophospholipids and Their Receptors Lysophosphatidic Acid (LPA) receptors Sphingosine 1-Phosphate (S1P) receptors S1P fingolimod in MS Effects in the immune system Effects in the CNS involving astrocytes Department of Molecular Biology Dorris Neuroscience Center The Scripps Research Institute jchun@scripps.edu Google or Yahoo: Chun Lab
- 20. Jerold Chun, MD, Ph.D., jchun@scripps.edu l An autoimmune disease that is the most common cause of disability in young adults, associated with neurodegeneration. l Lysophospholipid signaling occurs in most cell types relevant to MS: immunological and neural lineages. l Receptors provided a foundation to understand a fungal biochemical derivative discovered in Japan known as “FTY720,” which is now known by its generic name “fingolimod” and trade name, “Gilenya®”, “Imusera®”. Multiple Sclerosis (MS) Relevance
- 21. Jerold Chun, MD, Ph.D., jchun@scripps.edu • MS affects up to 2.5 million people worldwide1 o Caucasian people are at greater risk2 o Greater frequency in temperate la:tudes o 60-‐100 per 100,000 for Northern EU, USA, Canada, Australia and New Zealand o Up to 20 per 100,000 in Central and South America o 5 per 100,000 in Asia • Women are almost twice as likely to develop MS as men1 • MS generally affects people in the prime of their life – onset typically begins between 20-‐40 years of age3,4 MS, Multiple Sclerosis 1. World Health Organization. Neurology atlas 2004; 2. World Health Organization. Multiple sclerosis resources in the world atlas, 2008; ! 3. Noseworthy JH et al. N Engl J Med 2000; 4. Confavreux C et al. Brain 1980! Global prevalence of MS per 100,000 population2! Mul:ple sclerosis (MS): a global prevalence problem
- 22. Jerold Chun, MD, Ph.D., jchun@scripps.edu Immune and CNS mechanisms characterize MS1-3 BBB, blood-brain barrier; CNS, central nervous system; MS, multiple sclerosis.! 1. Frohman E et al. Arch Neurol. 2005;62:1345-1356; 2. Kerschensteiner M et al. J Exp Med. 2004;200:1027-1038; 3. Compston A et al. Lancet. 2002;359:1221-1231; Neuron image adapted from Waxman SG et al. N Engl J Med. 1998;338:323-325. ! BBB! MS untreated! Circulating autoaggressive lymphocytes! Demyelination! Neuro-degeneration (axonal / neuronal loss, gliosis)! Injury!CNS! Repair! INFLAMMATION! GILENYATM (fingolimod) Advisory Board CONFIDENTIAL
- 23. Jerold Chun, MD, Ph.D., jchun@scripps.edu Agent Approved Indication Betaseron® (β interferon [IFN] 1b) FDA:1993 EU: 1997 For the treatment of relapsing forms of MS (US), CIS pending For RRMS and SPMS with active disease (relapses) in EU Avonex® (β IFN 1a-intramuscular [im]) FDA: 1996 EU: 1997 For the treatment of relapsing forms of MS, and for a single clinical episode if MRI features consistent with MS are also present Copaxone® (glatiramer acetate) FDA:1996 EU: 2002 For the treatment of relapsing-remitting MS Novantrone® (mitoxantrone) FDA: 2000 not approved for MS in EU For treatment of worsening relapsing-remitting MS and for progressive-relapsing or secondary-progressive MS; 2nd line therapy Rebif® (β IFN 1a-subcutaneous [sc]) FDA: 2002 EU: 1998 For the treatment of relapsing forms of MS (US) For MS patients with 2 or more relapses in last 2 yrs (EU) Tysabri® (natalizumab) FDA: 2004* FDA EU: 2006 For the treatment of patients with relapsing forms of MS to reduce disability and slow progression; 2nd line therapy *Voluntarily withdrawn from the market on 2/28/2005 due to PML; US FDA March 2006 Advisory Panel recommended for reapproval Approved disease modifying drugs for MS
- 24. Jerold Chun, MD, Ph.D., jchun@scripps.edu Sphingosine is a major brain lipid that gives rise to sphingosine 1-phosphate (S1P) § Sphingolipids were first discovered in lipid extracts from brain by Johann Ludwig Wilhelm Thudichum in the 1870s § Named for its enigmatic function like the Sphinx of Egyptian mythology S1P, sphingosine 1-phosphate! 1. Brinkmann V et al. Curr Opin Immunol. 2002;14:569-575; 2. Anliker B et al. J Biol Chem. 2004; 279:20555-20558.!
- 25. Jerold Chun, MD, Ph.D., jchun@scripps.edu Species: (Cordyceps) Isaria sinclairii Common Name: Vegetable cicada (nymph) Found: Mixed Forest Substrate: Ground partly buried Spores: White Height: 40 mm Width: 4 mm Season: ? Edible: No Fungal Photos by Clive Shirely, with permission! New Zealand Encyclopedia! Fungal precursor (myriocin) led to fingolimod (FTY720) Winter worm, Summer grass!
- 26. Jerold Chun, MD, Ph.D., jchun@scripps.edu FTY720 (Fingolimod), derived from myriocin in Japan during studies modified fungal compounds FTY720-PFTY720
- 27. Jerold Chun, MD, Ph.D., jchun@scripps.edu Fingolimod is a sphingosine analogue that is phosphorylated and acts via sphingosine 1-phosphate (S1P) receptors Sphingolipid metabolism Ceramide Sphingosine S1P Ceraminidases Sphingosine kinases Sphingomyelinases S1P receptor O!H! N!H!2! O!H! Sphingosine O!H! O!H! N!H!2! Outside Inside Fingolimod-P / S1P! Sphingomyelin Fingolimod! Fingolimod-P! Fingolimod! Adapted from 1. Brinkmann and Lynch. Curr Opin Immunol 2002;14:569!
- 28. Jerold Chun, MD, Ph.D., jchun@scripps.edu Receptor activities of fingolimod: phosphorylation produces a nonselective S1P receptor AGONIST fingolimod-P S1P1 S1P2 S1P3 S1P4 S1P5 (0.3 nM) (3.1 nM) (0.6 nM) (0.3 nM)(10 µM) Mandala et al. Science 293:346 (2002) Brinkmann et al., JBC 277:21453 (2002)!
- 29. Jerold Chun, MD, Ph.D., jchun@scripps.edu Alterations in S1P levels in the CNS of patients with MS1 **p0.01 vs control; CSF, cerebrospinal fluid; EDSS, Expanded Disability Status Scale; NAWM, normal appearing white matter 1. Wheeler et al. Brain 2008; 2. Kulakowska A et al. Neurosci Lett 2010; 3. Van Doorn R et al. Glia 2010; 4. Van Doorn R et al. Poster P662 presented at ECTRIMS 2010
- 30. Jerold Chun, MD, Ph.D., jchun@scripps.edu FTY 720, discovered in Japan (Yoshitomi, Taito, Kyoto University) was thought to be an immunosuppressive agent. However, it did not provide immunosuppression as a mono-therapy and did not provide advantages as an adjuvant therapy (with Cyclosporine; Novartis Phase III trials for renal transplantation rejection). Could it have other uses?
- 31. Jerold Chun, MD, Ph.D., jchun@scripps.edu Clinical Stage Scoring 0 healthy mouse 1 flaccid tail 2 hind limb weakness 3 hind limb paralysis 4 fore limb paralysis (euthanize) 5 death from disease EAE: Experimental Autoimmune Encephalomyelitis, an animal model for MS!
- 32. Jerold Chun, MD, Ph.D., jchun@scripps.edu FTY720 ameliorates the EAE clinical score J. Neuroimmunol., 153, 108-121 (2004) !
- 33. Jerold Chun, MD, Ph.D., jchun@scripps.edu ! Fingolimod targets S1P receptors FDA approved (9/2010) as a first oral MS therapy, with ~65,000 patients now on treatment Acts on immune but also central nervous systems (astrocytes) ! ! ! ! Lysophospholipid (S1P) receptors as bona fide drug targets
- 34. Jerold Chun, MD, Ph.D., jchun@scripps.edu Gilenya use world-wide! 92,000!39,000!
- 35. Jerold Chun, MD, Ph.D., jchun@scripps.edu ! CNS mechanisms of sphingosine 1-phosphate (S1P) receptor signalling in animal models of multiple sclerosis (MS)! Lysophospholipids and Their Receptors Lysophosphatidic Acid (LPA) receptors Sphingosine 1-Phosphate (S1P) receptors S1P fingolimod in MS Effects in the immune system Effects in the CNS involving astrocytes Department of Molecular Biology Dorris Neuroscience Center The Scripps Research Institute jchun@scripps.edu Google or Yahoo: Chun Lab
- 36. Jerold Chun, MD, Ph.D., jchun@scripps.edu ! CNS mechanisms of sphingosine 1-phosphate (S1P) receptor signalling multiple sclerosis (MS)! Lysophospholipids and Their Receptors Lysophosphatidic Acid (LPA) receptors Sphingosine 1-Phosphate (S1P) receptors S1P fingolimod in MS Effects in the immune system Effects in the CNS involving astrocytes Department of Molecular Biology Dorris Neuroscience Center The Scripps Research Institute jchun@scripps.edu Google or Yahoo: Chun Lab
- 37. Jerold Chun, MD, Ph.D., jchun@scripps.edu Development of lymphocytes!
- 38. Jerold Chun, MD, Ph.D., jchun@scripps.edu Maturation of T and B cells!
- 39. Jerold Chun, MD, Ph.D., jchun@scripps.edu Circulation of lymphocytes!
- 40. Jerold Chun, MD, Ph.D., jchun@scripps.edu S1P signaling regulates the egress of lymphocytes from lymph nodes: involvement of S1P1 1. Am J Transplant 2004;4:1019-25; 2. Nat Immunol 2007;8:1295-301; S1P, sphingosine 1-phosphate; 3. Science 2007 316: 295-298! Blood! Efferent lymph! Lymphoid organ! S1P1 lymphocytes egress from lymphoid organs via S1P1 signalling! Lymphocyte! S1P gradient! Normal! Lymphocytes circulate between blood and lymphoid tissue!
- 41. Jerold Chun, MD, Ph.D., jchun@scripps.edu Fingolimod prevents lymphocyte egress from lymph nodes via S1P Receptor functional antagonism (this is important!) 1. Am J Transplant 2004;4:1019-25 CNS, central nervous system; S1P, sphingosine 1-phosphate FTY720-P functional antagonism of S1P1: lymphocytes are unable to egress! S1P gradient! Efferent lymph!Blood! Lymphoid organ!
- 42. Jerold Chun, MD, Ph.D., jchun@scripps.edu Receptor activities of fingolimod: phosphorylation produces a nonselective S1P receptor AGONIST fingolimod-P S1P1 S1P2 S1P3 S1P4 S1P5 (0.3 nM) (3.1 nM) (0.6 nM) (0.3 nM)(10 µM) Mandala et al. Science 293:346 (2002) Brinkmann et al., JBC 277:21453 (2002)!
- 43. Jerold Chun, MD, Ph.D., jchun@scripps.edu Fingolimod, when continuously present, down- modulates the S1P1 receptor S1P, sphingosine 1-phosphate; S1P1, sphingosine 1-phosphate receptor type 1! Model based on Brinkmann V et al. J Biol Chem. 2002;277(24):21453-21457; Matloubian M et al. Nature. 2004;427:355-360; Pham TH et al. Immunity 2008;28:122-133; Oo ML et al. J Biol Chem 2007;282:9082-9089. Müllershausen F et al. Nat Chem Biol. 2009;5:428-434.! S1P1 receptor! S1P! S1P1 signal! termination! Cell ! membrane!Fingolimod-P! Fingolimod-P! Internalises and degrades S1P1 ! S1P! Internalises and recycles S1P1! GILENYATM (fingolimod) Advisory Board
- 44. Jerold Chun, MD, Ph.D., jchun@scripps.edu ! CNS mechanisms of sphingosine 1-phosphate (S1P) receptor signalling multiple sclerosis (MS)! Lysophospholipids and Their Receptors Lysophosphatidic Acid (LPA) receptors Sphingosine 1-Phosphate (S1P) receptors S1P fingolimod in MS Effects in the immune system Effects in the CNS involving astrocytes Department of Molecular Biology Dorris Neuroscience Center The Scripps Research Institute jchun@scripps.edu Google or Yahoo: Chun Lab
- 45. Jerold Chun, MD, Ph.D., jchun@scripps.edu l S1P and S1P receptors are widespread in the CNS, and LP receptors act in the brain l A large range of observations support the existence of non-immunological mechanisms Direct CNS effects of S1P and fingolimod in MS?
- 46. Jerold Chun, MD, Ph.D., jchun@scripps.edu Fingolimod dose-dependence of peripheral blood lymphocyte levels *There were two examinations at each of weeks 2, 3 and 4 ! 1. Tedesco-Silva et al. Transplantation 2004;77:1826-33; 2. Schmouder R et al. Poster presented at ECTRIMS 2006! Fingolimod 0.5 mg! Fingolimod 2.5 mg! 0.0! 1.2! 1.4! 1.6! 1.8! Days! 1.0! 0.8! 0.6! 0.4! 0.2! Absolutelymphocyte count(109/L)! Weeks! 0! 1! 2! 3! 5! 7! 2*! 3*! 4*! 5! 10! 15!16!20! End of! treatment! End of ! study!
- 47. Jerold Chun, MD, Ph.D., jchun@scripps.edu Clinical Score Lymphopenia (%) Post-immunization Day (DPI) 20 0.5 30 DPI 29 1.5 30 Table 2. Mismatch between clinical score and lymphopenia Could there be a non-immunological perhaps neural mechanism for fingolimod efficacy? J Neuroimmunol 2004;153:108-121!
- 48. Jerold Chun, MD, Ph.D., jchun@scripps.edu Phase III data (Cohen et al., 2010) report major endpoints (e.g., ARR, time to first relapse) with better efficacy at the LOWEST (not highest) fingolimod dose that contrasts with the dose-dependent lymphocyte depletion in peripheral blood.! ! Fingolimod also reduced brain atrophy, constrasting with other immune-targeted DMTs, ! suggesting CNS effects.! ! ! ! !
- 49. Jerold Chun, MD, Ph.D., jchun@scripps.edu Fingolimod is present in the CNS following oral administra:on Foster CA et al. J Pharmacol Exp Ther. 2007;323:469-475.! Light microscopy autoradiography in rats following multiple oral doses of [14C]-fingolimod (7.5 mg/kg p.o. once a day for 7 days)! GILENYATM (fingolimod) Advisory Board
- 50. Jerold Chun, MD, Ph.D., jchun@scripps.edu S1P1 (S1pr1) is expressed in the CNS: especially on astrocytes S1P1 in situ GFAP immunolabelling Double-labelling
- 51. Jerold Chun, MD, Ph.D., jchun@scripps.edu -Structural -Metabolic -Blood-brain barrier -Neurotransmitter uptake and release -Regulation of extracellular ions -Modulation of synaptic transmission -Vasomodulation -Promotion of oligodendrocyte myelinating activity -Nervous system repair -Long-term potentiation (LTP: learning and memory) Roles for Astrocytes
- 52. Jerold Chun, MD, Ph.D., jchun@scripps.edu Control! S1P! Astrocytes (primary culture, labeled for f-actin)!
- 53. Jerold Chun, MD, Ph.D., jchun@scripps.edu l Remove S1P1 from astrocytes, but not other neural cells or lymphocytes and test by EAE l S1P1 removal should alter (reduce, if like fingolimod) EAE-relevant endpoints l S1P1 removal could reduce or eliminate the effects of fingolimod on EAE signs To assess direct CNS actions of fingolimod in MS models, use a conditional mouse mutant:
- 54. Jerold Chun, MD, Ph.D., jchun@scripps.edu GFAP In situ
- 55. Jerold Chun, MD, Ph.D., jchun@scripps.edu In situ hybridization for S1pr1 identifies astrocytes as a primary CNS locus
- 56. Jerold Chun, MD, Ph.D., jchun@scripps.edu Baseline effects of S1P1 removal on EAE: reduced disease
- 57. Jerold Chun, MD, Ph.D., jchun@scripps.edu Fingolimod efficacy is LOST after S1P1 removal from astrocyte lineages, despite continued interruption of normal lymphocyte trafficking
- 58. Jerold Chun, MD, Ph.D., jchun@scripps.edu Adoptive transfer; maintained protection of neural S1P1 loss
- 59. Jerold Chun, MD, Ph.D., jchun@scripps.edu S1P and cytokine levels are altered by S1P1 removal from astrocytes! ! !
- 60. Jerold Chun, MD, Ph.D., jchun@scripps.edu S1P1-‐dele:on from astrocytes protects CNS parenchyma CNS, central nervous system; EAE, experimental autoimmune encephalomyelitis; GFAP, glial fibrillary acidic protein; KO, knockout; S1P, sphingosine 1-phosphate; S1P1, sphingosine 1-phosphate receptor type 1! Choi et al. Proc Natl Acad Sci USA. 2011! § Deletion of S1P1 from astrocytes reduces astrogliosis (GFAP expression), demyelination and axonal damage! Astrogliosis! (GFAP)! Myelin! (Fluoromyelin)! Axons! (Neurofilament)! EAE! EAE-Neural cell S1P1 KO! Immunolabelling in lumbar region of spinal cord!
- 61. Jerold Chun, MD, Ph.D., jchun@scripps.edu Fingolimod similarly supports protec:on of CNS parenchyma consistent with S1P1 loss CNS, central nervous system; EAE, experimental autoimmune encephalomyelitis; GFAP, glial fibrillary acidic protein! Choi et al. Proc Natl Acad Sci USA. 2011! § FTY720 treatment reduces astrogliosis (GFAP expression), demyelination and axonal damage! Astrogliosis! (GFAP)! Myelin! (Fluoromyelin)! Axons! (Neurofilament)! EAE! EAE + fingolimod! Immunolabelling in lumbar region of spinal cord! GILENYATM (fingolimod) Advisory Board CONFIDENTIAL
- 62. Jerold Chun, MD, Ph.D., jchun@scripps.edu Receptor Mechanism: Functional antagonism of S1P1 Primary culture, S1P1-null astrocyte rescue experiments These data support astrocyte S1P receptors as mechanistic CNS targets for EAE and for fingolimod activity. !
- 63. Jerold Chun, MD, Ph.D., jchun@scripps.edu Immune and neural components are therapeutically accessible thru S1P signaling in EAE and likely MS Demyelination! Neuro-degeneration (axonal / neuronal loss, gliosis)! Injury! BBB! CNS! Repair! MS untreated! Circulating autoaggressive lymphocytes! INFLAMMATION! S1P1 signaling in astrocytes! S1P5 signaling in oligodendrocytes?!
- 64. Jerold Chun, MD, Ph.D., jchun@scripps.edu ! CNS mechanisms of sphingosine 1-phosphate (S1P) receptor signalling multiple sclerosis (MS)! Lysophospholipids and Their Receptors Lysophosphatidic Acid (LPA) receptors Sphingosine 1-Phosphate (S1P) receptors S1P fingolimod in MS Effects in the immune system Effects in the CNS involving astrocytes Department of Molecular Biology Dorris Neuroscience Center The Scripps Research Institute jchun@scripps.edu Google or Yahoo: Chun Lab
- 65. Jerold Chun, MD, Ph.D., jchun@scripps.edu Acknowledgements Jonathan Hecht (UCSF) Joshua Weiner (U. Iowa) James “Rocky” Contos Nobuyuki Fukushima (Kinki U., Osaka) Isao Ishii (Keio U., Tokyo) Yuka Kimura (Natl. Inst. Of Neuroscience, Tokyo) Eric Birbauer (Winthrop U.) Christine McGiffert Adrienne Dubin Chang-Wook Lee Rich Rivera Ji Woon Choi Shannon Gardell Deron Herr Rich Rivera Siew Teng (Keira) Teo Grace Kennedy Melissa Lu Kyoko Noguchi Mike Webb (Merck) Tadimeti Rao (Merck) Volker Brinkmann (Novartis) Supported by the NIH Additional support: Novartis, AG Pfizer, Inc.