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US 20140038949A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2014/0038949 A1 Schultz et al. (43) Pub. Date: Feb. 6, 2014

(54) DIRECTED DIFFERENTATION OF Publication Classification OLGODENIDROCYTE PRECURSORCELLS TO A MYELINATING CELL FATE (51) Int. Cl. A63/545 (2006.01) (75) Inventors: Peter Schultz, La Jolla, CA (US); Luke A613 L/439 (2006.01) Lairson, San Diego, CA (US); Vishal A63L/38 (2006.01) Deshmukh, La Jolla, CA (US); Costas A613 L/40 (2006.01) Lyssiotis, Boston, MA (US) (52) U.S. Cl. CPC ...... A6 IK3I/5415 (2013.01); A61K 31/40 (73) Assignees: IRM LLC, Hamilton (BM); The (2013.01); A61 K3I/439 (2013.01); A61 K Scripps Research Institute, La Jolla, 3 1/138 (2013.01) CA (US) USPC ...... 514/225.8: 514/428: 514/304: 514/653 (21) Appl. No.: 13/985,342 (57) ABSTRACT (22) PCT Filed: Feb. 17, 2012 The present invention provides methods of inducing differ (86) PCT NO.: PCT/US12/25712 entiation of oligodendrocyte progenitor cells to a mature myelinating cell fate with a modu S371 (c)(1), lating agent. The present invention also provides methods of (2), (4) Date: Oct. 18, 2013 stimulating increased myelination in a Subject in need thereof by administering said neurotransmitter receptor modulating Related U.S. Application Data agent. Methods of treating a subject having a demyelinating (60) Provisional application No. 61/444,666, filed on Feb. disease using a neurotransmitter receptor modulating agent 18, 2011. are also provided. Patent Application Publication Feb. 6, 2014 Sheet 1 of 52 US 2014/0038949 A1

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DIRECTED DIFFERENTATION OF G. Wolswijk, J Neurosci, 18, 601 (1998); A. Chang et al., N OLGODENIDROCYTE PRECURSORCELLS Engl J Med, 346, 165 (2002); T. Kuhlmann et al., , 131, TO A MYELINATING CELL FATE 1749 (2008)). 0005. There is no known cure for MS. For treating acute CROSS-REFERENCES TO RELATED inflammatory attacks, intravenous are typi APPLICATIONS cally administered. Other treatments for MS involve the 0001. The present application claims benefit of priority to administration of an immunomodulator. Although immuno U.S. Provisional Application No. 61/444,666, filed Feb. 18, modulators are able to reduce the frequency and severity of 2011, the entire content of which is incorporated by reference attacks or accumulation of lesions, they do not promote remy in its entirety. elination of damaged axons.

BACKGROUND OF THE INVENTION BRIEF SUMMARY OF THE INVENTION 0002 Multiple sclerosis (MS) afflicts approximately 400, 0006. In one aspect, the present invention provides for 000 people in the United States and 2.5 million worldwide. methods of stimulating increased myelination of nerves in a MS is an inflammatory disease in which myelin sheaths subject in need thereof. In some embodiments, the method around the axons of the brain and spinal cord are damaged. In comprises administering to the Subject a therapeutically MS as well as other demyelinating diseases, autoimmune effective dose of a neurotransmitter receptor modulating inflammatory attack against myelin and oligodendrocytes agent selected from a muscarinic , a causes demyelination. The thinning or loss of myelin Sur receptor antagonist, a antago rounding axons impairs the ability of the axons to effectively nist, a beta , and an conduct signals and results in progressive neuronal damage. receptor modulator; thereby stimulating increased myelina 0003 Remyelination is the process by which new myelin tion of nerves in the subject. sheaths are generated around axons. Remyelination persists 0007. In another aspect, the present invention provides for throughout adulthood in the CNS and involves the generation methods of treating a Subject having a demyelinating disease. of new myelinating oligodendrocytes (C. Ffrench-Constant, In some embodiments, the method comprises administering M. C. Raff, Nature, 319, 499 (1986)). Despite controversy to the subject atherapeutically effective dose of a neurotrans regarding their intrinsic in vitro and in vivo lineage potential mitter receptor modulating agent selected from a muscarinic (M. C. Nunes et al., Nat Med, 9, 439 (2003); S. Belachew et receptor antagonist, a antagonist, a hista al., J Cell Biol, 161, 169 (2003); T. Kondo, M. Raff, Science, mine receptor antagonist, a beta modula 289, 1754 (2000); Jackson, 2006: Zhu, 2011; Richardson, tor, and an modulator, thereby treating the 2011; R. J. Franklin, C. Ffrench-Constant, Nat Rev Neurosci, demyelinating disease. 9, 839 (2008)), compelling evidence indicates that a wide 0008. In yet another aspect, the present invention provides spread proliferating population of nerve/glial antigen-2 for methods of enhancing the therapeutic effect of an immu (NG2), platelet-derived growth factor receptor ( sub nomodulatory agent in a Subject in need thereof. In some unit, PDGFRa) positive cells, termed NG2-glia or oligoden embodiments, the method comprises administering to the drocyte precursor cells (OPCs), are the major source of newly Subject the immunomodulatory agent and a neurotransmitter formed mature oligodendrocytes required for remyelination receptor modulating agent; thereby enhancing the therapeutic (P. J. Homer et al., JNeurosci, 20, 2218 (2000); M. C. Nunes effect of the immunomodulatory agent. et al., Nat Med, 9,439 (2003); J. M. Gensert, J. E. Goldman, 0009. In some embodiments, the neurotransmitter recep Neuron, 19, 197 (1997); M. S. Windrem et al., Nat Med 10, tor modulating agent is a muscarinic receptor antagonist. In 93 (2004): R. J. Franklin, C. Ffrench-Constant, Nat Rev Neu Some embodiments, the muscarinic receptor antagonist is a rosci, 9,839 (2008); Richarson, 2011). muscarinic receptor modulator compound listed in Table 1. In 0004 Remyelination can occur following the loss of Some embodiments, the muscarinic receptor is selected from myelin in diseases such as MS, thus restoring neurological benztropine, carbetapentane, clemastine, ipratropium, atro function to axons. However, although remyelination can pine, and salts thereof. occur in the early stages of MS, oligodendrocytes are unable to completely rebuild the myelin sheath, and repeated inflam 0010. In some embodiments, the neurotransmitter recep matory attacks ultimately lead to fewer effective remyelina tor modulating agent is a dopamine receptor antagonist. In tions until plaques build up around the damaged axons. A Some embodiments, the dopamine receptor antagonist is a primary cause of remyelination failure is the progressive dopamine receptor modulator compound listed in Table 1. In inability of somatic oligodendrocyte precursor cells to differ Some embodiments, the dopamine receptor antagonist is entiate at the sites of injury. Thus, remission in MS is largely selected from benztropine, GBR12935, trifluoperazine, and dependent upon OPCs migrating to sites of injury, and Sub salts thereof. sequently differentiating to a mature cell fate capable of 0011. In some embodiments, the neurotransmitter recep repair (J. R. Patel, R. S. Klein, FEBS Lett, 585, 3730 (2011): tor modulating agent is a histamine receptor antagonist. In D. Kremer et al., Ann Neurol, 69,602 (2011); A. Changet al., Some embodiments, the histamine receptor antagonist is a NEngll Med, 346, 165 (2002)). Studies aimed at evaluating histamine receptor modulator compound listed in Table 1. In the presence and relative densities of OPCs at sites of chroni Some embodiments, the histamine receptor antagonist is cally demyelinated MS lesions indicate that it is not a failure clemastine or a salt thereof. of repopulation or migration of OPCs, but rather inhibition of 0012. In some embodiments, the neurotransmitter recep OPC differentiation at sites of injury that contributes to dis tor modulating agent is a beta adrenergic receptor modulator. ease progression (D. M. Chari, W. F. Blakemore, Glia, 37, In some embodiments, the beta adrenergic receptor modula 307 (2002); D. M. Charietal.J Neurosci Res, 73,787 (2003); toris a beta adrenergic receptor modulator compound listed in US 2014/0038949 A1 Feb. 6, 2014

Table 1. In some embodiments, the beta adrenergic receptor cally. In some embodiments, the method comprises adminis modulator is selected from , , Salbutamol. tering the neurotransmitter receptor modulating agent and the albuterol, and salts thereof. immunomodulatory agent sequentially. In some embodi 0013. In some embodiments, the neurotransmitter recep ments, the method comprises administering the neurotrans tor modulating agent is an opioid receptor modulator. In some mitter receptor modulating agent concurrently. embodiments, the opioid receptor modulator is an opioid 0020. In another aspect, the present invention provides for receptor modulator compound listed in Table 1. In some compositions for use in treating a subject having a demyeli embodiments, the opioid receptor modulator is carbetapen nating disease. In some embodiments, the composition com tane, Snc-80, BD-1047, or salts thereof. prises: 0014. In some embodiments, the neurotransmitter recep 0021 a neurotransmitter receptor modulating agent; tor modulating agent is benztropine, carbetapentane, clemas and tine, pindolol, ipratropium, atropine, GBR12935, Snc-80, 0022 an immunomodulatory agent. BD-1047, salmeterol, albuterol, trifluoperazine, or a salt 0023. In yet another aspect, the present invention provides thereof. In some embodiments, the neurotransmitter receptor for kits for use in treating a Subject having a demyelinating modulating agent is benztropine, clemastine, salmeterol, disease. In some embodiments, the composition comprises: salbutamol, trifluoperazine, or a salt thereof. In some embodi 0024 a neurotransmitter receptor modulating agent; ments, the neurotransmitter receptor modulating agentis ben and Ztropine or a salt thereof (e.g., benztropine mesylate). 0.025 an immunomodulatory agent. 0015. In some embodiments, the subject has a demyelinat 0026. In some embodiments, the composition or kitcom ing disease. In some embodiments, the demyelinating disease prises a neurotransmitter receptor modulating agent as is multiple Sclerosis, idiopathic inflammatory demyelinating described herein and an immunomodulatory agent as disease, transverse myelitis, Devic's disease, progressive described herein. In some embodiments, the neurotransmitter multifocal leukoencephalopathy, optic neuritis, leukoystro receptor modulating agent is selected from a muscarinic phy, Guillain-Barre Syndrome, chronic inflammatory demy receptor antagonist, a dopamine receptor antagonist, a hista elinating polyneuropathy, autoimmune peripheral neuropa mine receptor antagonist, a beta adrenergic receptor modula thy, Charcot-Marie-Tooth disease, acute disseminated tor, and an opioid receptor modulator. In some embodiments, encephalomyelitis, adrenoleukodystrophy, adrenomyelo the neurotransmitter receptor modulating agent is benz neuropathy, Leber's hereditary optic neuropathy, or tropine, clemastine, Salmeterol, Salbutamol, trifluoperazine, T-cell lymphotropic virus (HTLV)-associated myelopathy. In or a salt thereof. In some embodiments, the neurotransmitter Some embodiments, the demyelinating disease is multiple receptor modulating agent is benztropine or a salt thereof. In Sclerosis. In some embodiments, the demyelinating disease is Some embodiments, the immunomodulatory agent is fingoli relapsing-remitting multiple sclerosis (RRMS). In some mod (FTY720), interferon beta-1a, interferon beta-1b, glati embodiments, the demyelinating disease is secondary pro ramer acetate, mitoxantrone, or natalizumab. In some gressive multiple sclerosis (SPMS). In some embodiments, embodiments, the neurotransmitter receptor modulating the demyelinating disease is primary progressive multiple agent is benztropine, clemastine, Salmeterol, Salbutamol, tri sclerosis (PPMS). In some embodiments, the demyelinating fluoperazine, or a salt thereof and the immunomodulatory disease is progressive relapsing multiple sclerosis (PRMS). agent is fingolimod (FTY720), interferon beta-1a, or inter 0016. In some embodiments, the subject is a human. In feron beta-1b. Some embodiments, the Subject is a non-human mammal. 0027. In some embodiments, the neurotransmitter recep 0017. In some embodiments, the method further com tor modulating agent is formulated as a therapeutically effec prises administering to the Subject an immunomodulatory tive or optimal dose and the immunomodulatory agent is agent. In some embodiments, the immunomodulatory agent formulated as a therapeutically effective or optimal dose. In is fingolimod (FTY720), interferon beta-1a, interferon beta Some embodiments, the neurotransmitter receptor modulat 1b, glatirameracetate, mitoxantrone, or natalizumab. In some ing agent is formulated as a therapeutically effective or opti embodiments, the immunomodulatory agent is fingolimod mal dose and the immunomodulatory agent is formulated as a (FTY720), interferon beta-1a, or interferon beta-1b. Subtherapeutic dose. In some embodiments, the immuno 0018. In some embodiments, the method comprises modulatory agent is formulated as a therapeutically effective administering to the subject a therapeutically effective or or optimal dose and the neurotransmitter receptor modulating optimal dose of one or both of the neurotransmitter receptor agent is formulated as a Subtherapeutic dose. In some modulating agent and the immunomodulatory agent. In some embodiments, the neurotransmitter receptor modulating embodiments, the method comprises administering to the agent is formulated as a subtherapeutic dose and the immu subject a subtherapeutic dose of one or both of the neurotrans nomodulatory agent is formulated as a subtherapeutic dose. mitter receptor modulating agent and the immunomodulatory 0028. In yet another aspect, the present invention also agent. In some embodiments, the method comprises admin provides for use of a composition as described herein for the istering to the subject a therapeutically effective dose or opti manufacture of a medicament for the treatment of a demyeli mal dose of the neurotransmitter receptor modulating agent nating disease. and a subtherapeutic dose of the immunomodulatory agent. In Some embodiments, the method comprises administering to DEFINITIONS the subject a therapeutically effective dose or optimal dose of 0029. As used herein, the term “neurotransmitter receptor the immunomodulatory agent and a Subtherapeutic dose of modulating agent” refers to an agent that inhibits or activates the neurotransmitter receptor modulating agent. the activity of a neurotransmitter receptor. In some embodi 0019. In some embodiments, the method comprises ments, the term refers to a compound that modulates the administering one or both of the neurotransmitter receptor activity of a muscarinic receptor (e.g., a muscarinic receptor modulating agent and the immunomodulatory agent systemi antagonist), a dopamine receptor (e.g., a dopamine receptor US 2014/0038949 A1 Feb. 6, 2014 antagonist), a histamine receptor (e.g., a histamine receptor nervous system or a disease affecting the peripheral nervous antagonist), a beta adrenergic receptor (e.g., a beta adrenergic system. Examples of demyelinating diseases include, but are receptor antagonist), or an opioid receptor (e.g., an opioid not limited to, multiple Sclerosis, idiopathic inflammatory receptor modulator). For any compound that is identified as a demyelinating disease, transverse myelitis, Devic's disease, neurotransmitter receptor modulating agent (e.g., a com progressive multifocal leukoencephalopathy, optic neuritis, pound described in Table 1 herein), it is also contemplated leukoystrophy, Guillain-Barre Syndrome, chronic inflamma that any pharmaceutically acceptable salts, , racemic tory demyelinating polyneuropathy, autoimmune peripheral mixtures, conformational and/or optical isomers, crystalline neuropathy, Charcot-Marie-Tooth disease, acute dissemi polymorphs and isotopic variants of the compound may also nated encephalomyelitis, adrenoleukodystrophy, adrenomy be used. In some embodiments, the neurotransmitter receptor eloneuropathy, Leber's hereditary optic neuropathy, or modulating agent is a small molecule, e.g., a molecule having HTLV-associated myelopathy. In some embodiments, the a molecular weight of less than 800 kDa. In some embodi demyelinating disease is multiple Sclerosis. ments, the neurotransmitter receptor modulating agent is a 0033. As used herein, the term “subject” refers to animals small molecule that is able to cross the blood-brain barrier. Such as mammals, including, but not limited to, primates 0030. As used herein, the term "oligodendrocyte precur (e.g., ), cows, sheep,goats, horses, dogs, cats, rabbits, sor cell' or “OPC refers to an undifferentiated progenitor , mice and the like. In some embodiments, the Subject is a cell with the capacity to self-renew and differentiate into a human. myelinating oligodendrocyte. A "mature myelinating cell 0034. As used herein, the term “compound” refers to any fate' refers to cell that is capable of forming myelin, e.g., a molecule, either naturally occurring or synthetic, e.g., pep myelinating oligodendrocyte. “Differentiation” refers to the tide, protein, oligopeptide (e.g., from about 5 to about 50 process by which a specialized cell type is formed from a less amino acids in length), Small organic molecule, polysaccha specialized cell type, for example, a myelinating oligoden ride, , circular peptide, peptidomimetic, lipid, fatty drocyte from an OPC. In some embodiments, an OPC is acid, siRNA, polynucleotide, oligonucleotide, etc., to be identified by morphology and/or by the presence of a biom tested for the capacity to induce OPC differentiation. The arker, e.g., PDGFR-C. or NG2. In some embodiments, a compound to be tested can be in the form of a library of test myelinating oligodendrocyte is identified by morphology compounds, Such as a combinatorial or randomized library and/or by the presence of a marker, e.g., myelin basic protein that provides a sufficient range of diversity. Test compounds (MBP), myelin oligodendrocyte glycoprotein (MOG), 23'- are optionally linked to a fusion partner, e.g., targeting com cyclic-nucleotide 3' phosphodiesterase (CNP), galactocebro pounds, rescue compounds, dimerization compounds, stabi side (GalC), O1 antigen (O1), or O4 antigen (O4). lizing compounds, addressable compounds, and other func 0031. As used herein, the terms “stimulating increased tional moieties. Conventionally, compounds are screened by myelination' or “stimulate increased myelination” refer to identifying a test compound (called a 'screening hit) with inducing an increased amount of myelin Surrounding an axon, Some desirable property or activity, e.g., inducing activity, e.g., by administering an agent that induces the differentiation and Screening hits are confirmed and validated using in vitro of oligodendrocyte precursor cells to a mature myelinating and in vivo assays. Often, high throughput screening (HTS) cell fate, as compared to the amount of myelin Surrounding methods are employed for Such an analysis. the axon in the absence of the agent being administered. In 0035 An “” refers to an agent that stimulates, Some embodiments, an agent stimulates "increased myeli increases, activates, or enhances activation of a neurotrans nation when the amount of myelin Surrounding the axon in a mitter receptor (e.g., muscarinic receptor, dopamine receptor, sample (e.g., a brain tissue sample from a Subject having a histamine receptor, beta adrenergic receptor, and/or opioid demyelinating disease) Subsequent to administration of an receptor) of the invention. agent that induces the differentiation of OPCs to a mature 0036 An “antagonist” refers to an agent that partially or myelinating cell fate is at least about 5%, 10%, 15%, 20%, totally blocks stimulation, decreases, prevents, inactivates, or 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more as compared delays activation of a neurotransmitter receptor (e.g., musca to the amount of myelin Surrounding the axon in the sample rinic receptor, dopamine receptor, histamine receptor, beta prior to administration of the agent. The amount of myelin adrenergic receptor, and/or opioid receptor) of the invention. Surrounding an axon can be measured by any method known 0037. As used herein, the terms “therapeutically effective in the art, e.g., using magnetic resonance imaging (MRI). In amount or dose' or “therapeutically sufficient amount or Some embodiments, an agent stimulates increased myelina dose” or “effective or sufficient amount or dose” refer to a tion when one or more characteristics of a demyelinating dose that produces therapeutic effects for which it is admin disease (e.g., multiple Sclerosis) improves Subsequent to istered when it is administered on its own. The exact dose will administration of an agent that induces differentiation of depend on the purpose of the treatment, and will be ascertain OPCs to a mature myelinating cell fate as compared to the able by one skilled in the art using known techniques (see, characteristic of the diseases prior to administration of the e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, agent. As a non-limiting example, an agentis said to stimulate 1992); Lloyd, The Art, Science and Technology of Pharma increased myelination in a Subject having multiple Sclerosis ceutical Compounding (1999); Pickar, Dosage Calculations when the frequency and/or severity of inflammatory attacks (1999); and Remington. The Science and Practice of Phar decreases Subsequent to administration of an agent as com macy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams pared to the frequency and/or severity of inflammatory & Wilkins). attacks prior to administration of the agent. 0038. As used herein, the terms “administer” or “admin 0032. As used herein, the term “demyelinating disease s istering refer to any type of administration, including but not refers to a disease or condition of the nervous system charac limited to oral administration, administration as a Supposi terized by damage to or loss of the myelin sheath of neurons. tory, topical contact, parenteral, intravenous, intraperitoneal, Ademyelinating disease can be a disease affecting the central intramuscular, intralesional, intranasal or Subcutaneous US 2014/0038949 A1 Feb. 6, 2014

administration, intrathecal administration, or the implanta MOG expression was quantified using specific probes and tion of a slow-release device e.g., a mini-osmotic pump, to the Taqman-based qRT-PCR. Expression was normalized to the Subject. internal controls f-actin and GAPDH. Fold change in gene 0039. As used herein, the terms “treat” or “treating” or expression over DMSO treated control cells is plotted for “treatment” refer to any indicia of success in the treatment or MBP (B) and MOG (C). Results are displayed as mean+/- amelioration of an injury, pathology, condition, or symptom standard deviation, n=3. (e.g., pain), including any objective or Subjective parameter 0043 FIG. 3. Immunofluorescence analysis of compound Such as abatement; remission; diminishing of symptoms or treated OPCs using mature oligodendrocyte specific markers. making the symptom, injury, pathology or condition more Immunofluorescence staining using specific antibodies for tolerable to the patient; decreasing the frequency or duration MBP, MOG, and 2',3'-cyclic nucleotide 3' phosphodiesterase of the symptom or condition; or, in some situations, prevent (CNP). Nuclei were identified using DAPI. Compound treat ing the onset of the symptom or condition. The treatment or ment of OPCs at EC concentrations for 6 days induces the amelioration of symptoms can be based on any objective or robust expression of mature oligodendrocyte markers. Subjective parameter, including, e.g., the result of a physical 0044 FIG. 4. Immunofluorescence analysis of compound examination. treated OPCs using mature oligodendrocyte specific markers. 0040. As used herein, the term “subtherapeutic dose' Immunofluorescence staining using specific antibodies for refers to a dose of a pharmacologically active agent(s), either galactocereberoside (GalC), oligodendrocyte marker O1 as an administered dose of pharmacologically active agent, or (O1) and oligodendrocyte marker O4(O4). Nuclei were iden actual level of pharmacologically active agent in a subject, tified using DAPI. Compound treatment of OPCs at EC that functionally is insufficient to elicit the intended pharma concentrations for 6 days induces the robust expression of cological effect in itself, or that quantitatively is less than the mature oligodendrocyte markers. established therapeutic dose for that particular pharmacologi 0045 FIG. 5. Representative neurotransmitter receptor cal agent (e.g., as published in a reference consulted by a modulating agents benzatropine and trifluoperazine amelio person of skill, for example, doses for a pharmacological rate symptoms in a PLP induced relapsing EAE mouse agent published in the Physicians' Desk Reference, 66th Ed., model. SJL mice were immunized with proteolipid peptide 2012, PDR Network, LLC; or Brunton, et al., Goodman & (PLP) and pertussis toxin and monitored daily with scoring Gilman's The Pharmacological Basis of Therapeutics, 12th on the standard clinical EAE scale (0-5). Compounds were edition, 2011, McGraw-Hill Professional) when adminis administered daily for the duration of the study in saline at 10 tered on its own. A “subtherapeutic dose” can be defined in mg/kg via intra-peritoneal injection (0.1 ml), starting on the relative terms (i.e., as a percentage amount (less than 100%) day of the appearance of clinical EAE Symptoms (day 10). of the amount of pharmacologically active agent convention Sub-optimal dosing of mycophenolate mofetil at 20 mg/kg in ally administered). For example, a Subtherapeutic dose sterile saline (pH 5) was administered in combination with amount can be about 1% to about 75% of the amount of benZatropine and trifluoperazine respectively, starting on the pharmacologically active agent conventionally administered. day of peak clinical EAE symptoms (day 14) post PLP injec In some embodiments, a subtherapeutic dose can be about tion. Mean clinical EAE score and standard error of mean 75%, 50%, 30%, 25%, 20%, 10% or less, of the amount of (SEM) are shown for each study group. Mice in the vehicle pharmacologically active agent conventionally administered. control group, n=6 (FIG.5A-E, open boxes) show appearance ofacute phase (mean maximal clinical EAE score of 2+0.8 on BRIEF DESCRIPTION OF THE DRAWINGS day 11) followed by remission (mean maximal clinical EAE 0041 FIG. 1. Neurotransmitter receptor modulating score of 0.3-0.3 on day 19) and relapse of EAE symptoms agents dose dependently induce OPC differentiation. (A) (mean maximal clinical EAE score of 1.5+0.4 on day 25). Chemical structures of representative identified OPC differ Mycophenolate mofetil, n=6 (FIG. 5A closed boxes) shows entiating molecules. The calculated ECso values were deter partial reduction in the severity of relapse (mean maximal mined using appropriate Graphpad Prism 5.0 curve fitting EAE score 0.7+0.3 on day 25, p value <0.05). Benzatropine, software. (B) Dose dependent compound induced OPC dif n=5 (FIG.5B closed boxes) and trifluoperazine, n=6 (FIG.5D ferentiation. OPCs were plated in 384 well plates at 1000 cells closed boxes) show a significant reduction in the severity of per well in OPC media containing basal PDGF-C.C. (2 ng/mL) relapse (mean maximal clinical EAE score 0.2-0.2 (day 25) and treated with serial dilutions of compounds. Following six and 0.4+0.4 (day 26) respectively, p values <0.001). Benzat days of compound treatment, cells were fixed and Subjected ropine and trifluoperazine in combination with mycopheno to immunofluorescence analysis using anti-myelin basic pro late mofetil, n-6 for both (FIG.5C and FIG. 5E respectively, tein (MBP) antibody. Image acquisition and quantification of closed boxes) show complete Suppression of relapse (mean MBP staining was performed using the OPERA imaging maximal clinical EAE score 0+0 for both on day 25, p values system. (C) Maximal OPC differentiation inducing activity of <0.001). identified compounds compared to DMSO control. 0046 FIG. 6. Muscarinic receptor antagonism induces 0042 FIG. 2. Neurotransmitter receptor modulating OPC differentiation but is not the sole pharmacological agents induce the differentiation of OPCs to a myelinating mechanism of all identified differentiation inducing agents. oligodendrocyte cell fate. (A) Western blot analysis of OPCs (A) The muscarinic receptor agonist carbachol antagonizes treated with compounds at EC concentrations for 6 days. compound induced OPC differentiation in some cases. OPCs Total protein was isolated from cell pellets and probed for were plated in 384-well plates at 1000 cells per well in media myelin basic protein (MBP) and myelin oligodendrocyte gly containing compounds at EC concentrations and treated coprotein (MOG) using specific antibodies. (B-C) Quantita with 1:3 serial dilutions of carbachol. Following 6 days of tive RT-PCR (qRT-PCR) analysis of OPCs treated with com treatment, cells were fixed and stained for MBP. Plates were pounds at EC concentrations for 6 days. Total RNA was imaged using the OPERA high content Screening system and isolated from cell pellets and reverse transcribed. MBP and MBP staining was quantified as described. Carbachol treat US 2014/0038949 A1 Feb. 6, 2014

ment results in dose dependent inhibition of the differentia 0050 FIG. 10. Combination with benztropine improves tion activity of benzatropine, carbapentane and clemastine, efficacy and allows for a reduction in the dose of FTY720 and while no effect is observed on the differentiation activity of interferon-?3. (A) Clinical EAE scores for mice treated with salmeterol, GBR12935 and trifluoperazine. (B) Carbachol FTY720 (1 mg/kg) in combination with benztropine (BA; 2.5 induced calcium (Ca") influx is blocked by the muscarinic mg/kg) show a significantly decreased clinical severity as receptor antagonist activity of some compounds. OPCs were compared to mice treated with FTY720 (1 mg/kg) or benz plated in 384-well plates at 5000 cells per well in media tropine (2.5 mg/kg) alone. (B) Clinical EAE scores for mice containing basal PDGF-C. Cells were equilibrated with Ca" treated with Interferon-B (IFN: 10,000 U/mouse) in combi sensitive Fluo-3AMR) dye in HBSS for 30 min. Multiple nation with benztropine (BA, 2.5 mg/kg) show a significantly concentrations of OPC differentiation-inducing compounds decreased clinical severity as compared to mice treated with (3 times the EC, EC and ECs) were added followed by Interferon-Y (IFN: 10,000 U/mouse) or Benztropine (2.5 addition of 1:3 serial dilutions of the muscarinic receptor mg/kg) alone. (C) Clinical EAE scores for mice treated with agonist Carbachol. Ca" influx was immediately measured FTY720 (0.1 mg/kg) or FTY720 (0.1 mg/kg) or benztropine using the FLIPRTETRAR) system. Shown are representative (BA; 2.5 mg/kg). (D) Clinical EAE scores for mice treated data for carbachol treatment at 100 uM and compound treat with interferon-B (IFN: 3000U/mouse) or interferon-?3(IFN: ment at EC Relative light units (RLU) indicating Ca" 10,000 U/mouse) or benztropine (BA; 2.5 mg/kg). (E) Clini influx are plotted on the y-axis and time (in sec) after addition cal EAE scores for mice treated with FTY720 (0.1 mg/kg) in of carbachol plotted on the x-axis. GBR12935, trifluopera combination with benztropine (BA, 2.5 mg/kg) show a com Zine, and salmeterol (top) show no effect on carbachol parable decrease in clinical severity Scores as compared to induced Ca" influx while clemastine, benzatropine, and car FTY720 (1 mg/kg), facilitating the reduction in the dose of bapentane (bottom) inhibit Ca" influx induced by carbachol. FTY720. (F) Clinical EAE scores for mice treated with inter 0047 FIG. 7. A high throughput screen identified musca feron-B (IFN: 3000 U/mouse) in combination with benz rinic receptor antagonist benztropine as an inducer of OPC tropine (BA, 2.5 mg/kg) do not show a comparable decrease differentiation. (A) Benztropine 1.5uM and positive control in clinical severity scores as interferon-?3 (IFN: 10,000 thyroid hormone 1 uM treated OPCs were cultured under U/mouse). Error bars indicate standard deviation of the mean basal differentiation conditions (2 ng/ml PDGF) for 6 days within each group of 8 mice. and stained for MBP (green). The structure of benztropine is 0051 FIG. 11. Screen to identify inducers of OPC differ also shown. (B) Benztropine 1.5 uM treated OPCs were entiation. (A) OPCs were maintained as proliferating A2B5 analyzed for MBP and MOG expression after 6 days in cul positive cells under basal growth conditions (Neurobasal ture using qRT-PCR. (C) OPCs were co-treated with benz medium, B27 supplement without Vitamin A, non-essential tropine 2.3 uM and carbachol OuM, 0.6 uMand 4.7uM for amino acids, L-Glutamine, 30 ng/ml PDGF). OPCs were 6 days under basal differentiation conditions and stained for plated in basal differentiation media (Neurobasal medium, MBP. B27 supplement without Vitamin A, non-essential amino acids, L-Glutamine, 2 ng/ml PDGF), treated with DMSO 0048 FIG. 8. Benztropine decreases the clinical severity (<0.1%) or thyroid hormone (1 uM), fixed after 6 days in of disease in the PLP induced EAE model for MS. (A) Ben culture, and stained using antibodies for CNP. O4, or MBP. Ztropine decreased the clinical severity of disease in the PLP A2B5-positive OPCs differentiate into immature oligoden induced EAE model when dosed prophylactically (starting on drocytes that express CNP and O4, but not MBP, upon with the day of PLP injection) as well as therapeutically (at the drawal of PDGF. Addition of thyroid hormone induces the start of EAE symptoms) and showed efficacy comparable to differentiation of OPCs into mature oligodendrocytes that FTY720 (1 mg/kg) and interferon-B (10,000 U/mouse). (B) express MBP (B) Screen strategy using A2B5 positive OPCs Quantification of confocal images of spinal cord sections cultured under basal differentiation conditions and treated from EAE mice treated with benztropine and stained with with compounds for 6 days to identify small molecules that specific antibodies for GST-It (a marker of mature oligoden induce the differentiation of OPCs to mature, MBP express drocytes) and NG2 (a marker of OPCs) showed increased ing oligodendrocytes. GST-IL positive cells as compared to vehicle treated mice, 0052 FIG. 12. Primary hit confirmation. (A) Dose with no change in the number of NG2 positive cells. (C) response assay used to confirm primary screening hits and Representative confocal images of spinal cord sections from determine (ECs). (ECs) is defined as the concen EAE mice treated with benztropine and stained with specific tration that results in a half maximal increase in the percent antibodies for GST-It (mature oligodendrocytes) and NG2 age of total cells that express MBP as detected by immun (OPCs). ostaining OPCs were cultured in differentiation medium and 0049 FIG. 9. Benztropine treatment induces remyelina treated with benztropine or control (DMSO <0.01%) for 6 tion in vivo in the cuprizone model. (A) Representative days. Cells were fixed and immunostained using antibodies images from the corpus callosum region of the brain at vari for MBP. Error bars represent standard deviations from 3 ous time points show increased remyelination observed in replicate experiments. (B) Representative images show dose benZatropine treated mice as compared to vehicle treated dependent activity of benztropine. mice 2 weeks after cuprizone withdrawal and commence 0053 FIG. 13. Benztropine induces differentiation of ment of administration. (B) Quantification of the myeli OPCs to mature oligodendrocytes. OPCs were plated in dif nated areas in the corpus callosum region shows a significant ferentiation medium (Neurobasal medium, B27 supplement (~2-fold) increase in myelin staining in benztropine treated without Vitamin A, non-essential amino acids, L-Glutamine, mice as compared vehicle controls 2 weeks after drug treat 2 ng/ml PDGF) and treated with DMSO (<0.01%), benz ment. Data is represented in terms of threshold bins on the tropine 1.5 uM or thyroid hormone 1 uM. After 6 days in grey scale (0-50) as described. Error bars represent standard culture, cells were analyzed for MBP and MOG expression deviations of at least 6 corpus callosum regions. by Western blot. US 2014/0038949 A1 Feb. 6, 2014

0054 FIG. 14. Compound treatment induces the differen Vitamin A, non-essential amino acids, L-Glutamine, 2 ng/ml tiation of OPCs to mature oligodendrocytes. OPCs were PDGF) and co-treated with various concentrations of benz plated in differentiation medium (Neurobasal medium, B27 tropine and the dopamine receptor (A) agonist quinpirole or Supplement without Vitamin A, non-essential amino acids, (B) antagonist . Error bars indicate standard L-Glutamine, 2 ng/ml PDGF) and treated with DMSO (<0. deviations of 3 replicate measurements. 01%), benztropine 1.5uM or thyroid hormone 1 uM for 6 0061 FIG. 21. Muscarinic antagonists induce differentia days. Cells were fixed and immunostained for MBP, MOG, tion of OPCs to oligodendrocytes. OPCs were plated in dif CNP. GalC, O1, or O4. Representative images of DMSO, ferentiation medium (2 ng/ml PDGF) and treated with various benztropine and thyroid hormone treated cells show expres concentrations of compounds for 6 days. Cells were fixed and sion of mature oligodendrocyte markers in benztropine and immunostained for MBP. Selective muscarinic antagonists thyroid hormone treated cells, but not in DMSO treated cells. oxybutynin, atropine, ipratropium, propiverine, and Scopola 0055 FIG. 15. Gene expression profile of OPCs differen mine induced differentiation of OPCs in a dose dependent tiated to oligodendrocytes by benztropine treatment. OPCs manner. ECso values for compound induced differentiation were plated in differentiation medium and treated with are indicated. DMSO (<0.1%), benztropine 2.3 uMorthyroid hormone 1 0062 FIG. 22. OPCs express muscarinic receptors and uM for 6 days. OPCs from the same passage were also acetyl transferase. Total RNA was isolated from pelleted and frozen. Total RNA was isolated from the cells OPCs treated with DMSO (<0.1%) or thyroid hormone 1 and gene expression analysis was performed using rat uM for 6 days, or from whole rat brain. RNA was reverse genome arrays from Affymetrix. Global clustering analysis of transcribed to cDNA and gene expression of muscarinic mRNA expression probe sets that displayed a >2-fold change receptors M. M., or M was detected by PCR using gene in expression across samples shows clustering of benztropine specific primers. treated samples with thyroid hormone treated samples while 0063 FIG. 23. Antagonism of M/M muscarinic signal gene expression profiles of DMSO treated cells cluster with ing pathway by benztropine primes OPCs for differentiation. OPCs. Data are represented as fold change over DMSO (A) OPCs were treated with benztropine 10 uM for the treated controls. Increased expression of mature oligodendro indicated times and pelleted for Western blot analysis of total cyte genes was seen in compound treated cells as compared to protein. Benztropine inhibits signaling proteins downstream DMSO treated cells. of M/M muscarinic receptors by down-regulating phospho 0056 FIG. 16. Gene expression profiles of benztropine rylation of Akt, p42MAP Kinase, and increasing phosphory treated cells show a downregulation of OPC genes and an lation of p38 MAP Kinase and CREB. (B) OPCs were plated upregulation of mature oligodendrocyte genes. (A) Increased in basal differentiation conditions and treated with benz expression (fold change) of mature oligodendrocyte genes in tropine 1.5 uM), thyroid hormone 1 uM or DMSO (<0. compound treated cells as compared to DMSO treated cells. 1%). OPCs from the same passage were also pelleted and (B) Decreased expression (fold change) of OPC genes in frozen. Total RNA was isolated from the cells, reverse tran compound treated cells as compared to DMSO treated cells. scribed to first strand cDNA and used as a template for qRT 0057 FIG. 17. Timing of compound treatment determines PCR. Gene specific FAM labeled probes were used to detect the efficiency of differentiation. (A) OPCs were plated in expression levels of various genes, with probes for beta-actin differentiation medium on day 0 and treated with compounds and GAPDH as internal controls. Gene expression shows a on various days. Cells were fixed and immunostained for downregulation in cell cycle genes such as Cyclin D1, Cyclin MBP on day 6 after plating. (B) OPCs were plated in differ D2, c-Fos, c-Jun indicating an exit from cell cycle. (C) Sig entiation medium on day 0 and treated with compounds 12 naling pathway downstream of the M/M muscarinic recep hours later. Cells were fixed on various days following com tOrS. pound treatment and immunostained for MBP (C) Treatment 0064 FIG. 24. Benztropine antagonizes the M/M mus with benztropine within 48 hours of plating in differentiation carinic receptors but not the M/M muscarinic receptors. medium induced efficient differentiation of OPCs to mature OPCs were plated in differentiation media for 12 hours. oligodendrocytes. Compound treatment 72 hours or more Media was changed to Hank's Balanced Salt Solution after plating in differentiation medium reduced the efficiency (HBSS) with 4-(2-hydroxyethyl)-1-piperazineethane of differentiation of OPCs. (D) Benztropine treatment for a sulfonic acid (HEPES) and cells were treated with benz minimum of 5 days was necessary to induce efficient differ tropine at various concentrations for 1 hour. Carbachol was entiation of OPCs to mature oligodendrocytes. added and calcium flux was measured for 186 seconds using 0058 FIG. 18. Carbachol antagonizes benztropine the FLIPRTETRAR) system. (A) Carbachol induced a dose induced OPC differentiation. OPCs were plated in basal dif dependent increase in intracellular Ca" levels. (B) Benz ferentiation media and co-treated with benztropine 2.3 uM tropine dose dependently blocked carbachol induced calcium and carbachol OuM, 0.6 uM or 4.7uM for 6 days and stained influx through antagonism of M/M muscarinic receptors. for MBP (green). (C) Atropine, a , serves as a positive 0059 FIG. 19. Benztropine has no effect on histamine control. (D) OPCs were plated in differentiation medium for receptor signaling. OPCs were plated in basal differentiation 12 hours. A cAMP-HTRF assay was performed using the medium (Neurobasal medium, B27 supplement without Vita cAMP dynamic 2 kit. Benztropine had no effect on the levels min A, non-essential amino acids, L-Glutamine, 2 ng/ml of cAMP, IBMX was added as a cAMP stabilizer, and for PDGF) and co-treated with various concentrations of benz Skolin was a positive control. tropine and (A) histamine or (B) the histamine receptor ago 0065 FIG. 25. Dose dependent activity of prophylactic nist histamine trifluoromethyltoluidide (HTMT). benztropine in the EAE model. EAE was induced in mice 0060 FIG. 20. Benztropine has no effect on dopamine D2 using PLP and pertussis toxin. Benztropine dissolved in and D3 receptor signaling. OPCs plated in basal differentia saline at various doses was injected via I.P. injection daily tion medium (Neurobasal medium, B27 supplement without using the prophylactic mode followed by scoring clinical US 2014/0038949 A1 Feb. 6, 2014 symptoms. Prophylactic dosing is defined as administration no effect on cytokine production measured as the number of of compound commenced on the day of PLP injection. Error IL2 producing CD4 T-cells (G), number of IL 10 producing bars indicate standard deviation of the mean within each CD4"T-cells (H), number of TNF-Y producing CD4"T-cells group of 8 mice. and number of IFN-Y producing CD4 T-cells. Error bars 0066 FIG. 26. Benztropine treatment induces remyelina indicate standard deviation of the mean within each group of tion in vivo in the PLP induced EAE models for MS. BenZ 5 mice. tropine does not block lymphocyte infiltration in EAE mice, (0070 FIG. 30. Benztropine does not suppress T-cell but leads to significantly increased LFB staining, indicating dependent and independent immune responses. Mice were the presence of myelin in areas infiltrated by lymphocytes as injected with Keyhole Limpet Hemocyanin protein conju compared to vehicle treated mice. EAE was induced in mice gated to 2,4,6, trinitrophenylhapten (TNP-KLH). by injecting PLP and pertussis toxin and the mice were treated lipopolysaccharide conjugated to 2,4,6, trinitrophenylhapten with benztropine (10 mg/kg) or vehicle controls at the first (TNP-LPS), or TNP (2,4,6-Trinitrophenyl)-FICOLL conju appearance of EAE Symptoms. Spinal cords were isolated gate (TNP-Ficoll) in appropriate adjuvants and treated with from mice representative of the average group scores during vehicle or benztropine (10 mg/kg). Serum was isolated at the relapse phase of EAE, sectioned and stained with Luxol various time points and IgG and IgM levels were measured by Fast Blue and H&E (left panel) and Luxol Fast Blue only ELISA. (A, B) Benztropine showed no effect on TNP-LPS (right panel). Arrows point to regions of lymphocyte infiltra induced T-cell independent B-cell responses measured as tion. serum IgM and IgG levels. (C, D) Benztropine showed no 0067 FIG. 27. Benztropine has no effect on T-cell activa effect on TNP-Ficol induced T-cell independent B-cell tion and proliferation in vitro. Total splenocytes were isolated responses measured as serum IgM and IgG levels. (E., F) from mice and stimulated with CD3 and analyzed by flow Benztropine showed no effect on TNP-KLH induced T-cell cytometry for expression of T-cell activation markers CD69 dependent B-cell responses measured as serum IgM and IgG and CD25 and T-cell proliferation using CFSE. (A) Unstimu levels. Error bars represent standard deviations from 3 repli lated cells. (B) DMSO treated cells. (C) Mycophenolate Sup cate ELISAs performed on samples from 5 mice in each presses the activation and proliferation of T-cells as compared treatment group. to DMSO. (D) Benztropine has no effect on T-cell activation. 0071 FIG. 31. Quantification of myelination staining in (E) Mycophenolate suppresses T-cell proliferation. (F) Ben the cuprizone model. (A) Luxol Fast Blue staining was per Ztropine has no effect on T-cell proliferation. The numbers formed on sections from the corpus callosum region of the represent percentage gated populations positive for the given isolated from mice treated either with benztropine (10 marker. mg/kg) or vehicle control after 7 weeks of exposure to cupri 0068 FIG. 28. Benztropine shows no immunosuppressive Zone. (B) Images were converted to a 256 shade grey Scale. effects in vivo after induction of EAE in mice. EAE was (C) The 256 shades of grey were divided into 5 bins of 50 induced in mice by injecting PLP and pertussis toxin. Benz shades each. Number of objects in the corpus callosum region tropine (10 mg/kg) and saline (vehicle control) were injected in each bin were counted using Image-Pro plus. (D) Repre intraperitoneally in the therapeutic mode for 14 days. Total sentative images of Image-Pro rendering of the quantification splenocytes were isolated from the mice, stimulated with of objects in each bin. PMA and ionomycin, and analyzed for various populations of (0072 FIG. 32. Western blot analysis of cleaved caspase immune cells and cytokine secretion after 48 hours. Protein activity in differentiated OPCs. OPCs were plated in basal transport was blocked in cells used for cytokine analysis differentiation media and treated with benztropine 1.5 uM. using Monoeiosin. Benztropine treatment had no effect on the thyroid hormone 1 uM or DMSO<0.1% for 6 days. Total number of total splenocytes (A), number of B cells (B), num protein was isolated and analyzed by Western blot using a ber of CD4"T-cells (C), number of CD8"T-cells (D), number specific antibody for the expression of caspase 3 and cleaved of CD4"/CD44HiT-cells (E), and number of CD8"/CD44Hi caspase 3. No expression of cleaved caspase 3 was detected in T-cells (F). Benztropine also had no effect on cytokine pro the compound treated cells or in untreated OPCs. duction measured as the number of IL2 producing CD4" (0073 FIG. 33. Combination with benztropine improves T-cells (G), number of IL10 producing CD4 T-cells (H), efficacy and allows for a reduction in the dose of FTY720 and number of TNF-C. producing CD4 T-cells and number of interferon-?3. EAE was induced in mice using PLP and per IFN-Y producing CD4 T-cells. Error bars indicate standard tussis toxin. Benztropine (2.5 mg/kg) and FTY720 (various deviation of the mean within each group of 5 mice. Repre doses) and interferon (various does) were injected via intra sentative flow cytometry scatter plots (K) show similar num peritoneal injections in the therapeutic mode at the start of bers of CD4", CD8", and CD44" cells in spleens isolated from EAE symptoms. (A) Clinical EAE scores for mice treated vehicle treated mice and benztropine treated mice. with FTY720 at doses of 1 mg/kg, 0.1 mg/kg and 0.01 mg/kg 0069 FIG. 29. Benztropine shows no immunosuppressive show a dose dependent activity for FTY720. (B) Clinical effects in vivo in normal mice. BenZitropine (10 mg/kg) and EAE scores for mice treated with interferon-?3 at doses of saline (vehicle control) were injected intraperitoneally in nor 10,000 U, 3000 U and 1000 U per mouse shows a dose mal mice for 14 days. Total splenocytes were isolated from dependent activity for interferon-?3(C) Combinations of ben the mice, stimulated with PMA and ionomycin and analyzed Ztropine (2.5 mg/kg) with FTY720 (1 mg/kg, 0.1 mg/kg and for various populations of immune cells and cytokine secre 0.01 mg/kg). (D) Combinations of benztropine (2.5 mg/kg) tion after 48 hrs. Protein transport was blocked in cells used with interferon-B (10,000U,3000 U and 1000 Uper mouse). for cytokine analysis using monoeiosin. Benztropine treat (E) Clinical EAE scores for mice treated with FTY720 (0.01 ment had no effect on the number of total splenocytes (A), mg/kg) in combination with benztropine (BA, 2.5 mg/kg) number of B cells (B), number of CD4 T-cells (C), number of does not show a significantly decreased clinical severity as CD8" T-cells (D), number of CD4"/CD44Hi T-cells (E) and compared to mice treated with FTY720 (0.01 mg/kg) or ben number of CD8"/CD44Hi T-cells (F). Benztropine also had Ztropine (2.5 mg/kg) alone. (F) Clinical EAE scores for mice US 2014/0038949 A1 Feb. 6, 2014

treated with interferon-B (IFN: 1000 U/mouse) in combina tion or recruitment, but rather is due to impaired differentia tion with benztropine (BA, 2.5 mg/kg) does not show a sig tion of OPCs (reviewed in Chong and Chan, J. Cell Biol. nificantly decreased clinical severity as compared to mice 188:305-312 (2010)). Accordingly, the present invention fur treated with interferon-B (IFN: 1000 U/mouse) or benz ther provides for methods of stimulating increased myelina tropine (2.5 mg/kg) alone. Error bars indicate standard devia tion of nerves in a subject in need thereof by administering to tion of the mean within each group of 8 mice. a subject a neurotransmitter receptor modulating agent. Such 0074 FIG. 34. Identified compounds decrease clinical as a muscarinic receptor antagonist, a dopamine receptor severity in the EAE model. EAE was induced in mice by antagonist, a histamine receptor antagonist, a beta adrenergic injecting PLP in complete Freund's adjuvant (CFA) and per receptor antagonist, or an opioid receptor modulator. The tussis toxin, and the animals were scored daily for clinical present invention also provides methods of treating a subject severity of disease on a scale of 0-5. (A) Clinical EAE scores having a demyelinating disease by administering to a subject for mice treated with benztropine (10 mg/kg) in the therapeu a neurotransmitter receptor modulating agent. mode (injections starting at the first appearance of EAE II. Agents that Stimulate Increased Myelination of Nerves symptoms on day 8-10) showed a significantly decreased 0077 A. Neurotransmitter Receptor Modulating Agents clinical severity in the relapse phase of the disease as com 0078. A neurotransmitter receptor modulating agent is an pared to vehicle treated mice. (B) Clinical EAE scores for agent that induces oligodendrocyte precursor cell (OPC) dif mice treated with benztropine (10 mg/kg) in the prophylactic ferentiation to a mature myelinating cell fate (e.g., myelinat mode (injections starting on day 0) showed a significantly ing oligodendrocytes) and/or stimulates increased myelina decreased clinical severity in both acute and relapse phase of tion. In some embodiments, a neurotransmitter receptor the disease as compared to vehicle treated mice. (C) Benz modulating agent is selected from a muscarinic receptor tropine showed a dose dependent efficacy in decreasing clini antagonist, a dopamine receptor antagonist, a histamine cal severity scores in the EAE model, with 12.5 mg/kg being receptor antagonist, a beta adrenergic receptor modulator, the most effective dose and 0.1 mg/kg showing no effect. and an opioid receptor modulator. As shown in the Examples Clinical EAE scores for mice treated with (D) trifluoperazine section below, exemplary members of each of these classes of (10 mg/kg), (E) clemastine (10 mg/kg), or (E) salbutamol (10 compounds has been shown to induce OPC differentiation to mg/kg) in the therapeutic mode showed a significantly a myelinating oligodendrocyte cell fate, and thus stimulate decreased clinical severity in the relapse phase of the disease increased myelination. Based on the data showing this activ as compared to vehicle treated mice. Error bars indicate stan ity of exemplary muscarinic receptor antagonists, dopamine dard deviation of the mean within each group of 8-10 mice. receptor antagonists, histamine receptor antagonists, beta adrenergic receptor antagonists, and opioid receptor modula DETAILED DESCRIPTION OF THE INVENTION tors, other compounds in each of these classes and having similar pharmacological mechanisms to the exemplified I. Introduction compounds, such as the compounds listed in Table 1, are also 0075. The present invention is based, in part, on the dis predicted to be useful for inducing OPC differentiation to a covery that compounds that modulate various classes of neu mature myelinating cell fate (e.g., myelinating oligodendro rotransmitter receptors, such as muscarinic receptor antago cytes) and/or stimulating increased myelination. nists, dopamine receptor antagonists, histamine receptor 0079. In some embodiments, a compound that is identified antagonists, beta adrenergic receptor antagonists, and opioid as an agent that stimulates increased myelination has “selec receptor modulators, promote the differentiation of oligoden tive' activity for one of these classes of neurotransmitter drocyte precursor cells (OPCs) into a mature myelinating cell receptors (i.e., has an agonistic or antagonistic effect against fate (e.g., myelinating oligodendrocytes). Accordingly, in one one of a muscarinic receptor, a dopamine receptor, a hista aspect, the present invention provides for methods of induc mine receptor, a beta adrenergic receptor, or an opioid recep ing OPC differentiation to myelinating oligodendrocytes. tor, or a subtype of any of these receptors, and has a weaker 0076. Without being bound by a particular theory, it is effector substantially no effect against the other receptors). In believed that in demyelinating diseases such as multiple scle Some embodiments, a compound that is identified as an agent rosis, OPCs are present and able to migrate to demyelinated that stimulates increased myelination has activity against two regions, Suggesting that the progressive decrease in remyeli or more of these classes of neurotransmitter receptors or nation in these diseases is not due to defects in OPC popula Subtypes of neurotransmitter receptors. TABLE 1 Neurotransmitter receptor modulating agents Neurotransmitter Receptor(s) Modulated Low Molecular Weight Compound Muscarinic Adrenergic Opioid Antitussive (-)-Norephedrine (-)-Quinpirole hydrochloride (-)-Terbuclomine (+)- (+)-BUTACLAMOL HYDROCHLORIDE (+-)- (+)-Lappaconitine (+)- e (+-)-Trimethoduinol (+/-)- US 2014/0038949 A1 Feb. 6, 2014

TABLE 1-continued Neurotransmitter receptor modulating agents Neurotransmitter Receptor(s) Modulated Low Molecular Weight Compound Muscarinic Dopaminergic Histaminergic Adrenergic Opioid Antitussive Serotonergic (1r,2r)-cyclohexane-1,2-dicarboxylic acid e (3,4-dihydroxyphenylamino)-2-imidazoline e .DELTA.9- e 11CMNPA e e -((6,7-Dimethoxy-3-methyl-2- e benzofuranyl)carbonyl)-4-methylpiperazine monohydrochloride -(3-bromo-5-isoxazolyl)-2-(tert- e butylamino) hydrochloride -(3-Chlorophenyl) e -(3-Chlorophenyl)piperazine dihydrochloride e -(3-Chlorophenyl)piperazine hydrochloride e -(4-Hydroxyphenyl)-2-aminoethanol e 2,9,10-tetramethoxyaporphine e e 4-bis(2-(4-methoxynaphthalen-1- e yl)methylidenehydrazinyl-phthalazine 5-Trimethylenetetrazole e -3-(Trifluoromethyl) e 21524-08-1 e 25-71-3 (Parent) e 27-35-5 e 7-Hydroxy-2,3-cyclopropanoandrostane e 9-Propylorvinol e -Benzylimidazole e -M-Thiq e -PHENYLPIPERAZINE e -Propanamine, 3-dibenzob.ethiepin-11 (6H)- e ylidene-N,N-dimethyl 2-((2-Ethoxyphenoxy)phenylmethyl)morpholine e methanesulfonate 2-(1-Piperazinyl)pyrimidine e 2-(2-Aminoethyl)pyridine e 2-(2-Aminoethyl)pyridine dihydrochloride e 2-Detpq. e 2-Methoxyidazoxan e 2-METHYL-1,3- e 2-Thiazoleethanamine e 3-iodopindolol e 3-Quinuclidinyl benzilate e 4-DAMP e 4-Damp methiodide e 4-DO e 4-NMPB e 5,6-Dihydroxytryptamine e 5,7-DIHYDROXYTRYPTAMINE e 5-Carboxamidotryptamine e 5-Fhdbat e 5-Hmdptime e 5-Hydroxypropafenone e 5-Methylfurmethide e 5-Methylurapidil e 6-Dta e 6-Hydroxydopamine hydrobromide e 6-Hydroxydopamine hydrochloride e 6-nitroquipazine e 76-57-3 e e 7-Ohdpat e 87-00-3 e 8-Artp e 8-OH-Dpat e A-38SO3 e A-77636 hydrochloride e e e Acebutolol hydrochloride e e Aceclidine hydrochloride e maleate e Aceroxatidine e acetaminophen e Acetylpromazine e Acide tolfenamique INN-French e US 2014/0038949 A1 Feb. 6, 2014 10

TABLE 1-continued Neurotransmitter receptor modulating agents Neurotransmitter Receptor(s) Modulated Low Molecular Weight Compound Muscarinic Dopaminergic Histaminergic Adrenergic Opioid Antitussive Serotonergic

ACRIVASTINE e Actifed e ACTINOQUINOL SODIUM e Adobiol e DRAFINIL e drenaline bittartrate e DTN e erolone e erOwent e dx 384 e 76 e ktamin hydrochloride e aproclate e aproclate hydrochloride e esion e eudrin e fenta e LFENTANIL e fentanil hydrochloride e uZosin e ginor e golysin e e imeZine (TN) e legra e lergival e lococaine e motriptan e nespiron e e nespirone (INN) e e omide (TN) e LOSETRONHYDROCHLORIDE e oxi e oxi (TN) e alpha-Ergocryptine e alpha- e alpha-Methylhistamine e alpha-Methyl-L-dopa e alpha-Methylserotonin e Alphaprodin e e Alprenolol hydrochloride e alrestatin e Altat e e e e Amantadine hydrochloride e e e Ambenonium chloride e Amerge e Amfebutamone e amfebutamonum e Ami begron hydrochloride e e e e e Amitriptyline hydrochloride e e e amosulalol hydrochloride e e e e e Amperozide hydrochloride e e e AMPHETAMINE SULFATE e e Amsulosin e Anatran e Andantol e Anemet e e e e Anplag e e Antazoline hydrochloride e Antazoline phosphate e US 2014/0038949 A1 Feb. 6, 2014 11

TABLE 1-continued Neurotransmitter receptor modulating agents Neurotransmitter Receptor(s) Modulated Low Molecular Weight Compound Muscarinic Dopaminergic Histaminergic Adrenergic Opioid Antitussive Serotonergic

Antergan e e Antussan e Anzemet e e e Apomorphine HCI e e e APRACLONDINEHYDROCHLORIDE e a- e Aprofen hydrochloride e Aprofene e Ara-putp e Arbutamina e e Arbutamine hydrochloride e Arc. 239 e e e e arotinolol hydrochloride e Artane e Arterenolbitartrate e Artex e Astelin e e Astomin e Astramorph e AtaraX e altenolol e e e ATROPINE e Atropine iodomethylate e Atropine methyl nitrate e Atropine sulfate e Atrovent e Auteral e Avacan e Avapyrazone e Avinza e Axert e e e e AZepexole e Azepexole hydrochloride e e Banistyl e Banthine e Batebulast e Batebulast hydrochloride e BD1047 e BE2254 e Beforal e e e Beldavrin e e e e e Benactyzine hydrochloride e benalfocin e Benextramine e Benfuran e e Benoxathian hydrochloride e e e e Bentanidol e Benzetimide e BENZETIMIDEHYDROCHLORIDE e Benzfetamine e e Benzhexol e e US 2014/0038949 A1 Feb. 6, 2014 12

TABLE 1-continued Neurotransmitter receptor modulating agents Neurotransmitter Receptor(s) Modulated Low Molecular Weight Compound Muscarinic Dopaminergic Histaminergic Adrenergic Opioid Antitussive Serotonergic benztropine Benztropinum Berachin beta-Cft beta-ENDORPHIN beta-Endorphin (1-31) beta-Flupenthixol Betahistine dihydrochloride Betahistine mesilate Betaxolol hydrochloride Betazole hydrochloride Bethanechol chloride MESYLATE Biperiden hydrochloride Bisguanidinium phosphate Bisoprolol fumarate Bladderon Bmy-7378 Bonamine BrAAM tosylate Birl 15572 Bril 26830 Br37344A Br 48553 Br-15572 Brocadisipal Brolamfetamine Bromopride hydrochloride Brompheniramine maleate Broncaspin Bronitin Mist Bronkometer Broxalterol BTCP Buccastem Bucindolol hydrochloride Buclodin Budipine hydrochloride Bufotenine Bunolol Bunolol hydrochloride bupranolol hydrochloride Buprenorfina INN-Spanish BUPRENORPHINE HYDROCHLORIDE Buprenorphine hydrochloride solution US 2014/0038949 A1 Feb. 6, 2014 13

TABLE 1-continued Neurotransmitter receptor modulating agents Neurotransmitter Receptor(s) Modulated Low Molecular Weight Compound Muscarinic Dopaminergic Histaminergic Adrenergic Opioid Antitussive Serotonergic Bupropion hydrochloride e e Buscapine e e Buspirone hydrochloride e BUTACLAMOLEHYDROCHLORIDE e butanoic acid e Butaxamina e e e e Butorphanol tartrate e e Butoxamine (INN) e BUTOXAMINEHYDROCHLORIDE e Butylhyoscine e Butylscopolamine e C11796 e

C19H2SNOOHC e

C5976 SIGMA e e e benzoate e Calcium fusarate e e e carbachol e e e Carbamazepine dihydrate e Carbamylcholine e e carbetapentane e e Carbetapentane citrate e e e e Carbidopa hydrate e e Carbidopa Monohydrate e e Carbidopa, (S)-Isomer e e Carbidopa-levodopa e e e CARBINOXAMINEMALEATE e Carebastine e e e carteolol hydrochloride e carvedillol e CCRIS 3490 e CEC dihydrochloride e celliprolol e Centralvet e e Cerocral e e e CGP 20712A e CGP 20712A methanesulfonate e CGS 1206.6B e CGS 12066B dimaleate e CH38O83 e CHEBI: 104181 e CHEBI: 117275 e CHEBI: 124645 e CHEBI: 126213 e CHEBI: 136626 e CHEBI: 142179 e CHEBI: 1488.98 e CHEBI: 159721 e e CHEBI: 161127 e CHEBI: 178303 e CHEBI: 2O1827 e CHEBI: 238.638 e CHEBI: 268876 e US 2014/0038949 A1 Feb. 6, 2014 14

TABLE 1-continued Neurotransmitter receptor modulating agents Neurotransmitter Receptor(s) Modulated Low Molecular Weight Compound Muscarinic Dopaminergic Histaminergic Adrenergic Opioid Antitussive Serotonergic

HEBI: 334862 e HEBI: 3SOS46 e HEBI: 36796 e e e : 399928 e : 40751 e HEBI: 431080 e HEBI: 471632 e

: 48295 e : S17861 e : 583615 e HEBI: S84626 e HEBI: 623294 e HEBI: 648957 e HEBI: 702837 e HEMBL446167 e HEMBL93361 e HLORAZINE e hlorethylclonidine e hloropyramine e hloropyramine hydrochloride e C hlorpheniramine e hlorpheniramine maleate e hlorpromazine e hlorpromazine hydrochloride e hlorprothixene e ANOPRAMINE e 105105 e DS122 e D517557 e e e e imetidine hydrochloride e NANSERIN e inanserin hydrochloride e e e e e Cisapride monohydrate e e italopram hydrobromide e L316.243 e L316243 e -Apb e earnal e emastine e e emastine (USAN) e e LEMASTINE FUMARATE e e lenbuterol e hydrochloride e clidinium e CLIDINIUMBROMIDE e clobempropit e e e Clomipramine hydrochloride e e LONIDINEHYDROCHLORIDE e LOPENTHIXOL e opiXol e oranolol e ovoxamine e ovoxamine fumarate e e cocaethylene e Cocaethyline e Cocain-chlorhydrat German e e COCAINE HYDROCHLORIDE e Cocaine muriate e e e US 2014/0038949 A1 Feb. 6, 2014 15

TABLE 1-continued Neurotransmitter receptor modulating agents Neurotransmitter Receptor(s) Modulated Low Molecular Weight Compound Muscarinic Dopaminergic Histaminergic Adrenergic Opioid Antitussive Serotonergic Cogentin Cogentin mesylate Cognex Compazine Concerta Concordin Congesteze Contristamine Corindolan Corlopam Corynanthin Corynanthine hydrochloride CP 93.129 Crispin CV 705 Cyclizine hydrochloride Cyclogyl cyproheptadine hydrochloride Cystospaz d1- DAGO Daipin Dalcipran Dalgan Dalmee DAMGO Dapiprazole hydrochloride Darifenacin hydrobromide Darwon D-Chlorpheniramine D-Dopa Debridat DEBRISOQUIN SULFATE deisopropyldisopyramide C Deprenalin Deprenil Deptropine citrate (INN) Deramciclane fumarate Desipramine hydrochloride Desoratadine Desoxedrine Detrol DEXBROMPHENIRAMINEMALEATE maleate Dexmedetomidinum (INN-Latin DEXPROPRANOLOL Dexpropranolol hydrochloride US 2014/0038949 A1 Feb. 6, 2014 16

TABLE 1-continued Neurotransmitter receptor modulating agents Neurotransmitter Receptor(s) Modulated Low Molecular Weight Compound Muscarinic Dopaminergic Histaminergic Adrenergic Opioid Antitussive Serotonergic Dextromethorphan hydrobromide monohydrate e e e Dextrostat e e Diacetylmonoxime e Diacetylmorphine e Diamaprit-2HCl e Dicetel e Dicodethal e e dicyclomine e Dicyclomine hydrochloride e Difril e e Dihydro-alpha-ergocryptine meSylate e e e Dihydrocodeine bitartrate e Dihydroergocornine e Dihydroergocristine e DIHYDROERGOCRISTINE MESYLATE e e e e e Dihydroergotamine mesilate e e e e e Dihydroquinidine e Dihydroquinine e dihydroxyphenylalanine e Dilaudid e Dilevalol e Dilevalol hydrochloride e e e Dimemorfan (INN) e e Dimethindene maleate e DIMETHYLTRYPTAMINE e e Diphemanil e e Diphenhydramine citrate e DIPEHENHYDRAMINEHYDROCHLORIDE e DIPEHENOXYLATE e HCl e diplivefrin e Dipivefrin hydrochloride e Dironyl e Ditropan e e DL- e |-Desoxyephedrine e e DL-DOPA e l-Narcotine e DL- e D L-threo-3,4-Dihydroxyphenylserine e D L-threo-DOPS e obutamine e hydrochloride e ocarpamine e olasetron e DOLASETRONMESYLATE e Dolasetronum (INN-Latin e Domin e e Omperidone e Maleate e Dopabain e e opamine e Dopamine hydrochloride e opazinol e e e US 2014/0038949 A1 Feb. 6, 2014 17

TABLE 1-continued Neurotransmitter receptor modulating agents Neurotransmitter Receptor(s) Modulated Low Molecular Weight Compound Muscarinic Dopaminergic Histaminergic Adrenergic Opioid Antitussive Serotonergic

DOPEXAMINEHYDROCHLORIDE e e hydrochloride e (INN) e Dothiepin e e Doxazosin meSylate e e Doxepin Hydrochloride e Doxepine e DOXOFYLLINE e e Doxylamine Succinate e DPDPE e Dramamine e Drixoral e e e e drotaverine e e DSP 4 e DSP-4 hydrochloride e DU-29373 e dulloxetine e e e HYDROCHLORIDE e e e DuP 734 e Duremesin e 1-13 e e e e Edronax e EEDQ e e e e Efaroxan hydrochloride e Effexor e Effortilwet e Eldoral e e Eletriptan hydrobromide e e Eltoprazine hydrochloride e Emadine e e Emepronum e Emergil e Enantio-PAF C-16 e 1 e Endomorphin 2 e Endovalpin e e e eperisone e e Ephedrine hydrochloride e EPHEDRINE SULFATE e Ephetonine e Epibatidine e e epinephrine e Epinephrine hydrochloride e fumarate e Ergocryptine e Ergocryptine mesylate e Ergocryptine-alpha e mesylate e Ergomar e e Ergotamin e e e e e ergotamine tartrate e Esbuphon e e US 2014/0038949 A1 Feb. 6, 2014 18

TABLE 1-continued Neurotransmitter receptor modulating agents Neurotransmitter Receptor(s) Modulated Low Molecular Weight Compound Muscarinic Dopaminergic Histaminergic Adrenergic Opioid Antitussive Serotonergic Escitallopram Escitallopram oxalate Esmolol hydrochloride ethaverine Ethaverine hydrochloride ethopropazine Ethopropazine hydrochloride ETHYLKETOCYCLAZOCINE Eticlopride hydrochloride etillefrine hydrochloride Etintidine Etintidine hydrochloride EU-010O372 Eupaverina Euspirol Evoxac Exaprolol hydrochloride Famotidina amotidine HCI Fastin Femoxetinum (INN-Latin Fencarbamide hydrochloride Fencarol Fenclonine enfluramine Fenistill enoldopam enoterol hydrobromide Fenoverine entanyl Fentora exofenadine FG 4963 Finaten Finibron lawOxate avoxate hydrochloride b. 457 esinoxan esinoxan hydrochloride LESTOLOL LESTOLOL SULFATE uanxol depot (TN) lunarizine lunarizine hydrochloride uorofen LUPENTHIXOL DECANOATE upentixol LUPHENAZINE uphenazine hydrochloride uspirilene FLUVOXAMINEMALEATE Focalin FONAZINE ormoterol US 2014/0038949 A1 Feb. 6, 2014 19

TABLE 1-continued Neurotransmitter receptor modulating agents Neurotransmitter Receptor(s) Modulated Low Molecular Weight Compound Muscarinic Dopaminergic Histaminergic Adrenergic Opioid Antitussive Serotonergic

Frovatriptan e Frovatiriptan Succinate e e e Galantamin e e Galantamine hydrobromide e Ganglefene e Ganglerone e Gastrozepin e Gbr 12783 e GBR12935 e Geodon e e e GEPIRONEHYDROCHLORIDE e Gewatran e e e e Glauconex e GlaxoWellcome brand of e GLYCOPYRROLATE e Gnoscopine e Gotensin e GR 113808 e GR-127935 e e Granisetron hydrochloride e e Guanabenz acetate e e guanethidine Sulfate e e guanfacine hydrochloride e e Guanidine bromide e Guanidine hydrochloride e Guanidine nitrate e GUANIDINIUM e Gynergen e e Hag-PC e haloperidol e aymine e HEAT e emicholinium e emicholinium-3 e eroin hydrochloride e hsi-difenidol e igenamine e imbacine e histamine e istamine dihydrochloride e istamine diphosphate e istamine hydrochloride e istantin e Oe-893d e OMATROPINE e omatropine hydrobromide (RS) e omocodeine e Hycodan e e Hydergine e e Hydriatine e Hydroaminacrine e e e e ydromorphone hydrochloride e hydroquinidine e ydroquinidine hydrochloride e HYDROQUININE e e Hydroxyzine pamoate e yoscine hydrobromide e US 2014/0038949 A1 Feb. 6, 2014 20

TABLE 1-continued Neurotransmitter receptor modulating agents Neurotransmitter Receptor(s) Modulated Low Molecular Weight Compound Muscarinic Dopaminergic Histaminergic Adrenergic Opioid Antitussive Serotonergic Hyoscine Methobromide e hyoscyamine e Hyoscyamine (D)- e Hyoscyamine Sulfate e Hyoscyamine sulfate (USP) e Hypostamine e Hysco e bopamine e e BZM e catibant e catibant acetate e c 118551 e CI-894O6 e DAZOXAN e DAZOXAN HYDROCHLORIDE e fenprodil e tartrate e HEAT e ldamen e e metit dihydrobromide e midacloprid e e mipramine hydrochloride e MPROMIDINE e mpromidine hydrochloride e napetyl e e NDALPINE e e e inderal e indolophenanthridine e e NDORAMIN e Hydrochloride e nopamil e insidon e intropin e odocyanopindolol e ipratropium e e pratropium bromide monohydrate e prazochrome e ps-339 e PSAPIRONE e psapirone hydrochloride e Smelin e Soaminile e Socodeine e e isoetharine e isoproterenol e SOPROTERENOL HYDROCHLORIDE e soproterenol sulfate e Sospaglumic acid e Sothipendyl e e SOXSuprine hydrochloride e tro e animine e etrium tartrate e Kadian e Kerlone e e Ketanserin tartrate e e Ketogan e e KETOTIFENFUMARATE e Kinichron e e Kytril e L 657743 e US 2014/0038949 A1 Feb. 6, 2014 21

TABLE 1-continued Neurotransmitter receptor modulating agents Neurotransmitter Receptor(s) Modulated Low Molecular Weight Compound Muscarinic Dopaminergic Histaminergic Adrenergic Opioid Antitussive Serotonergic

L-741,626 e abetalol e hydrochloride e afutidine e L-alpha-Acetyl-N-normethadol e e appaconitine e e Legatrin e Leoplexamin e -Ephedrine e Lethidrone e e e Levamfetamine e e Levcromakalim e Levetimide e e LevobunololeHCI e evocabastine e HYDROCHLORIDE e e evodopa e e e Levomeprazine e e e e e Levomethorphan hydrobromide e evorphanol e e Levospasme e Levsinex e LIDAMIDINE e LIDAMIDINEHYDROCHLORIDE e Lilly 53857 e - chloride e e e e isuride maleate e e e L-Noradrenaline bittartrate e LODOXAMIDE TROMETHAMINE e e Lofentaniloxalate e Longifene e Lopac0 000714 e Lopac-A-164 e Lopac-C-130 e Lopressor e oratadine e LOrcet e LotrOneX e oxapine e hydrochloride e Loxapine Succinate e Loxtidine e L- e LSD tartrate e LUPITIDINEHYDROCHLORIDE e LY 2.35959 e LY 277359 maleate e Ly-165163 e Lysergide e Lysivane e e e e madopar e Malexi e e Maprotiline hydrochloride e Marzine e Maxolon e e e Mci9042 e US 2014/0038949 A1 Feb. 6, 2014 22

TABLE 1-continued Neurotransmitter receptor modulating agents Neurotransmitter Receptor(s) Modulated Low Molecular Weight Compound Muscarinic Dopaminergic Histaminergic Adrenergic Opioid Antitussive Serotonergic

McIl S652 e McN-A-343 e mCPBG e m-CPBG hydrochloride e MDL-100907 e MDMA e e e MEBEVERINE HYDROCHLORIDE e Meclastine e e Meclizine hydrochloride e Meclizine Mixture With Niacin e Mecloprodine e e e Medetomidine hydrochloride e e e Medroxalol hydrochloride e e e e MEMANTINE HYDROCHLORIDE e e meperidine e Mepindolo e Meptazino e HYDROCHLORIDE e e Merital e e Mescomine e e e e e Mesulergine hydrochloride e e e Metabolites (street) e e metaproterenol e Metaproterenol hemisulfate e bittartrate e Metatsin e e e e Methacholine chloride e e e Methadyl acetate e e e Methamphetamine hydrochloride e e e methalpyrilene e METHAPYRILENEHYDROCHLORIDE e Metharsinat e methiothepin e Methiothepin maleate e e Metholes e e e METHOXAMINEHYDROCHLORIDE e e methyldopa e METHYLDOPA SESQUIHYDRATE e Methylfurmetide e methyloctatropine bromide e e Methylscopolamine e e Methysergide maleate e e e e Metoclopramide dihydrochloride e e Metoprolol fumarate e e e e Mianserin hydrochloride e e e US 2014/0038949 A1 Feb. 6, 2014 23

TABLE 1-continued Neurotransmitter receptor modulating agents Neurotransmitter Receptor(s) Modulated Low Molecular Weight Compound Muscarinic Dopaminergic Histaminergic Adrenergic Opioid Antitussive Serotonergic Mictonorm Midaglizole Midaglizole hydrochloride MIDODRINE HYDROCHLORIDE Mifentidine Minipress Mintussin Minusine Mirapex MK-212 MK-912 M-Mptp Mofegiline hydrochloride MONATEPILMALEATE Morphine hydrochloride MORPHINE SULFATE morphinesulfate Mosapride citrate Moxaverine moxisyllyte hydrochloride MPTP Muscarin Myonal Myophedrine N,3-Dimethylmorphinan Naaxia N-acetylaspartylglutamate N-Acetyl-Asp-Glu nafronyl Nafronyl oxalate malbuphine Nalorphine hydrochloride Naloxone hydrochloride NAN-190 hydrobromide NAPHAZOLINEHYDROCHLORIDE Naphazoline nitrate Naphthisen Nargoline Narphen Navaron NaZasetron NCGCOOO15261-01 inchembio873-comp43 inchembio873-comp53 inchembio873-compé7 Ncq 298 Nebracetam Nebracetam fumarate Nedeltran Nefopam hydrochloride neostigmine bromide US 2014/0038949 A1 Feb. 6, 2014 24

TABLE 1-continued Neurotransmitter receptor modulating agents Neurotransmitter Receptor(s) Modulated Low Molecular Weight Compound Muscarinic Dopaminergic Histaminergic Adrenergic Opioid Antitussive Serotonergic Neostigmine methylsulfate e e NIH-8805 e Nipradillol e Nisentil e e nizatioline e N-Methylspiroperidol e Noleptan e e Norephedrine e e e Norfenfloramine e Norflex e Norprolac e e Nortriptyline hydrochloride e Norzine Ampuls e NoScapalin e e Novopropoxyn e n-Propylapomorphine e e NSC10004 e e e NSC11433S e NSC28.9336 e NSC61391 e NSC61806 e e NSC69886 e NSC79303 e Nubain e nylidrin e Nylidrin hydrochloride e Octatropine e e Oils, peppermint e e e e ONDANSETRONHYDROCHLORIDE e Opana e Opcon e e Oprea1 021650 e e Orphenadrine hydrochloride e e Otillonium Bromide e e e e Oxifedirinum e e e Oximetidine hydrochloride e e Oxolamine citrate e e oxotremorine methiodide e e Oxprenolol hydrochloride e oxybutynin e Oxybutynin chloride e e Oxycodone hydrochloride e e e oxyphenonium e OXYPHENONIUMBROMIDE e US 2014/0038949 A1 Feb. 6, 2014 25

TABLE 1-continued Neurotransmitter receptor modulating agents Neurotransmitter Receptor(s) Modulated Low Molecular Weight Compound Muscarinic Dopaminergic Histaminergic Adrenergic Opioid Antitussive Serotonergic oZagrel oZagrel hydrochloride Palfadonna Palladone Palomosetron hydrochloride PAPP Paracodin Paracymethadol Parasan Parlodel Paroxetine hydrochloride Paroxetine maleate Pataday Paxil PBPO p-Chloramphetamine Pemilaston Pentalgine pentazocine Peracon maleate PERGOLIDE MESYLATE Perhydrohistrionicotoxin Periactin Pernazine Pernovine periphenazine hydrochloride FHHSD henacetin HENAZOCINE hencarbamide henindamine heniramine HENIRAMINEMALEATE HENOPERIDINE henoperidine hydrochloride henopropamine (OXcle henoxybenzamine henoxybenzamine hydrochloride Phentolamine mesylate PHENYLEPHRINE HYDROCHLORIDE PHENYLPROPANOLAMINE HYDROCHLORIDE PHOLCODNE Phospholine iodide Picumast Picumast dihydrochloride Pilocarpine Pilocarpine hydrochloride Pilocarpine nitrate Pilocarpine nitrate salt bimozide Pinaverium bindolol Pipazethate US 2014/0038949 A1 Feb. 6, 2014 26

TABLE 1-continued Neurotransmitter receptor modulating agents Neurotransmitter Receptor(s) Modulated Low Molecular Weight Compound Muscarinic Dopaminergic Histaminergic Adrenergic Opioid Antitussive Serotonergic Pirenzepine dihydrochloride Piribedill hydrochloride Piribedil mesylate PIZOTYLINE Plegine 3-MPPI Pinu 99194A Pondimin Pramipexol Spanish Prazosin hydrochloride PRBCM Precedex Preclamol Preclamollum Latin 21-pivalate Prestwick 144 Prialt Privine Prizidio procaterol hydrochloride Prochlorperazine dimaleate Prochlorperazine edisylate Prochlorperazine maleate Procyclidine hydrochloride Prolixin PROMAZINEHYDROCHLORIDE Promedol PROMETHAZINE HYDROCHLORIDE propantheline Propitan Propiverine propiverine hydrochloride Propranolol hydrochloride Proroxan INN) Proscomide Prothiaden protopine PROTOPINE HYDROCHLORIDE Protriptyline hydrochloride Proxicromil Prozac PSXeladine Psychostyl pyrilamine PYRILAMINEMALEATE Pyrroxane Quadramet Quinagollide Quinidex US 2014/0038949 A1 Feb. 6, 2014 27

TABLE 1-continued Neurotransmitter receptor modulating agents Neurotransmitter Receptor(s) Modulated Low Molecular Weight Compound Muscarinic Dopaminergic Histaminergic Adrenergic Opioid Antitussive Serotonergic Quinidine sulfate Quinine Quinine hydrochloride quinine Sulfate QUININESULFATE DIHYDRATE QUINPIROLE QUIPAZINE MALEATE R(-)-Denopamine R-SOS47 Raclopride C11 Raclopridum Latin Ractopamine hydrochloride Ramosetron hydrochloride Ranitidine bismuth citrate ranitidine hydrochloride Rapimine Reboxetine mesylate Reboxetine mesylate hydrate Redux Reglan Relaspium REMIFENTANILEHYDROCHLORIDE REMOXIPRIDE HYDROCHLORIDE Renzapride hydrochloride repirinast REPROTEROL HYDROCHLORIDE Respilene Restenacht Rexigen Rexolate Rilmenidine phosphate Rimiterol Hydrobromide Ritodrine hydrochloride Rizatriptan benzoate Ro 363 Robinal Robinul Ropinirole hydrochloride Rotenolone Roti gotine RS 86, hydrobromide RS 86HB RS-2S259-197 RU 24969 RU-24213 S(-)Eticlopride hydrochloride S-2OSOO Salbutamol Saligren Salmeterol Salmeterol Xinafoate US 2014/0038949 A1 Feb. 6, 2014 28

TABLE 1-continued Neurotransmitter receptor modulating agents Neurotransmitter Receptor(s) Modulated Low Molecular Weight Compound Muscarinic Dopaminergic Histaminergic Adrenergic Opioid Antitussive Serotonergic

Samarium Sm 153 lexidronam e e Savella e e SB 206SS3 e Sch. 23982 e Sch-23982 e Scopolamine e Scopolamine butylbromide e Scopolamine hydrobromide e Sdz 205,557 e Secoverine e SECOVERINEHYDROCHLORIDE e Selecal e e Selegiline hydrochloride e Selozok e Serc e Serotone e e e Sertraline hydrochloride e e Sgd 101-75 e Silomat e Sinemet e e SKF 38393 e SKF 38393 hydrochloride e SKF 81297 e Skif 83566 e SKF 91.488 e SKF 91488 dihydrochloride e Sm-Edtmp e SMROOO449272 e Snc80 e e Solifenacin Succinate e Sordinol e e Sotalol hydrochloride e Soventol e Soventol (TN) e Soventol hydrochloride e Spasril e Spectrum OO1815 e e piriva e piriva Handihaler e piroXatrine e Q 10643 e RS923OA e T91 e e adol e e elazine e e STP () e Strattera e Subecholine e Suberyldicholine e

Sulpiride e e Sultopride hydrochloride e e SUMATRIPTAN SUCCINATE e Suplatast Tosilate e Sympatholytin e e T123 SIGMA e e Tacrine hydrochloride e US 2014/0038949 A1 Feb. 6, 2014 29

TABLE 1-continued Neurotransmitter receptor modulating agents Neurotransmitter Receptor(s) Modulated Low Molecular Weight Compound Muscarinic Dopaminergic Histaminergic Adrenergic Opioid Antitussive Serotonergic acrine hydrochloride hydrate e tageflar e Talacen e Tallinolol e e e Talwin e Talwin 50 e e citrate e Tbhpbo e TCMDC-12SSO9 e maleate e e Teletux e TEMELASTINE e Tempium e Tenamfetamine e e Terazosin hydrochloride e e TERBUTALINE HEMISULFATE e Terbutaline sulfate e e e e TERODILINEHYDROCHLORIDE e Tersigat e e Tesmilifene e e TETRAHYDROCANNABINOL e e e e Tetraspasmin-Lefa e FMP C e Thecodinum e hephorin hydrochloride e heratuSS e hiethylperazine e maleate e e hioridazine e hydrochloride e hiothixene e honzylamine e THONZYLAMINEHYDROCHLORIDE e e e Tillisolol e timolo e hemihydrate e imolol maleate e TIOTIDINE e e Tiotropium e TIOTROPIUMBROMIDE e TIPP e Tiropramide e e e Tizanidine hydrochloride e e Tobanum e Tocodilydrin e e Tolazoline hydrochloride e e e e Tolterodine tartrate e Torecan e e tramadol hydrochloride e tranilast e US 2014/0038949 A1 Feb. 6, 2014 30

TABLE 1-continued Neurotransmitter receptor modulating agents Neurotransmitter Receptor(s) Modulated Low Molecular Weight Compound Muscarinic Dopaminergic Histaminergic Adrenergic Opioid Antitussive Serotonergic

Traxanox e Traxanox sodium e e Trazodone hydrochloride e Tremblex e e Tricodein e e trifluoperazine e Trifluoperazine dihydrochloride e e e Triflupromazine hydrochloride e e e Trimebutine maleate e e Trimethoduinol e e Trimipramine maleate e e Tripelennamine citrate e Tripelennamine hydrochloride e e Triprolidine hydrochloride e e Tropine tropate e e TROPISETRONHCI e Tropyl 3,5-dichlorobenzoate e e e Tulobuterol hydrochloride e Tuscodin e e Tyr-D-Ala-Gly-N-Methyl-Phe-Gly-ol e Tyr-tic-phe-phe-OH e e e Oroxatral e Valoron e Vanoxeamine e e e Ventipulmin e Ventolin e Verton e e Viloxazine hydrochloride e Vistaril e e Vf 9153 e Way 100635 e Way-100135 e Wiin-3S428 e e Wyamine sulfate e e Xamoterol hemifumarate e e Xanomeline tartrate e e Xylamidine tosylate e e Xylazine hydrochloride e XyZall e e YOHIMBINEHYDROCHLORIDE e e ZACOPRIDE HYDROCHLORIDE e e ZATOSETRONMALEATE e US 2014/0038949 A1 Feb. 6, 2014

TABLE 1-continued Neurotransmitter receptor modulating agents Neurotransmitter Receptor(s) Modulated Low Molecular Weight Compound Muscarinic Dopaminergic Histaminergic Adrenergic Opioid Antitussive Serotonergic Zebeta Zelmac Zelnorm ZD7114 Zetidoline e Zimeldine Zimeldine hydrochloride Zimelidine dihydrochloride ZINTEROL HYDROCHLORIDE e Ziprasidone hydrochloride e Ziprasidone mesylate e Zofran e Zyrtec

0080) 1. Muscarinic Receptor Antagonists inhibit one or more characteristic responses of a dopamine 0081. A muscarinic receptor is a G-protein coupled ace receptor or receptor Subtype. As a non-limiting example, an tylcholine receptor. There are five known subtypes of musca antagonist may competitively or non-competitively bind to rinic receptors: M receptors, M receptors, M receptors, Ma (1) a dopamine receptor, (2) an agonist or partial agonist (or receptors, and Ms receptors. A muscarinic receptor antago other ) of the dopamine receptor, and/or (3) a down nist is an agent able to inhibit one or more characteristic stream signaling molecule to inhibit the dopamine receptors responses of a muscarinic receptor or receptor Subtype. As a function. As shown in the Examples, benztropine, non-limiting example, an antagonist may competitively or GBR12935, and trifluoperazine have been shown to antago non-competitively bind to (1) a muscarinic receptor, (2) an nize the function of a dopamine receptor and/or are known agonist or partial agonist (or other ligand) of the muscarinic antagonists of a dopamine receptor. Therefore, in some receptor, and/or (3) a downstream signaling molecule to embodiments, the dopamine receptor antagonist is a com inhibit the muscarinic receptor's function. As shown in the pound selected from benztropine, GBR12935, trifluopera Examples, benztropine, carbetapentane, clemastine, ipratro Zine, and salts, prodrugs, racemic mixtures, conformational pium, and atropine have been shown to antagonize the func and/or optical isomers, crystalline polymorphs, and isotopic tion of a muscarinic receptor and/or are known antagonists of variants thereof. Alternatively, any of the dopamine receptor a muscarinic receptor. Therefore, in Some embodiments, the modulators listed in Table 1 can be used to antagonize a muscarinic receptor antagonist is a compound selected from dopamine receptor. Thus, in some embodiments, the dopam benztropine, carbetapentane, clemastine, ipratropium, atro ine receptor antagonist is a dopamine receptor modulator pine, and salts, prodrugs, racemic mixtures, conformational compound listed in Table 1. The compounds described in and/or optical isomers, crystalline polymorphs, and isotopic Table 1 are readily available. variants thereof. Alternatively, any of the muscarinic receptor I0085. In some embodiments, the neurotransmitter recep modulators listed in Table 1 can be used to antagonize a tor modulating agent is benztropine or a salt thereof (e.g., muscarinic receptor. Thus, in some embodiments, the mus benztropine mesylate). In some embodiments, the neu carinic receptor antagonist is a muscarinic receptor modula rotransmitter receptor modulating agentistrifluoperazine or a tor compound listed in Table 1. The compounds described in salt thereof (e.g., trifluoperazine hydrochloride). Table 1 are readily available. 0086 3. Histamine Receptor Antagonists 0082 In some embodiments, the neurotransmitter recep I0087. A histamine receptor is a G-protein coupled recep tor modulating agent is benztropine or a salt thereof (e.g., tor, for which the neurotransmitter histamine is the primary benztropine mesylate). In some embodiments, the neu endogenous ligand. There are four known Subtypes of hista rotransmitter receptor modulating agent is clemastine orasalt mine receptors: H receptors, H2 receptors, H receptors, and thereof (e.g., clemastine fumarate). He receptors. A histamine receptor antagonist is an agentable 0083. 2. Dopamine Receptor Antagonists to inhibit one or more characteristic responses of a histamine 0084. A dopamine receptor is a G-protein coupled recep receptor or receptor Subtype. As a non-limiting example, an tor, for which the neurotransmitter dopamine is the primary antagonist may competitively or non-competitively bind to endogenous ligand. There are five known Subtypes of dopam (1) a histamine receptor, (2) an agonist or partial agonist (or ine receptors: D and Ds receptors, the D-like receptors, other ligand) of the histamine receptor, and/or (3) a down activate adenylyl cyclase, while the D, D, and D receptors, stream signaling molecule to inhibit the histamine receptors the D-like receptors, inhibit adenylyl cyclase and activate K function. As shown in the Examples, clemastine has been channels. A dopamine receptor antagonist is an agent able to shown to antagonize the function of a histamine receptor. US 2014/0038949 A1 Feb. 6, 2014 32

Therefore, in some embodiments, the histamine receptor opioid receptor. Therefore, in some embodiments, the is clemastine or a salt, , racemic mixture, receptor antagonist is a compound selected from carbetapen conformational and/or optical isomer, crystalline polymorph, tane, Snc-80, BD-1047, and salts, prodrugs, racemic mix or isotopic variant thereof. Alternatively, any of the histamine tures, conformational and/or optical isomers, crystalline receptor modulators listed in Table 1 can be used to antago polymorphs, and isotopic variants thereof. Alternatively, any nize a histamine receptor. Thus, in Some embodiments, the of the opioid receptor modulators listed in Table 1 can be used histamine receptor antagonist is a histamine receptor modu to modulate an opioid receptor. Thus, in Some embodiments, lator compound listed in Table 1. The compounds described the opioid receptor modulator is an opioid receptor modulator in Table 1 are readily available. compound listed in Table 1. The compounds described in 0088. In some embodiments, the neurotransmitter recep Table 1 are readily available. tor modulating agent is clemastine or a salt thereof (e.g., 0094 B. Identification of Modulating Agents clemastine fumarate). 0.095 A number of different screening protocols can be 0089 4. Beta Adrenergic Receptor Modulators utilized to identify agents that stimulate increased myelina 0090 Abeta adrenergic receptor is a subtype of the adr tion of nerves. In general terms, the screening methods energic receptor, a G-protein coupled receptor, for which involve screening a plurality of agents to identify an agent that (e.g., epinephrine and norepinephrine) are increases the number of cells in a sample having a differen the primary endogenous ligand. There are three known Sub tiated, myelinating cell fate (e.g., mature myelinating oligo types of beta adrenergic receptors: B receptors, B receptors, dendrocyte). In some embodiments, an agent promotes or and f receptors. A beta adrenergic receptor antagonist is an increases OPC differentiation when it increases the percent agent able to inhibit one or more characteristic responses of a age of OPCs (e.g., in a sample comprising a plurality of beta adrenergic receptor or receptor Subtype. As a non-limit OPCs) that differentiate to a mature myelinating cell fate by at ing example, an antagonist may competitively or non-com least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or petitively bind to (1) a beta adrenergic receptor, (2) an agonist more as compared to the percentage of OPCs that differenti or partial agonist (or other ligand) of the beta adrenergic ate to a mature myelinating cell fate in the absence of the receptor, and/or (3) a downstream signaling molecule to agent. inhibit the beta adrenergic receptor's function. As a non (0096 1. Marker Assays limiting example, pindolol is able to antagonize the function 0097. In some embodiments, agents that stimulate of a beta adrenergic receptor. A beta adrenergic receptor increased myelination of nerves are identified by Screening agonist is an agent able to induce or stimulate one or more for induction of markers of mature myelinating oligodendro characteristic responses of a beta adrenergic receptor or cytes. In some embodiments, samples comprising a plurality receptor Subtype. As shown in the Examples, pindolol, sal of OPCs are contacted with a candidate agent, incubated meterol, salbutamol, and albuterol have been shown to ago under conditions suitable for the differentiation of OPCs, and nize the function of a beta adrenergic receptor and/or are evaluated for the presence or absence of one or more markers known of a beta adrenergic receptor. Therefore, in of mature myelinating oligodendrocytes. Examples of mark Some embodiments, the beta adrenergic receptor modulatoris ers of mature myelinating oligodendrocytes include, but are a compound selected from pindolol, Salmeterol, salbutamol. not limited to, myelin basic protein (MBP), myelin oligoden albuterol, and salts, prodrugs, racemic mixtures, conforma drocyte glycoprotein (MOG), 23'-cyclic-nucleotide 3' phos tional and/or optical isomers, crystalline polymorphs, and phodiesterase (CNP), GalC, O1, or O4. isotopic variants thereof. Alternatively, any of the beta adren 0.098 Markers of mature myelinating oligodendrocytes ergic receptor modulators listed in Table 1 can be used to can be detected using any number of established analytical modulate a beta adrenergic receptor. Thus, in some embodi techniques. For example, detection can be accomplished by ments, the beta adrenergic receptor modulator is a beta adr detecting nucleic acid (e.g., by in situ hybridization or RT energic receptor modulator compound listed in Table 1. The PCR) or protein (e.g., by immunoassay or Western blot analy compounds described in Table 1 are readily available. sis) levels, followed by visualization and/or quanitification 0091. In some embodiments, the neurotransmitter recep using any one of a variety of methods known in the art. In tor modulating agent is salmeterol or a salt thereof (e.g., Some embodiments, a marker of mature myelinating oligo salmeterol Xinfoate). In some embodiments, the neurotrans dendrocytes is detected by in situ hybridization. In situ mitter receptor modulating agent is salbutamol or a salt hybridization techniques are generally described in In Situ thereof (e.g., Salbutamol hemisulfate). Hybridization: A Practical Approach (Wilkinson, D.G., ed.), 0092 5. Opioid Receptor Modulators Oxford University Press, 1992. In some embodiments, a 0093. An opioid receptor is a G-protein coupled receptor, marker of mature myelinating oligodendrocytes is detected for which are the primary endogenous ligand. An by immunoassay. Immunoassay techniques and protocols are opioid receptor antagonist is an agent able to inhibit one or generally described in Price and Newman, “Principles and more characteristic responses of an opioid receptor or recep Practice of Immunoassay, 2nd Edition, Grove’s Dictionar tor Subtype. As a non-limiting example, an antagonist may ies, 1997; and Gosling, “Immunoassays: A Practical competitively or non-competitively bind to (1) an opioid Approach, Oxford University Press, 2000. In some embodi receptor, (2) an agonist or partial agonist (or other ligand) of ments, the immunoassay is an immunofluorescence assay. a receptor, and/or (3) a downstream signaling molecule to 0099. A detectable moiety can be used in the assays inhibit a receptor's function. An opioid receptoragonist is an described herein. A wide variety of detectable moieties can be agent able to induce or stimulate one or more characteristic used, with the choice of label depending on the sensitivity responses of an opioid receptor or receptor Subtype. For required, ease of conjugation with the antibody, stability example, an agonist may activate an opioid receptor. As requirements, and available instrumentation and disposal shown in the Examples, carbetapentane, Snc-80, and provisions. Suitable detectable moieties include, but are not BD-1047 have been shown to modulate the function of an limited to, radionuclides, fluorescent dyes (e.g., fluorescein, US 2014/0038949 A1 Feb. 6, 2014

fluorescein isothiocyanate (FITC), Oregon GreenTM, of the agents can be desirable because Smallermolecules have rhodamine, Texas red, tetrarhodimine isothiocynate a higher likelihood of having physiochemical properties com (TRITC), Cy3, Cy5, etc.), fluorescent markers (e.g., green patible with good pharmacokinetic characteristics, including fluorescent protein (GFP), phycoerythrin, etc.), auto oral absorption than agents with higher molecular weight. For quenched fluorescent compounds that are activated by tumor example, agents less likely to be successful as based on associated proteases, (e.g., luciferase, horseradish permeability and solubility were described by Lipinski et al. peroxidase, alkaline phosphatase, etc.), nanoparticles, biotin, as follows: having more than 5 H-bond donors (expressed as digoxigenin, and the like. the sum of OHs and NHs); having a molecular weight over 0100 2. Cells and Reagents 500; having a LogP over 5 (or MLogP over 4.15); and/or 0101 The primary screens for identifying agents that having more than 10 H-bond acceptors (expressed as the Sum induce OPC differentiation and/or stimulate increased myeli of Ns and Os). See, e.g., Lipinski et al., Adv. Drug Delivery nation of nerves can be performed in cell-based assays using Res 23:3-25 (1997). Compound classes that are substrates for cultured OPC cell lines or OPCs derived from a subject (e.g., biological transporters are typically exceptions to the rule. from a mammal). 0102 OPCs can be derived from any of a variety of 0107. In some embodiments, the agents are from a com sources. In some embodiments, OPCs are harvested from a binatorial chemical or peptide library containing a large num tissue, for example, brain tissue, spinal cord tissue, or optic ber of potential therapeutic compounds (potential modulator nerve tissue. The tissue can be from a rodent (e.g., rat or or ligand compounds). Such “combinatorial chemical librar mouse), chicken, dog, cat, rabbit, cow, sheep,goat, or primate ies' or “ligand libraries' are then screened in one or more (e.g., a monkey, a chimpanzee, or a human). In some embodi assays, as described herein, to identify those library members ments, OPCs are derived from fetal tissue. In some embodi (particular chemical species or Subclasses) that display a ments, OPCs are derived from adult tissue. Alternatively, desired characteristic activity. The compounds thus identified OPCs can be derived from culturing stem cells (e.g., neural can serve as conventional “lead compounds' or can them stem cells or embryonic stem cells) or from other cells that selves be used as potential or actual therapeutics. can be induced to give rise to OPCs (e.g., bone marrow stromal cells). 0108. A combinatorial chemical library is a collection of (0103 Examples of conditions suitable for OPC differen diverse chemical compounds generated by either chemical tiation are described in the Examples section below. Cell synthesis or biological synthesis, by combining a number of culture conditions are described in more detail, e.g., in Picot, chemical “building blocks.” For example, a linear combina Human Cell Culture Protocols (Methods in Molecular Medi torial chemical library such as a polypeptide library is formed cine) 2010 ed., and in Davis, Basic Cell Culture 2002 ed. by combining a set of chemical building blocks (amino acids) OPCs are cultured with growth factor, for example, PDG in every possible way for a given compound length (i.e., the FC.C. As a non-limiting example, OPCs are proliferated in number of amino acids in a polypeptide compound). Millions culture on poly-D- coated cell culture dishes using of chemical compounds can be synthesized through Such OPC media (Neurobasal media, B27 supplement without combinatorial mixing of chemical building blocks. Vitamin A, non-essential amino acids) containing 30 ng/mL 0109 Preparation and screening of combinatorial chemi PDGFC.C. For differentiation, OPCs are seeded on poly-D- cal libraries is well known to those of skill in the art. Such Lysine coated cell culture dishes using OPC media containing combinatorial chemical libraries include, but are not limited 2 ng/mL PDGFC.C. and treated with compounds dissolved in to, peptide libraries (see, e.g., U.S. Pat. No. 5,010, 175, Furka, DMSO (<1% final concentration). Differentiating OPCs are Int. J. Pept. Prot. Res. 37:487-493 (1991) and Houghton et al., incubated at 37°C., 5% CO for 6 days. At the end of 6 days, Nature 354:84-88 (1991)). Other chemistries for generating cells are fixed with 4% paraformaldehyde for immunofluo chemical diversity libraries can also be used. Such chemis rescence analysis or are harvested for biochemical analysis. tries include, but are not limited to: peptoids (e.g., PCT Pub 0104 3. Candidate Agents lication No. WO 91/19735), encoded (e.g., PCT 0105. The agents that are screened for the ability to pro Publication WO93/20242), random bio-oligomers (e.g., PCT mote OPC differentiation can be any small chemical com Publication No. WO92/00091), (e.g., U.S. pound, or a biological entity. Such as a polypeptide, Sugar, Pat. No. 5.288,514), diversomers such as , benzo nucleic acid or lipid. Typically, test compounds will be small diazepines and dipeptides (Hobbs et al., Proc. Nat. Acad. Sci. chemical molecules and peptides. Essentially any chemical USA 90:6909-6913 (1993)), vinylogous polypeptides (Hagi compound can be used as a potential modulator or ligand in hara et al., J. Amer: Chem. Soc. 114:6568 (1992)), nonpeptidal the assays of the invention, although most often compounds peptidomimetics with glucose scaffolding (Hirschmann et that can be dissolved in aqueous or organic (especially al., J. Amer: Chem. Soc. 114:9217-9218 (1992)), analogous DMSO-based) solutions are used. The assays are designed to organic syntheses of small compound libraries (Chen et al., J. screenlarge chemical libraries by automating the assay steps Amer: Chem. Soc. 116:2661 (1994)), oligocarbamates (Cho et and providing compounds from any convenient source to al., Science 261: 1303 (1993)), and/or peptidyl phosphonates assays, which are typically run in parallel (e.g., in microtiter (Campbell et al., J. Org. Chem. 59:658 (1994)), nucleic acid formats on microtiter plates in robotic assays). It will be libraries (see Ausubel, Berger and Sambrook, all Supra), pep appreciated that there are many suppliers of chemical com tide nucleic acid libraries (see, e.g., U.S. Pat. No. 5,539,083), pounds, including Sigma (St. Louis, Mo.), Aldrich (St. Louis, antibody libraries (see, e.g., Vaughn et al., Nature Biotech Mo.), Sigma-Aldrich (St. Louis, Mo.), Fluka Chemika-Bio nology, 14(3):309-314 1522 (1996) and U.S. Pat. No. 5,593, chemica Analytika (Buchs, Switzerland) and the like. 853), Small organic molecule libraries (see, e.g., benzodiaz 0106. In some embodiments, the agents have a molecular epines, Baum C&EN, January 18, page 33 (1993); weight of less than 1,500 daltons, and in Some cases less than isoprenoids, U.S. Pat. No. 5,569.588; thiazolidinones and 1,000, 800, 600, 500, or 400 daltons. The relatively small size metathiazanones, U.S. Pat. No. 5,549,974; pyrrolidines, U.S. US 2014/0038949 A1 Feb. 6, 2014 34

Pat. Nos. 5,525,735 and 5,519,134; morpholino compounds, Some embodiments, the neurotransmitter receptor modulat U.S. Pat. No. 5,506,337; benzodiazepines, U.S. Pat. No. ing agent is a compound selected from a muscarinic receptor 5,288,514, and the like). antagonist, a dopamine receptor antagonist, a histamine 0110 Devices for the preparation of combinatorial librar receptor antagonist, a beta adrenergic receptor modulator, ies are commercially available (see, e.g.,357 MPS,390 MPS, and an opioid receptor modulator. Advanced Chem Tech, Louisville Ky., Symphony, Rainin, 0116. In some embodiments, a subject in need of a method Woburn, Mass., 433A Applied Biosystems, Foster City, of stimulating increased myelination of nerves is a subject Calif., 9050 Plus, Millipore, Bedford, Mass.). In addition, having a demyelinating disease. Thus, in yet another aspect, numerous combinatorial libraries are themselves commer the present invention provides methods of treating and/or cially available (see, e.g., ComGenex, Princeton, N.J., Tripos, ameliorating a subject having a demyelinating disease. In Inc., St. Louis, Mo., 3D Pharmaceuticals, Exton, Pa., Martek Some embodiments, a subject in need of a method of stimu Biosciences, Columbia, Md., etc.). lating increased myelination of nerves is a subject at risk of 0111. In some embodiments, candidate agents are able to having a demyelinating disease. Thus, in yet another aspect, penetrate the blood-brain barrier. In some embodiments, can the present invention provides methods of preventing a demy didate agents have a low molecular weight (i.e., a molecular elinating disease or delaying the occurrence of a demyelinat weight of no more than 800 kDa). In some embodiments, ing disease. candidate agents are screened for one or more other criteria, 0117. In some embodiments, the method comprises Such as or brain . administering to the Subject a neurotransmitter receptor 0112 4. Validation modulating agent selected from a muscarinic receptor 0113 Agents that are initially identified by any of the antagonist, a dopamine receptor antagonist, a histamine foregoing screening methods can be further tested to validate receptor antagonist, a beta adrenergic receptor modulator, the apparentactivity. In some embodiments, validation assays and an opioid receptor modulator. In some embodiments, the are in vitro assays. In some embodiments, such studies are neurotransmitter receptor modulating agent is a compound conducted with suitable animal models. The basic format of listed in Table 1 (e.g., a muscarinic receptor modulator com Such methods involves administering a lead compound iden pound, dopamine receptor modulator compound, histamine tified during an initial screen to an animal that serves as a receptor modulator compound, beta adrenergic receptor disease model for humans and then determining if the disease modulator compound, or opioid receptor modulator com (e.g., a demyelinating disease) is in fact modulated and/or the pound listed in Table 1). In some embodiments, the neu disease or condition is ameliorated. The animal models uti rotransmitter receptor modulating agent is benztropine, cer lized in validation studies generally are mammals of any kind betapentane, clemastine, pindolol, ipratropium, atropine, Specific examples of Suitable animals include, but are not GBR12935, Snc-80, BD-1047, salmeterol, albuterol, or trif limited to, primates, mice, rats and Zebrafish. luoperazine, or a salt thereof. In some embodiments, the neurotransmitter receptor modulating agent is benztropine, III. Methods Using Neurotransmitter Receptor Modulating clemastine, Salmeterol, Salbutamol, trifluoperazine, or a salt Agents thereof. In some embodiments, the neurotransmitter receptor 0114. The neurotransmitter receptor modulating agents modulating agent is benztropine or a salt thereof (e.g., benz described herein can be used in various therapeutic and/or tropine mesylate). prophylactic methods. In one aspect, the present invention 0118. In some embodiments, the demyelinating disease is provides methods of inducing oligodendrocyte precursor cell multiple Sclerosis, idiopathic inflammatory demyelinating (OPC) differentiation to a mature myelinating cell fate (e.g., disease, transverse myelitis, Devic's disease, progressive myelinating oligodendrocytes). In some embodiments, the multifocal leukoencephalopathy, optic neuritis, leukodystro method comprises contacting the OPC with a neurotransmit phy, Guillain-Barre Syndrome, chronic inflammatory demy ter receptor modulating agent as described herein and cultur elinating polyneuropathy, autoimmune peripheral neuropa ing the OPC under conditions suitable for OPC differentia thy, Charcot-Marie-Tooth disease, acute disseminated tion. In some embodiments, the neurotransmitter receptor encephalomyelitis, adrenoleukodystrophy, adrenomyelo modulating agent is selected from a muscarinic receptor neuropathy, Leber's hereditary optic neuropathy, or human antagonist, a dopamine receptor antagonist, a histamine T-cell lymphotropic virus (HTLV)-associated myelopathy. receptor antagonist, a beta adrenergic receptor modulator, 0119. In some embodiments, the demyelinating disease is and an opioid receptor modulator. In some embodiments, the multiple sclerosis (MS). There are several subtypes of MS, OPC is cultured in the presence of the neurotransmitter recep including relapsing-remitting multiple Sclerosis (RRMS), tor modulating agent for at least 2 days, at least 3 days, at least secondary progressive multiple sclerosis (SPMS), primary 4 days, at least 5 days, at least 6 days or longer under condi progressive multiple sclerosis (PPMS), and progressive tions suitable for OPC differentiation. Differentiation to a relapsing multiple sclerosis (PRMS). In some embodiments, mature myelinating cell fate can be determined by detecting the subject has RRMS. In some embodiments, the subject has the presence of one or more biological markers of mature SPMS. In some embodiments, the subject has PPMS. In some myelinating oligodendrocytes. Marker assays for detecting embodiments, the subject has PRMS. A subject may initially the presence or level of myelinating oligodendrocytes are be diagnosed as having one subtype of MS (e.g., RRMS), and described herein, for example in Section II.(B) above. subsequently the subtype of MS afflicting the subject may 0115. In another aspect, the present invention provides convert to another subtype of MS (e.g., from RRMS to methods of stimulating increased myelination of nerves in a SPMS). It is contemplated that the methods of the present subject in need thereof. In some embodiments, the method invention can be applied to treat a Subject whose Subtype of comprises administering to the Subject a neurotransmitter MS converts to another subtype of MS. receptor modulating agent as described herein; thereby I0120 In some embodiments, a subject in need thereof stimulating increased myelination of nerves in the Subject. In (e.g., a Subject having a demyelinating disease or at risk for US 2014/0038949 A1 Feb. 6, 2014 having ademyelinating disease) is administered a neurotrans istering each agent alone. Thus, in another aspect, the present mitter receptor modulating agent in combination with at least invention provides methods of treating a subject in need one other therapy. In some embodiments, the at least one thereof by administering an immunomodulatory agent and a other therapy is an immunomodulatory agent. As used herein, neurotransmitter receptor modulating agent, wherein the an “immunomodulatory agent” refers to a disease-modifying immunomodulatory agent is administered at a subtherapeutic drug which alters the course of a demyelinating disease (e.g., dose. In some embodiments, the Subject has a demyelinating multiple Sclerosis, idiopathic inflammatory demyelinating disease or is at risk of having a demyelinating disease. disease, transverse myelitis, Devic's disease, progressive 0.124. In some embodiments, the demyelinating disease is multifocal leukoencephalopathy, optic neuritis, leukodystro multiple Sclerosis, idiopathic inflammatory demyelinating phy, Guillain-Barre Syndrome, chronic inflammatory demy disease, transverse myelitis, Devic's disease, progressive elinating polyneuropathy, autoimmune peripheral neuropa multifocal leukoencephalopathy, optic neuritis, leukodystro thy, Charcot-Marie-Tooth disease, acute disseminated phy, Guillain-Barre Syndrome, chronic inflammatory demy encephalomyelitis, adrenoleukodystrophy, adrenomyelo elinating polyneuropathy, autoimmune peripheral neuropa neuropathy, Leber's hereditary optic neuropathy, or HTLV thy, associated myelopathy). 0.125 Charcot-Marie-Tooth disease, acute disseminated 0121. In some embodiments, an immunomodulatory encephalomyelitis, adrenoleukodystrophy, adrenomyelo agent is a disease-modifying drug that alters the course of neuropathy, Leber's hereditary optic neuropathy, or HTLV multiple sclerosis (e.g., RRMS, SPMS, PPMS, or PRMS). associated myelopathy. In some embodiments, the demyeli For example, a disease-modifying drug can reduce the fre nating disease is multiple Sclerosis, e.g., relapsing-remitting quency or severity of an MS relapse and/or reduce develop multiple sclerosis (RRMS), secondary progressive multiple ment of lesions or Scars at regions of demyelination. In some sclerosis (SPMS), primary progressive multiple sclerosis embodiments, an immunomodulatory agent is an immuno (PPMS), or progressive relapsing multiple sclerosis (PRMS). Suppressant (i.e., an agent that Suppresses or prevents an In some embodiments, the Subject is initially diagnosed as immune response). In some embodiments, an immunomodu having one subtype of MS (e.g., RRMS), and subsequently latory agent is an agent that modulates an immune response the subtype of MS afflicting the subject converts to another (e.g., by stimulating the induction of Suppressor T cells). subtype of MS (e.g., from RRMS to SPMS). Examples of immunomodulatory agents for the treatment of I0126. In some embodiments, the neurotransmitter recep MS include, but are not limited to, interferons (e.g., inter tor modulating agent is selected from a muscarinic receptor feron-?3, e.g., interferon beta-1a or interferon beta-1b), glati antagonist, a dopamine receptor antagonist, a histamine rameracetate, mitoxantrone, fingolimod (FTY720), or mono receptor antagonist, a beta adrenergic receptor modulator, clonal antibodies (e.g., natalizumab, rituximab, daclizumab, and an opioid receptor modulator, and the immunomodula oralemtuzumab). Thus, in some embodiments, the method of tory agent is selected from fingolimod (FTY720), interferon the present invention comprises administering to a subject beta-1a, interferon beta-1b, glatirameracetate, mitoxantrone, having MS a neurotransmitter receptor modulating agent natalizumab, rituximab, daclizumab, and alemtuzumab. In (e.g., a compound selected from a muscarinic receptor Some embodiments, the neurotransmitter receptor modulat antagonist, a dopamine receptor antagonist, a histamine ing agent is a muscarinic receptor modulator compound, a receptor antagonist, a beta adrenergic receptor antagonist, dopamine receptor modulator compound, a histamine recep and an opioid receptor modulator) in combination with fin tor modulator compound, a beta adrenergic receptor modu golimod (FTY720), interferon beta-1a, interferon beta-1b, lator compound, or an opioid receptor modulator compound glatiramer acetate, mitoxantrone, natalizumab, rituximab, listed in Table 1, and the immunosuppressant is fingolimod daclizumab, or alemtuzumab. (FTY720), interferon beta-1a, interferon beta-1b, glatiramer 0122. In another aspect, the present invention provides acetate, mitoxantrone, natalizumab, rituximab, daclizumab, methods of enhancing the therapeutic effect of an immuno oralemtuzumab. In some embodiments, the neurotransmitter modulatory agent in a Subject in need thereof. It has been receptor modulating agent is benztropine, clemastine, salme Surprisingly found that in mouse models of demyelinating terol, salbutamol, or trifluoperazine, or a salt thereof, and the disease, administering a combination of a neurotransmitter immunomodulatory agent is fingolimod (FTY720), inter receptor modulating agent and an immunomodulatory agent feron beta-1a, interferon beta-1b, glatiramer acetate, mitox results in a significantly larger decrease in the clinical severity antrone, natalizumab, rituximab, daclizumab, or alemtu of the demyelinating disease as compared to the decrease in Zumab. In some embodiments, the neurotransmitter receptor the clinical severity of the demyelinating disease that can be modulating agent is benztropine and the immunomodulatory achieved with either the neurotransmitter receptor modulat agent is fingolimod (FTY720), interferon beta-1a, or inter ing agent or the immunomodulatory agent alone. Thus, in feron beta-1b. Some embodiments, the method comprises administering to a I0127. Therapeutic doses for the immunomodulatory Subject an immunomodulatory agent and a neurotransmitter agents fingolimod (FTY720), interferon beta-1a, interferon receptor modulating agent; thereby enhancing the therapeutic beta-1b, glatiramer acetate, mitoxantrone, and natalizumab effect of the immunomodulatory agent in the Subject. In some are known in the art. See, e.g., Kappos et al., N Engll Med embodiments, the Subject has a demyelinating disease or is at 362:387-401 (2010); Cohen et al., N Engl J Med 362:402-15 risk of having a demyelinating disease. (2010); Gottesman et al., Mult Scler 12:271-80 (2006); Hur 0123. Furthermore, it has also surprisingly been found that witzet al., Clin Ther 30:1102-12 (2008); Gaindhet al., Expert administering a neurotransmitter receptor modulating agent, Opin Biol. Ther 8:1823-29 (2008): Koch-Henriksen et al., in combination with an immunomodulatory agent at a dose Neurology 66:1056-60 (2006); Benatar, Lancet 360:1428 that, on its own, is insufficient to be therapeutic for the treat (2008); Jacobs et al., Ann Neurol 39:285-94 (1996); Lancet ment of a demyelinating disease, results in a therapeutic 352:1498-1504 (1998); Johnsonet al., Neurology 45:1268-76 effect that is greater than the therapeutic effect from admin (1995); Johnson et al., Neurology 50:701-08 (1998); Comiet US 2014/0038949 A1 Feb. 6, 2014 36 al., Ann Neurol. 69:75-82 (2011); Calabresi Nat Clin Pract than about 25%, less than about 20%, less than about 15%, Neurol 3:540-1 (2007); and Polman et al., N Engl J Med less than about 10%, or less than about 5% of the dose that is 354:899-910 (2006); the contents of each of which are incor conventionally administered for the neurotransmitter recep porated by reference herein in their entirety. tor modulating agent. In some embodiments, the neurotrans 0128. In some embodiments, the immunomodulatory mitter receptor modulating agent that is administered at a agent (e.g., fingolimod, interferon beta-1a, interferon beta therapeutically effective dose or at a subtherapeutic dose is 1b, glatiramer acetate, mitoxantrone, or natalizumab) is benztropine, clemastine, Salmeterol, salbutamol, trifluopera administered at a therapeutically effective dose. In some Zine, or a salt thereof. In some embodiments, the neurotrans embodiments, the immunomodulatory agent (e.g., fingoli mitter receptor modulating agent that is administered at a mod, interferon beta-1a, interferon beta-1b, glatiramer therapeutically effective dose or at a subtherapeutic dose is acetate, mitoxantrone, or natalizumab) is administered at a benztropine or a salt thereof (e.g., benztropine mesylate). As Subtherapeutic dose, e.g., at a dose that is less than about 75%, a non-limiting example, a therapeutically effective dose of less than about 70%, less than about 60%, less than about benztropine may be from about 1 mg per day to about 10 mg 50%, less than about 40%, less than about 30%, less than per day (e.g., about 1, about 2, about 3, about 4, about 5, about about 25%, less than about 20%, less than about 15%, less 6, about 7, about 8, about 9, or about 10 mg per day). Thus, in than about 10%, or less than about 5% of the dose that is Some embodiments, a subtherapeutic dose of benztropine conventionally administered for the immunomodulatory may be from about 0.01 mg per day to about 0.75 mg per day agent. (e.g., about 0.01, about 0.05, about 0.10, about 0.015, about 0129. Fingolimod (FTY720) is conventionally adminis 0.20, about 0.25, about 0.30, about 0.35, about 0.40, about tered at a therapeutically effective dose of from about 0.5 mg 0.45, about 0.50, about 0.55, about 0.60, about 0.65, about per day to about 1.5 mg per day (e.g., about 0.5, about 0.6, 0.70, or about 0.75 mg per day). about 0.7, about 0.8, about 0.9, about 1.0, about 1.1, about 1.2, I0133. In some embodiments, a neurotransmitter receptor about 1.3, about 1.4, or about 1.5 mg per day). See, e.g., modulating agent (e.g., a muscarinic receptor antagonist, a Kappos et al., N Engl J Med 362:387-401 (2010). Thus, in dopamine receptor antagonist, a histamine receptor antago Some embodiments, a Subtherapeutic dose of fingolimod is nist, a beta adrenergic receptor modulator, oran opioid recep from about 0.005 mg per day to about 0.375 mg per day (e.g., tor modulator listed in Table 1, e.g., benztropine, clemastine, about 0.005, about 0.01, about 0.02, about 0.03, about 0.04, salmeterol, salbutamol, trifluoperazine, or a salt thereof) is about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, administered at a therapeutically effective dose and an immu about 0.1, about 0.15, about 0.2, about 0.25, about 0.3, about nomodulatory agent (e.g., fingolimod, interferon beta-la, 0.35, or about 0.375 mg per day). interferon beta-1b, glatiramer acetate, mitoxantrone, or 0130 Interferon beta-1a is conventionally administered at natalizumab) is administered at a therapeutically effective a therapeutically effective dose of about 30 ug per week. See dose. In some embodiments, the neurotransmitter receptor e.g., Jacobs et al., Ann Neurol 39:285-94 (1996). Thus, in modulating agent is benztropine or a salt thereof (e.g., benz some embodiments, a subtherapeutic dose of interferon beta tropine mesylate) and the immunomodulatory agent is fin 1a is from about 0.3 ug per week to about 23 Jug per week (e.g., golimod (FTY720), interferon beta-1a, or interferon beta-1b. about 0.3, about 0.5, about 0.75, about 1, about 2, about 3, In Some embodiments, benztropine is administered at a thera about 4, about 5, about 6, about 7, about 8, about 9, about 10, peutically effective dose of from about 1 mg per day to about about 11, about 12, about 13, about 14, about 15, about 16, 10 mg per day; fingolimod is administered at atherapeutically about 17, about 18, about 19, about 20, about 21, about 22, or effective dose of from about 0.5 mg per day to about 1.5 mg about 23 Jug per week). per day; interferon beta-1a is administered at a therapeuti 0131 Interferon beta-1b is conventionally administered at cally effective dose of about 30 ug per week; and/or interferon a therapeutically effective dose of from about 250 ug every beta-1b is administered at a therapeutically effective dose of other day to about 500 ug every other day. See, e.g., Gottes from about 250 ug every other day to about 500 ug every other man et al., Mult Scler 12:271-80 (2006). Thus, in some day. embodiments, a subtherapeutic dose of interferon beta-1b is I0134. In some embodiments, a neurotransmitter receptor from about 2 ug every other day to about 190 ug every other modulating agent (e.g., a muscarinic receptor antagonist, a day (e.g., about 2, about 5, about 10, about 20, about 30, about dopamine receptor antagonist, a histamine receptor antago 40, about 50, about 60, about 70, about 80, about 90, about nist, a beta adrenergic receptor modulator, oran opioid recep 100, about 110, about 120, about 130, about 140, about 150, tor modulator listed in Table 1, e.g., benztropine, clemastine, about 160, about 170, about 180, or about 190 ug every other salmeterol, salbutamol, trifluoperazine, or a salt thereof) is day). administered at a therapeutically effective dose and an immu 0.132. In some embodiments, the neurotransmitter recep nomodulatory agent (e.g., fingolimod, interferon beta-1a, tor modulating agent (e.g., a muscarinic receptor antagonist, interferon beta-1b, glatiramer acetate, mitoxantrone, or a dopamine receptor antagonist, a histamine receptor antago natalizumab) is administered at a subtherapeutic dose, e.g., at nist, a beta adrenergic receptor modulator, oran opioid recep a dose that is less than about 75%, less than about 70%, less tor modulator listed in Table 1) is administered at a therapeu than about 60%, less than about 50%, less than about 40%, tically effective dose. In some embodiments, the less than about 30%, less than about 25%, less than about neurotransmitter receptor modulating agent (e.g., muscarinic 20%, less than about 15%, less than about 10%, or less than receptor antagonist, dopamine receptor antagonist, histamine about 5% of the dose that is conventionally administered for receptor antagonist, beta adrenergic receptor modulator, or the immunomodulatory agent. In some embodiments, the opioid receptor modulator listed in Table 1) is administered at neurotransmitter receptor modulating agent is benztropine or a subtherapeutic dose, e.g., at a dose that is less than about a salt thereof (e.g., benztropine mesylate) and the immuno 75%, less than about 70%, less than about 60%, less than modulatory agent is fingolimod (FTY720), interferon beta about 50%, less than about 40%, less than about 30%, less 1a, or interferon beta-1b. In some embodiments, benztropine US 2014/0038949 A1 Feb. 6, 2014 37 is administered at a therapeutically effective dose of from dose of from about 0.005 mg per day to about 0.375 mg per about 1 mg per day to about 10 mg per day; fingolimod is day; interferon beta-1a is administered at a subtherapeutic administered at a subtherapeutic dose of from about 0.005 mg dose of from about 0.3 g per week to about 23 Jug per week; per day to about 0.375 mg per day; interferon beta-1a is and/or interferon beta-1b is administered at a subtherapeutic administered at a subtherapeutic dose of from about 0.3 ug dose of from about 2 ug every other day to about 190 ug every per week to about 23 Jug per week; and/or interferon beta-1b other day. is administered at a subtherapeutic dose of from about 2 ug 0.137 In some embodiments, a neurotransmitter receptor every other day to about 190 ug every other day. modulating agent (e.g., a muscarinic receptor antagonist, a 0135) In some embodiments, a neurotransmitter receptor dopamine receptor antagonist, a histamine receptor antago modulating agent (e.g., muscarinic receptor antagonist, nist, a beta adrenergic receptor modulator, oran opioid recep dopamine receptor antagonist, histamine receptor antagonist, tor modulator listed in Table 1, e.g., benztropine, clemastine, beta adrenergic receptor modulator, or opioid receptor modu salmeterol, salbutamol, trifluoperazine, or a salt thereof) is lator listed in Table 1, e.g., benztropine, clemastine, Salme administered at a therapeutically effective dose and an immu terol, Salbutamol, trifluoperazine, or a salt thereof) is admin nomodulatory agent (e.g., fingolimod, interferon beta-1a, istered at a Subtherapeutic dose, e.g., at a dose that is less than interferon beta-1b, glatiramer acetate, mitoxantrone, or about 75%, less than about 70%, less than about 60%, less natalizumab) is administered at a therapeutically effective than about 50%, less than about 40%, less than about 30%, dose for the treatment or prevention of a demyelinating dis less than about 25%, less than about 20%, less than about ease (e.g., multiple Sclerosis). In some embodiments, a neu 15%, less than about 10%, or less than about 5% of the dose rotransmitter receptor modulating agent (e.g., a muscarinic that is conventionally administered for the neurotransmitter receptor antagonist, a dopamine receptor antagonist, a hista receptor modulating agent, and an immunomodulatory agent mine receptor antagonist, a beta adrenergic receptor modula (e.g., fingolimod, interferon beta-1a, interferon beta-1b, glati tor, or an opioid receptor modulator listed in Table 1, e.g., ramer acetate, mitoxantrone, or natalizumab) is administered benztropine, clemastine, Salmeterol, salbutamol, trifluopera at a therapeutically effective dose. In some embodiments, the Zine, or a salt thereof) is administered at a therapeutically neurotransmitter receptor modulating agent is benztropine or effective dose and an immunomodulatory agent (e.g., fingoli a salt thereof (e.g., benztropine mesylate) and the immuno mod, interferon beta-1a, interferon beta-1b, glatiramer modulatory agent is fingolimod (FTY720), interferon beta acetate, mitoxantrone, or natalizumab) is administered at a 1a, or interferon beta-1b. In some embodiments, bentropine is subtherapeutic dose for the treatment or prevention of a administered at a subtherapeutic dose of from about 0.01 mg demyelinating disease (e.g., multiple sclerosis). In some per day to about 0.75 mg per day; fingolimod is administered embodiments, a neurotransmitter receptor modulating agent at a therapeutically effective dose of from about 0.5 mg per (e.g., a muscarinic receptor antagonist, a dopamine receptor day to about 1.5 mg per day; interferon beta-1a is adminis antagonist, a histamine receptor antagonist, a beta adrenergic tered at a therapeutically effective dose of about 30 ug per receptor modulator, or an opioid receptor modulator listed in week; and/or interferon beta-1b is administered at a therapeu Table 1, e.g., benztropine, clemastine, Salmeterol, salbuta tically effective dose of from about 250 ug every other day to mol, trifluoperazine, or a salt thereof) is administered at a about 500 ug every other day. Subtherapeutic dose and an immunomodulatory agent (e.g., fingolimod, interferon beta-1a, interferon beta-1b, glatiramer 0136. In some embodiments, a neurotransmitter receptor acetate, mitoxantrone, or natalizumab) is administered at a modulating agent (e.g., muscarinic receptor antagonist, therapeutically effective dose for the treatment or prevention dopamine receptor antagonist, histamine receptor antagonist, of a demyelinating disease (e.g., multiple Sclerosis). In some beta adrenergic receptor modulator, or opioid receptor modu embodiments, a neurotransmitter receptor modulating agent lator listed in Table 1, e.g., benztropine, clemastine, Salme (e.g., a muscarinic receptor antagonist, a dopamine receptor terol, Salbutamol, trifluoperazine, or a salt thereof) is admin antagonist, a histamine receptor antagonist, a beta adrenergic istered at a Subtherapeutic dose, e.g., at a dose that is less than receptor modulator, or an opioid receptor modulator listed in about 75%, less than about 70%, less than about 60%, less Table 1, e.g., benztropine, clemastine, Salmeterol, salbuta than about 50%, less than about 40%, less than about 30%, mol, trifluoperazine, or a salt thereof) is administered at a less than about 25%, less than about 20%, less than about Subtherapeutic dose and an immunomodulatory agent (e.g., 15%, less than about 10%, or less than about 5% of the dose fingolimod, interferon beta-1a, interferon beta-1b, glatiramer that is conventionally administered for the neurotransmitter acetate, mitoxantrone, or natalizumab) is administered at a receptor modulating agent, and an immunomodulatory agent subtherapeutic dose for the treatment or prevention of a (e.g., fingolimod, interferon beta-1a, interferon beta-1b, glati demyelinating disease (e.g., multiple Sclerosis). ramer acetate, mitoxantrone, or natalizumab) is administered at a subtherapeutic dose, e.g., at a dose that is less than about IV. Pharmaceutical Compositions 75%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less 0.138. In another aspect, the present invention provides than about 25%, less than about 20%, less than about 15%, pharmaceutical compositions for use in the treatment of a less than about 10%, or less than about 5% of the dose that is demyelinating disease (e.g., multiple Sclerosis, idiopathic conventionally administered for the immunomodulatory inflammatory demyelinating disease, transverse myelitis, agent. In some embodiments, the neurotransmitter receptor Devic's disease, progressive multifocal leukoencephalopa modulating agent is benztropine or a salt thereof (e.g., benz thy, optic neuritis, leukodystrophy, Guillain-Barre syndrome, tropine mesylate) and the immunomodulatory agent is fin chronic inflammatory demyelinating polyneuropathy, golimod (FTY720), interferon beta-1a, or interferon beta-1b. autoimmune peripheral neuropathy, Charcot-Marie-Tooth In some embodiments, bentropine is administered at a Sub disease, acute disseminated encephalomyelitis, adrenoleu therapeutic dose of from about 0.01 mg per day to about 0.75 kodystrophy, adrenomyeloneuropathy, Leber's hereditary mg per day; fingolimod is administered at a subtherapeutic optic neuropathy, or human T-cell lymphotropic virus US 2014/0038949 A1 Feb. 6, 2014

(HTLV)-associated myelopathy). In some embodiments, the the agent can be administered in a local rather than systemic composition comprises a mixture of a neurotransmitter recep manner, for example, in a depot or Sustained release formu tor modulating agent and an immunomodulatory agent. In lation. Some embodiments, the pharmaceutical composition com prises a neurotransmitter receptor modulating agent selected Formulations from a muscarinic receptor antagonist, a dopamine receptor 0.142 Suitable formulations for use in the present inven antagonist, a histamine receptor antagonist, a beta adrenergic tion are found in Remington. The Science and Practice of receptor modulator, and an opioid receptor modulator and an Pharmacy, 21st Ed., Gennaro, Ed., Lippencott Williams & immunomodulatory agent selected from interferon beta-1a, Wilkins (2003), which is hereby incorporated herein by ref interferon beta-1b, glatiramer acetate, mitoxantrone, fingoli erence. The pharmaceutical compositions described herein mod (FTY720), natalizumab, rituximab, daclizumab, and can be manufactured in a manner that is known to those of alemtuzumab. In some embodiments, the pharmaceutical skill in the art, i.e., by means of conventional mixing, dissolv composition comprises a neurotransmitter receptor modulat ing, granulating, dragee-making, levigating, emulsifying, ing agent selected from benztropine, clemastine, salmeterol, encapsulating, entrapping or lyophilizing processes. The fol salbutamol, trifluoperazine, and salts thereof and an immu lowing methods and excipients are merely exemplary and are nomodulatory agent selected from interferon beta-1a, inter in no way limiting. feron beta-1b, glatiramer acetate, mitoxantrone, fingolimod 0143. In some embodiments, an agent is prepared for delivery in a Sustained-release, controlled release, extended (FTY720), natalizumab, rituximab, daclizumab, and alemtu release, timed-release or delayed-release formulation, for Zumab. In some embodiments, the pharmaceutical composi example, in semipermeable matrices of Solid hydrophobic tion comprises benztropine or a salt thereof and an immuno polymers containing the therapeutic agent. Various types of modulatory agent selected from interferon beta-1a, interferon Sustained-release materials have been established and are beta-1b, and fingolimod (FTY720). well known by those skilled in the art. Current extended 0139. In some embodiments, one or both of the neu release formulations include film-coated tablets, multipar rotransmitter receptor modulating agent and the immuno ticulate or pellet systems, matrix technologies using hydro modulatory agent are formulated as a therapeutically effec philic or lipophilic materials and wax-based tablets with tive or optimal dose. In some embodiments, one or both of the pore-forming excipients (see, for example, Huang, et al. Drug neurotransmitter receptor modulating agent and the immuno Dev. Ind. Pharm. 29:79 (2003); Pearnchob, et al. Drug Dev. modulatory agent are formulated as a subtherapeutic dose. In Ind. Pharm. 29.925 (2003); Maggi, et al. Eur: J. Pharm. Some embodiments, the neurotransmitter receptor modulat Biopharm. 55:99 (2003); Khanvilkar, et al., Drug Dev. Ind. ing agent is formulated as a therapeutically effective or opti Pharm. 228:601 (2002); and Schmidt, et al., Int. J. Pharm. mal dose and the immunomodulatory agent is formulated as a 216:9 (2001)). Sustained-release delivery systems can, Subtherapeutic dose. In some embodiments, the immuno depending on their design, release the compounds over the modulatory agent is formulated as a therapeutically effective course of hours or days, for instance, over 4, 6, 8, 10, 12, 16, or optimal dose and the neurotransmitter receptor modulating 20, 24 hours or more. Usually, Sustained release formulations agent is formulated as a subtherapeutic dose. Suitable dosage can be prepared using naturally-occurring or synthetic poly ranges for therapeutically effective and Subtherapeutic doses mers, for instance, polymeric vinyl pyrrolidones, such as of neurotransmitter receptor modulating agents and immuno polyvinyl pyrrolidone (PVP); carboxyvinyl hydrophilic poly modulatory agents are described above. mers; hydrophobic and/or hydrophilic hydrocolloids, such as methylcellulose, ethylcellulose, hydroxypropylcellulose, 0140. An agent for use in any of the therapeutic methods of and hydroxypropylmethylcellulose; and carboxypolymethyl the present invention (e.g., an agent that stimulates increased CC. myelination as described herein, or an immunomodulatory 0144. The sustained or extended-release formulations can agent as described herein) may be in any pharmaceutically also be prepared using natural ingredients, such as minerals, acceptable form, including any pharmaceutically acceptable including titanium dioxide, silicon dioxide, oxide, and salts, prodrugs, racemic mixtures, conformational and/or clay (see, U.S. Pat. No. 6,638,521, herein incorporated by optical isomers, crystalline polymorphs and isotopic variants reference). Exemplified extended release formulations that of the neurotransmitter receptor modulating agents. can be used in delivering a compound of the present invention 0141. A combination of a neurotransmitter receptor include those described in U.S. Pat. Nos. 6,635,680; 6,624, modulating agent and an immunomodulatory agent can be 200; 6,613,361; 6,613,358, 6,596,308; 6,589,563; 6,562,375; incorporated into a variety of formulations for therapeutic or 6,548,084; 6,541,020; 6,537,579: 6,528,080 and 6,524,621, prophylactic administration. More particularly, a combina each of which is hereby incorporated herein by reference. tion of a neurotransmitter receptor modulating agent and an Controlled release formulations of particular interest include immunomodulatory agent can beformulated into pharmaceu those described in U.S. Pat. Nos. 6,607,751; 6,599,529; tical compositions, e.g., a single composition, by formulation 6,569,463; 6,565,883; 6,482,440; 6,403,597; 6,319,919: with appropriate pharmaceutically acceptable carriers or 6,150,354; 6,080,736; 5,672.356; 5,472,704; 5,445,829: diluents, and can be formulated into preparations in Solid, 5.312,817 and 5.296.483, each of which is hereby incorpo semi-solid, liquid orgaseous forms, such as tablets, capsules, rated herein by reference. Those skilled in the art will readily pills, powders, granules, dragees, gels, slurries, ointments, recognize other applicable Sustained release formulations. Solutions, Suppositories, injections, and aerosols. 0145 The pharmaceutical formulations of the invention AS Such, administration of an agent of the present invention can be provided as a salt and can be formed with many acids, can be achieved in various ways, including oral, buccal, including but not limited to hydrochloric, Sulfuric, acetic, parenteral, intravenous, intradermal (e.g., Subcutaneous, lactic, tartaric, malic, Succinic, etc. Salts tend to be more intramuscular), transdermal, etc., administration. Moreover, soluble in aqueous or other protonic solvents that are the US 2014/0038949 A1 Feb. 6, 2014 39 corresponding free base forms. In other cases, the preparation 0150 Pharmaceutical formulations for parenteral admin may be a lyophilized powder in 1 mM-50 mM , istration include aqueous solutions of the active agents in 0.1%-2% sucrose, 2%-7% at a pH range of 4.5 to water-soluble form. Additionally, suspensions of the active 5.5, that is combined with buffer prior to use. agents can be prepared as appropriate oily injection Suspen 0146 For oral administration, an agent of the present sions. Suitable lipophilic solvents or vehicles include fatty invention can be formulated readily by combining with phar oils such as sesame oil, or synthetic esters. Such as maceutically acceptable carriers that are well known in the ethyl oleate or triglycerides, or liposomes. Aqueous injection art. Such carriers enable the compounds to be formulated as Suspensions can contain Substances which increase the vis tablets, pills, dragees, capsules, emulsions, lipophilic and cosity of the Suspension, Such as Sodium carboxymethyl cel hydrophilic suspensions, liquids, gels, syrups, slurries, Sus lulose, Sorbitol, or dextran. Optionally, the Suspension can pensions and the like, for oral ingestion by a patient to be also contain Suitable stabilizers or agents which increase the treated. Pharmaceutical preparations for oral use can be solubility of the compounds to allow for the preparation of obtained by mixing the compounds with a solid excipient, highly concentrated solutions. Alternatively, the active ingre optionally grinding a resulting mixture, and processing the dient can be in powder form for constitution with a suitable mixture of granules, after adding Suitable auxiliaries, if vehicle, e.g., sterile pyrogen-free water, before use. desired, to obtain tablets or dragee cores. Suitable excipients 0151. Systemic administration can also be by transmu are, in particular, fillers such as Sugars, including lactose, cosal or transdermal means. For transmucosal or transdermal Sucrose, mannitol, or Sorbitol; cellulose preparations such as, administration, penetrants appropriate to the barrier to be for example, maize starch, wheat starch, rice starch, potato permeated are used in the formulation. For topical adminis starch, gelatin, gum tragacanth, methyl cellulose, hydrox tration, the agents are formulated into ointments, creams, ypropylmethyl-cellulose, Sodium carboxymethylcellulose, salves, powders and gels. In one embodiment, the transder and/or polyvinylpyrrolidone (PVP). If desired, disintegrating mal delivery agent can be DMSO. Transdermal delivery sys agents can be added. Such as a cross-linked polyvinyl pyrroli tems can include, e.g., patches. For transmucosal administra done, agar, or alginic acid or a salt thereof Such as sodium tion, penetrants appropriate to the barrier to be permeated are alginate. used in the formulation. Such penetrants are generally known 0147 Pharmaceutical preparations which can be used in the art. Exemplified transdermal delivery formulations that orally include push-fit capsules made of gelatin, as well as can find use in the present invention include those described soft, sealed capsules made of gelatin and a plasticizer, such as in U.S. Pat. Nos. 6,589,549; 6,544,548; 6,517,864; 6,512, glycerol or sorbitol. The push-fit capsules can contain the 010; 6,465,006; 6,379,696; 6,312,717 and 6,310,177, each of active ingredients in admixture with filler such as lactose, which are hereby incorporated herein by reference. binders such as starches, and/or lubricants such as talc or 0152 For buccal administration, the agents can take the magnesium Stearate and, optionally, stabilizers. In soft cap form of tablets or lozenges formulated in conventional man Sules, the active compounds can be dissolved or Suspended in Suitable liquids, such as fatty oils, liquid paraffin, or liquid 0153. In addition to the formulations described previously, polyethylene glycols. In addition, stabilizers can be added. an agent of the present invention can also be formulated as a All formulations for oral administration should be in dosages depot preparation. Such long acting formulations can be Suitable for Such administration. administered by implantation (for example Subcutaneously or 0148 Dragee cores are provided with suitable coatings. intramuscularly) or by intramuscular injection. Thus, for For this purpose, concentrated Sugar Solutions can be used, example, the agents can be formulated with Suitable poly which can optionally contain gum arabic, talc, polyvinyl pyr meric or hydrophobic materials (for example as an emulsion rolidone, carbopol gel, polyethylene glycol, and/or titanium in an acceptable oil) or ion exchange resins, or as sparingly dioxide, lacquer Solutions, and Suitable organic solvents or soluble derivatives, for example, as a sparingly soluble salt. solvent mixtures. Dyestuffs or pigments can be added to the 0154 The pharmaceutical compositions also can com tablets or dragee coatings for identification or to characterize prise Suitable Solid or gel phase carriers or excipients. different combinations of active compound doses. Examples of Such carriers or excipients include but are not 014.9 The agents can be formulated for parenteral admin limited to calcium carbonate, calcium phosphate, various istration by injection, e.g., by bolus injection or continuous Sugars, starches, cellulose derivatives, gelatin, and polymers infusion. For injection, the compound can be formulated into Such as polyethylene glycols. preparations by dissolving, Suspending or emulsifying them 0155 Pharmaceutical compositions suitable for use in the in an aqueous or nonaqueous solvent, such as Vegetable or present invention include compositions wherein the active other similar oils, syntheticaliphatic acid glycerides, esters of ingredients are contained in a therapeutically effective higheraliphatic acids or propylene glycol; and if desired, with amount. The present invention also contemplates pharmaceu conventional additives such as solubilizers, isotonic agents, tical compositions comprising the neurotransmitter receptor Suspending agents, emulsifying agents, stabilizers and pre modulating compounds as described herein with an effective servatives. In some embodiments, an agent of the invention amount of other therapeutic agents as combination partners, can be formulated in aqueous Solutions, preferably in physi particularly those used for treating demyelinating diseases, ologically compatible buffers such as Hanks’s solution, Ring Such as immunomodulary agents. An effective amount of the er's solution, or physiological saline buffer. Formulations for agent and/or combination partner will, of course, be depen injection can be presented in unit dosage form, e.g., in dent on the subject being treated, the severity of the affliction ampules or in multi-dose containers, with an added preserva and the manner of administration. Determination of an effec tive. The compositions can take Such forms as Suspensions, tive amount is well within the capability of those skilled in the Solutions or emulsions in oily or aqueous vehicles, and can art, especially in light of the detailed disclosure provided contain formulatory agents such as Suspending, stabilizing herein. Generally, an efficacious or effective amount of an and/or dispersing agents. agent is determined by first administering a low dose or Small US 2014/0038949 A1 Feb. 6, 2014 40 amount, and then incrementally increasing the administered and/or an immunomodulatory agent) is administered to a dose or dosages until a desired therapeutic effect is observed subject in need thereof over an extended period of time. The in the treated subject, with minimal or no toxic side effects. methods can be carried out for at least 20 days, in some Applicable methods for determining an appropriate dose and embodiments for at least 40, 60, 80 or 100 days, and in some dosing schedule for administration of the present invention embodiments for at least 150, 200,250,300,350 days, 1 year are described, for example, in Goodman and Gilman's The or longer. In some embodiments, a compound of the present Pharmacological Basis of Therapeutics, 11th Ed., Brunton, invention (e.g., a neurotransmitter receptor modulating agent Lazo and Parker, Eds. McGraw-Hill (2006), and in Reming and/or an immunomodulatory agent) is administered to a ton: The Science and Practice of Pharmacy, 21st Ed., subject in need thereof at or after the onset of one or more Gennaro, Ed., Lippencott Williams & Wilkins (2003), both of symptoms of ademyelinating disease. In some embodiments, which are hereby incorporated herein by reference. In some a compound of the present invention (e.g., a neurotransmitter embodiments, one or both of a neurotransmitter receptor receptor modulating agent and/or an immunomodulatory modulating agent and an immunomodulary agent are formu agent) is administered to a subject in need thereofprior to the lated in a therapeutically effective dose. In some embodi onset of symptoms of a demyelinating disease (i.e., prophy ments, one or both of a neurotransmitter receptor modulating lactically). agent and an immunomodulary agent are formulated in a Subtherapeutic dose. In some embodiments, a neurotransmit Co-Administration ter receptor modulating agent is formulated in a therapeuti cally effective dose and an immunomodulary agent is formu 0160. In some embodiments, the methods of the present lated in a subtherapeutic dose. In some embodiments, an invention comprise co-administering a neurotransmitter immunomodulary agent is formulated in a therapeutically receptor modulating agent and an immunomodulatory agent. effective dose and a neurotransmitter receptor modulating Co-administered agents can be administered together or sepa agent is formulated in a subtherapeutic dose. rately, simultaneously or at different times. When adminis 0156 The pharmaceutical preparation is preferably in unit tered, the neurotransmitter receptor modulating agent and the dosage form. In such form the preparation is Subdivided into immunomodulatory agent independently can be adminis unit doses containing appropriate quantities of the active tered once, twice, three, four times daily or more or less often, component. The unit dosage form can be a packaged prepa as needed. In some embodiments, the agents are administered ration, the package containing discrete quantities of prepara once daily. In some embodiments, the agents are adminis tion, such as packeted tablets, capsules, and powders in vials tered at the same time or times, for instance as an admixture. or ampoules. Also, the unit dosage form can be a capsule, One or more of the agents can be administered in a sustained , cachet, or lozenge itself, or it can be the appropriate release formulation. number of any of these in packaged form. 0.161. In some embodiments, co-administration includes administering one active agent within 0.5, 1, 2, 4, 6, 8, 10, 12. Administration 16, 20, or 24 hours of a second active agent. Co-administra tion includes administering two active agents simultaneously, 0157 Administration of a neurotransmitter receptor approximately simultaneously (e.g., within about 1, 5, 10, 15. modulating agent and/or an immunomodulatory agent can be 20, or 30 minutes of each other), or sequentially in any order. achieved in various ways, including oral, buccal, parenteral, In some embodiments, co-administration can be accom including intravenous, intradermal. Subcutaneous, intramus plished by co-formulation, i.e., preparing a single pharma cular, transdermal, transmucosal, intranasal, etc., administra ceutical composition including both active agents (i.e., the tion. When a neurotransmitter receptor modulating agent and neurotransmitter receptor modulating agent and the immuno an immunomodulatory agent are co-administered, the neu modulatory agent are administered as a single formulation). rotransmitter receptor modulating agent can be administered In other embodiments, the active agents can be formulated by the same or different route of administration as the immu separately (i.e., the neurotransmitter receptor modulating nomodulatory agent. In some embodiments, a compound of agent and the immunomodulatory agent are administered as the present invention (e.g., a neurotransmitter receptor modu separate formulations). In another embodiment, the active lating agent and/or an immunomodulatory agent) is adminis and/or adjunctive agents may be linked or conjugated to one tered systemically. another. 0158. The dosages and frequency of administration will (0162. In some embodiments, one or both of a neurotrans depend upon various factors generally appreciated by those of mitter receptor modulating agent and an immunomodulatory skill in the art, including, e.g., the severity of a subjects agent can be administered prophylactically to prevent unde disease. Generally, daily doses can range from about 0.001 sirable recurrence of the symptoms of the demyelinating dis 100 mg/kg total body weight, from about 0.01-50 mg/kg, or ease (e.g., to prevent or delay the recurrence of clinical attacks from about 0.01 mg/kg-10 mg/kg. However, doses in the in MS), or therapeutically to achieve a desired reduction in range of 10-500 mg/kg per day may be effective and well symptoms of the demyelinating disease and maintain Such tolerated. The principal determining factor in defining the reduction in Symptoms of the demyelinating disease for a appropriate dose is the amount of a particular compound necessary to be therapeutically effective in a particular con Sustained period of time. text. Repeated administrations may be required in order to achieve longer lasting immune tolerance. Single or multiple V. Kits administrations of the compositions can be carried out with 0163. In another aspect, the present invention provides kits the dose levels and pattern being selected by the treating for use in the treatment of a demyelinating disease (e.g., physician. multiple Sclerosis, idiopathic inflammatory demyelinating 0159. In some embodiments, a compound of the present disease, transverse myelitis, Devic's disease, progressive invention (e.g., a neurotransmitter receptor modulating agent multifocal leukoencephalopathy, optic neuritis, leukodystro US 2014/0038949 A1 Feb. 6, 2014

phy, Guillain-Barre Syndrome, chronic inflammatory demy EXAMPLES elinating polyneuropathy, autoimmune peripheral neuropa thy, Charcot-Marie-Tooth disease, acute disseminated 0166 The following examples are offered to illustrate, but encephalomyelitis, adrenoleukodystrophy, adrenomyelo not to limit the claimed invention. neuropathy, Leber's hereditary optic neuropathy, or human Example 1 T-cell lymphotropic virus (HTLV)-associated myelopathy). In some embodiments, the kit comprises a neurotransmitter receptor modulating agent selected from a muscarinic recep Small Molecule Screen to Identify Inducers of OPC tor antagonist, a dopamine receptor antagonist, a histamine Differentiation receptor antagonist, a beta adrenergic receptor modulator, 0.167 A large-scale small molecule screen was conducted and an opioid receptor modulator. In some embodiments, the to identify small molecules that promote the differentiation of kit comprises a neurotransmitter receptor modulating agent oligodendrocyte precursor cells (OPCs) to a mature myeli selected from a compound listed in Table 1. In some embodi nating fate. High content imaging was used to detect in-well ments, the kit comprises a neurotransmitter receptor modu differentiation of rat optic nerve derived OPCs. Rat optic lating agent selected from benztropine, clemastine, Salme nerve derived OPCs were expanded en masse in OPC media terol, Salbutamol, trifluoperazine, and salts thereof. In some containing 30 ng/mL PDGFC.C.. To screen for small molecule inducers of OPC differentiation, OPCs (cultured for fewer embodiments, the kit comprises benztropine or a salt thereof. than 15 passages) were plated in poly-D-lysine coated 384 0164. In some embodiments, a kit of the present invention well culture plates in OPC Media containing 2 ng/mL PDG further comprises an immunomodulatory agent. In some FC.C. and immediately treated with compounds at a final con embodiments, the kit comprises a neurotransmitter receptor centration of 6 uM (0.6% DMSO). Compound treated cells modulating agent and an immunomodulatory agent for the were incubated at 37°C., 5% CO, for 6 days. At the end of 6 treatment of MS (e.g., interferon-B, e.g., interferon beta-1a or days, cells were fixed in 4% paraformaldehyde and subjected interferon beta-1b, glatiramer acetate, mitoxantrone, fingoli to immunofluorescence analysis. Anti-Myelin Basic Protein mod (FTY720), natalizumab, rituximab, daclizumab, orale (a.a. 129-138, clone 1 monoclonal anti mtuzumab). In some embodiments, the kit comprises a neu body, Millipore) (1:1000 dilution) in blocking buffere solu rotransmitter receptor modulating agent selected from tion (3% BSA, 0.3% TritonX-100 in PBS) was added to fixed benztropine, clemastine, Salmeterol, salbutamol, trifluopera and washed cells and incubated at 4°C. overnight. The pri mary antibody solution was removed and cells were washed Zine, and salts thereof and an immunomodulatory agent with PBS prior to incubation with a blocking buffer solution selected from interferon beta-1a, interferon beta-1b, glati containing goat anti-mouse IgG Alexafluor488 (Invitrogen) ramer acetate, mitoxantrone, fingolimod (FTY720), natali secondary antibody (1:1000 dilution) and DAPI (2 ug/mL) Zumab, rituximab, daclizumab, and alemtuzumab. In some for 1 hr at 25° C. The secondary antibody solution was embodiments, the kit comprises benztropine or a salt thereof washed with PBS and plates were imaged using an OPERA and an immunomodulatory agent selected from interferon high content screening system (PerkinElmer). A cell scoring beta-1a, interferon beta-1b, and fingolimod (FTY720). based image analysis algorithm was used to identify and score 0.165. In some embodiments, a kit comprises a neurotrans MBP positive cells. Hits were identified as compounds that mitter receptor modulating agent as described herein and an induced >400 cells/field and >5% MBP positive cells/field. The average values for DMSO negative controls were con immunomodulatory agent in a single formulation. In some sistently <0.5% MBP positive. A total of 6058 compounds embodiments, a kit comprises a neurotransmitter receptor comprised of commercially available Small molecule screen modulating agent as described herein and an immunomodu ing collections (LOPAC, Tocris, Enzo) were screened. A total latory agent in separate formulations. Suitable formulations of 104 primary hits were identified. These consisted of a for the neurotransmitter receptor modulating agent and number of known OPC differentiating inducing agents (e.g., immunomodulatory agent are described herein. In some retenoids, sterols, nucleoside analogues, Rho kinase inhibi embodiments, a kit provides a neurotransmitter receptor tors), as well as a number of previously unidentified OPC modulating agent agent as described herein and one or more differentiation inducing agents (e.g., neurotransmitter modu additional therapeutic agents (e.g., an immunomodulatory lating agents). agent) independently in uniform dosage formulations 0168 Identified primary hits were subsequently evaluated throughout the course of treatment. In some embodiments, a by determining EC50 values in multiple replicate experi kit provides a neurotransmitter receptor modulating agent as ments using the primary assay format for determining OPC described herein and one or more additional therapeutic differentiation. Cells were treated with compounds serially agents (e.g., an immunomodulatory agent) independently in diluted in DMSO (10 dilutions of 1:3 across a range of 71 uM graduated dosages over the course of treatment, either to 4 nM). Staining and imaging was performed as described increasing or decreasing, but usually increasing to an effica for the primary screen. ECso values were determined using cious dosage level, according to the requirements of an indi appropriate curve fitting equations in Graphpad Prism 5.0. vidual. In some embodiments, a kit comprises a neurotrans FIG. 1A shows the EC50 for six identified hits (carbetapen mitter receptor modulating agent as described herein in a tane, clemastine, benztropine, trifluoperazine, salmeterol, therapeutically effective dose and an immunomodulatory and GBR12935), all of which are FDA-approved blood-brain agent in a therapeutically effective dose. In some embodi barrier-penetrating drugs. FIG. 1 also shows the ability of ments, a kit comprises a neurotransmitter receptor modulat these six identified hits to induce differentiation of OPCs (as ing agent as described herein in a therapeutically effective measured by the percentage of myelin basic protein (MBP)- dose and an immunomodulatory agent in a subtherapeutic positive cells) at varying concentrations (FIG. 1B). For each dose. of these agents, when the OPC sample was cultured with an US 2014/0038949 A1 Feb. 6, 2014 42 agent at maximum potency, the percentage of resulting MBP decrease in recovery time following the acute phase (FIG. positive cells was at least 10% (FIG.1C). For GBT12935, the 5D-E). Each of trifluoperazine and benztropine alone signifi percentage of resulting MBP-positive cells was over 15%. cantly reduced the intensity of relapse measured as signifi These results show that carbetapentane, clemastine, benz cantly lower EAE scores for compound treated animals com tropine, trifluoperazine, salmeterol, and GBR12935 all pro pared to vehicle controls (FIG. 5B, D). In the presence of mote OPC differentiation. mycophenolate motefil no relapse was observed in animals treated with compounds (FIG. 5C, E). Thus, trifluoperazine Example 2 and benztropine prevented relapse and improved motor and cognitive function as compared to the controls. Validation of Hits from Small Molecule Screen 0173. In another set of experiments, four compounds (ben 0169. Several classes of neurotransmitter receptor modu Ztropine, clemastine, trifluoperazine, and salbutamol) were lating agents that were identified by the primary Screen were tested in vivo in a EAE model. As shown in FIG. 34A, mice selected based on FDA approval status, known toxicity, and treated with benztropine (10 mg/kg) in a prophylactic mode brain pharmacokinetics for evaluation in Subsequent in vitro showed a significantly decreased clinical severity in both and in vivo Validation assays. The ability of compounds to acute and relapse phase of the disease as compared to vehicle induce robust differentiation of OPCs to a fully mature myeli treated mice. In a therapeutic mode, each of benztropine nating oligodendrocyte cell fate was evaluated in vitro using (FIG. 34C), trifluoperazine (FIG. 34D), clemastine (FIG. quantitative RT-PCR, western blotting and immunofluores 34E), and salbutamol (FIG. 34E) showed a significantly cence analysis techniques. The ability of compounds to decreased clinical severity in the relapse phase of the disease induceremyelination in Vivo (in the presence of inflammatory as compared to vehicle treated mice. insult) was evaluated using a proteolipid protein (PLP) 0.174. The pharmacological mechanisms of identified induced Experimental Autoimmune Encephalitis (EAE) OPC differentiation-inducing agents was also investigated. It mouse model of relapsing multiple Sclerosis. was determined that multiple pharmacological mechanisms (0170 Quantitative RT-PCR (“qRT-PCR) and Western exist for inducing OPC differentiation with identified agents. blot analysis were performed on OPC samples that had been For example, as shown in FIG. 6A, muscarinic receptor ago cultured with DMSO (negative control), T3 (positive con nism with carbachol inhibited the OPC differentiation trol), benztropine, carbetapentane, clemastine, trifluopera induced by benztropine, carbetapentane, and clemastine, Sug Zine, GBR12935, or salmeterol to measure the levels of gesting the mechanism at least some OPC differentiation expression of MBP and MOG, markers for myelinating oli inducing agents is muscarinic receptor antagonism. Other godendrocytes. As shown in FIG. 2A, OPC samples cultured muscarinic receptor antagonists, atropine and ipratropium, with benztropine, carbetapentane, clemastine, GBR12935, or also induce OPC differentiation. However, carbachol did not salmeterol all were positive for MBP and MOG expression. inhibit OPC differentiation induced by salmeterol, qRT-PCR showed two-fold induction of expression, or GBR12935, or trifluoperazine, Suggesting these agents greater, of MBP and MOG for OPC samples cultured with induce OPC differentiation by one or more pharmacological benztropine, carbetapentane, clemastine, trifluoperazine, mechanisms. GBR12935, or salmeterol (FIG. 2B-C). Example 3 0171 Immunofluorescence was also used to confirm that the hits were able to induce oligodendrocyte maturation. As shown in FIGS. 3 and 4, immunofluorescence analysis con A Stem Cell-Based Strategy for the Treatment of firmed the presence of multiple markers of mature myelinat Multiple Sclerosis ing oligodendrocytes (MBP, MOG, CNP. GalC, O1, and O4) 0.175 Studies aimed at evaluating the presence and rela following treatment of in vitro cultures with benztropine, tive densities of OPCs at sites of chronically demyelinated carbetapentane, clemastine, trifluoperazine, GBR12935, or MS lesions indicate that it is not a failure of repopulation or salmeterol. migration of OPCs, but rather inhibition of OPC differentia 0172. Two of the compounds, trifluoperazine and benz tion at sites of injury that contributes to disease progression tropine, were tested in vivo in proteolipid protein (PLP) (D. M. Chari, W. F. Blakemore, Glia, 37,307 (2002); D. M. induced Experimental Autoimmune Encephalitis (EAE) Chari et al., J Neurosci Res, 73,787 (2003); G. Wolswijk, J mice, a model of multiple sclerosis. 10 week old SJL mice Neurosci, 18, 601 (1998); A. Chang et al., NEngl.J Med, 346, were immunized with PLP emulsion and pertusis toxin to 165 (2002); T. Kuhlmann et al., Brain, 131, 1749 (2008)). As induce EAE. EAE symptoms developed in mice within 10 Such, the identification of drug-like Small molecules that days after immunization. Animals that developed EAE selectively induce differentiation of OPCs at sites of demy showed neurological deficits ranging from impaired tail elinated lesions would have a significant impact on the devel movements to paralysis in all 4 limbs with the most severe opment of new, effective treatments for MS (D. Kremer et al., symptoms lasting for about 4 days, followed by a period of Ann Neurol, 69, 602 (2011)). remission. Compounds were administered daily via intraperi 0176 Primary rodent and human OPCs proliferate in vitro toneal injection of 100 ul insterile saline at 10 mg/kg alone or when cultured in serum-free media containing PDGF (C. in combination with mycophenolate motefil insterilesaline at Ffrench-Constant, M. C. Raff, Nature, 319,499 (1986); M. C. pH 5 at 20 mg/kg. Compound administration was initiated at Raffet al., J Exp Biol, 132, 35 (1987)). Upon withdrawal of disease onset (day 10) while mycophenolate motefil admin PDGF, immature OPCs cease to proliferate, but also fail to istration was initiated on Day 14. The mice were scored daily efficiently differentiate to a mature myelin basic protein on a standard EAE scale of 0-5. MS-like symptoms were (MBP) positive fate (FIG. 11). Addition of triiodothryonine induced in 65-100% of the mice. Mycophenolate motefil, (T3), a known inducer of OPC differentiation (M.C. Nunes et used at a sub-optimal dose showed a decrease in severity of al., Nat Med, 9, 439 (2003); N. Billon et al., Dev Biol, 235, the relapse (FIG. 5A). Trifluoperazine showed a modest 110 (2001); N. Billon et al., EMBO.J. 21, 6452 (2002);Y. M. US 2014/0038949 A1 Feb. 6, 2014

Tokumoto, D. G. Tang, M. C. Raff, EMBOJ 20,5261 (2001): 5 days (FIG. 17), indicating that this drug likely acts on Fernandes, 2004; L. Calza, M. Fernandez, L. Giardino, J Mol immature A2B5 positive OPCs and not on an intermediate Endocrinol, 44, 13 (2010)), at the time of mitogen withdrawal “pre-oligodendrocyte stage of differentiation. results in the differentiation of OPCs to MBP positive oligo 0179 Benztropine is used clinically for the management dendrocytes following 6 days of culture (FIG. 11). Unfortu of Parkinson's disease and its pharmacological activity is nately, T3 has multiple physiological effects that make it thought to result from activity that decreases unattractive as an OPC differentiation-based therapeutic. the imbalance between dopamine and (A. J. 0177. To identify small drug-like molecules that induce Eshleman et al., Mol Pharmacol, 45, 312 (1994); R. Katsen OPC differentiation, we developed a high content imaging schlager et al., Cochrane Database Syst Rev. CD003735 assay that is based on the induction MBP expression in pri (2003); J. T. Coyle and S. H. Snyder, Science, 166, 899 mary rat optic nerve derived OPCs cultured for 6 days under (1969)). In addition to its anticholinergic activity, P Benz basal differentiation conditions. The assay was adapted to tropine is a centrally acting anti-histamine and dopamine 384 well format and used to screen collections of known re-uptake inhibitor (G. E. Agoston et al., J Med Chem, 40, biologically active compounds, as well as a collection of 4329 (1997); J. L. Katz et al., J Pharmacol Exp Therap, 309, ~50K structurally diverse drug-like molecules. This led to the 605 (2004); D. Simoniet al., J Med Chem, 48,3337 (2005)). identification of multiple previously identified inducers of In order to determine the of benztropine, OPC differentiation including retinoids, corticosteroids, we evaluated the ability of selective agonists of muscarinic nucleoside analogs, Rho-kinase inhibitors and ErbB inhibi acetylcholine receptors (mAChRs) (e.g., carbachol) or hista tors (M. J. Latasa et al., Glia, 58, 1451 (2010): C. E. Buckley minergic receptors (e.g., histamine and histamine triflorom et al., Neuropharmacology, 59, 149 (2010); L. Joubert et al., ethyl-toluidine (HTMT)) to block benztropine activity. J Neurosci Res, 88, 2546 (2010); A. S. Baer et al., Brain 132, Potent inhibition of benztropine induced OPC differentiation 465 (2009); A. S. Paintlia et al., Mol Pharmacol, 73, 1381 was observed in the presence of carbachol (FIGS. 7C and 18), (2008); C. Ibanez et al., Prog Neurobiol, 71, 49 (2003); L. whereas histamine or HTMT showed no observable effect Giardino, C. Bettelli, L. Calza, Neurosci Lett, 295, 17 (FIG. 19). In addition, neither the dopamine receptor antago (2000)), which have limited therapeutic potential due to off nist haloperidol, nor the dopamine receptor agonist quin target activities, toxicity, or a demonstrated lack of in vivo pirole affected the induction of OPC differentiation by ben efficacy. Surprisingly, among the most effective inducers of Ztropine (FIG. 20). Quinpirole did not induce significant OPC differentiation was benztropine (ECs-350 nM), for differentiation when used alone either (data not shown). We which OPC differentiation activity has not previously been therefore next evaluated a panel of mAChRantagonists (atro reported (FIGS. 7A and 12). We chose to further investigate pine, oxybutynin, Scopolamine, ipratropium, and propiver the activity of this compound, as it is an orally available, ine) and found that all induced OPC differentiation in a dose well-tolerated, FDA approved drug that readily crosses the dependent manner (FIG. 21). OPCs express maChRs, pre blood brain barrier, and therefore has the potential to move dominantly subtypes M. M., and Ms (F. Ragheb et al., J. rapidly to proof of concept studies as a new treatment for MS. Neurochem, 77, 1396 (2001)). We confirmed expression of 0.178 Benztropine-induced in vitro differentiation of these receptors, as well as the acetylcholine synthesizing rodent OPCs was confirmed by evaluating the transcription , choline acetyl transferase, in OPCs by RT-PCR and translation levels of the oligodendrocyte specific markers (FIG.22). Carbachol induced activation of mAChRs triggers MBP and myelin oligodendroglial glycoprotein (MOG) by protein kinase-C dependent activation of the MAPK/ERK RT-PCR and Western blot analysis, respectively (FIGS. 7B pathway leading to modulation of c-Fos expression (F. and 13). Additionally, in vitro OPC differentiation activity Ragheb et al., J Neurochem, 77, 1396 (2001)). Western blot was assessed by immunofluorescent analysis using multiple analysis of benztropine treated OPCs was consistent with markers specifically expressed in mature oligodendrocytes general inhibition of this pathway (i.e., decreased phospho (including MBP, MOG, 2',3'-cyclic-nucleotide 3'-phosphodi p42/44. MAPK and phospho-Akt and stimulated phosphory esterase (CNP), galactocerebrosidase (GalC), oligodendro lation of p38 MAPK and CREB) (FIG. 23A). Additionally, cyte marker O1 (O1) and oligodendrocyte marker O4 (O4)) transcript levels of cyclin D1, cyclin D2, c-Fos, and c-Jun following six days of compound treatment (FIG.14). Further were significantly decreased in benztropine treated OPCs, more, global gene expression profiles of OPCs treated for 6 consistent with a mechanism involving general inhibition of days with benztropine were found to cluster with those MAPK/ERK dependent cell cycle progression (FIG. 23B). obtained from T3 treated positive control cells (FIG. 15). Activation of M and M. maChRs is coupled to downstream Downregulation of OPC specific genes required for prolifera signal transduction events through phospholipase C, which tion (e.g., Idr2, Egr1, Sox11; V. A. Swiss et al., PLoS One, 6, results in increased intracellular calcium concentrations (F. e18088 (2011)), and upregulation of mature myelinatingoli Ragheb et al., JNeurochem, 77, 1396 (2001)) (FIG.23C). M godendrocyte specific genes (e.g., lipid , myelin and Ma mAChRactivation inhibits adenylatecyclase, leading related proteins (V. A. Swiss et al., PLoS One, 6, e18088 to decreased intracellular cAMP levels (C. C. Felder, FASEB (2011)) was observed following treatment of OPCs with ben J. 9, 619 (1995); M. Lopez-Ilasaca et al., Science, 275, 394 Ztropine (FIG. 16). In order to determine the stage of OPC (1997)). In OPCs, benztropine inhibits carbachol induced differentiation at which benztropine is active (Gard, Neuron, calcium influx, but has no effect on cAMP levels (FIG. 24). 3, 615 (1990); A. L. Gard, S. E. Pfeiffer, Dev Biol, 159, 618 Together, these results suggest that benztropine induces OPC (1993); D. Avossa, S. E. Pfeiffer, J Neurosci Res, 34, 113 differentiation by a mechanism involving direct antagonism (1993); R. Aharoni et al., Proc Natl AcadSci U.S.A., 102, of M/M muscarinic receptors. Acetylcholine is a known 19045 (2005)), we treated OPCs for differing durations start regulator of OPC proliferation and, as such, muscarinic ing at multiple time points (FIG. 17). Maximal induction of receptor Subtypes represent a reasonable class of therapeutic MBP expression was observed when compound was added targets for the modulation of OPC proliferation and differen within 48 hours of PDGF withdrawal and left to incubate for tiation (F. De Angelis et al., Dev Neurobiol, (2011)). US 2014/0038949 A1 Feb. 6, 2014 44

0180 We next examined the activity of benztropine in the al., J Immunol, 180, 6402 (2007), M. P. Pender and J. M. myelin proteolipid protein (PLP)-induced experimental Greer, curr Asthma Rep, 7, 285 (2007)). In order to autoimmune encephalomyelitis (EAE) rodent model of determine whether the efficacy of benztropine in the EAE relapsing-remitting MS (D. A. Sipkins, J Neuroimmunol, model results at least in part from a T-cell inhibitory activity, 104, 1 (2000); T. Owens, S. Sriram, Neurol Clin, 13, 51 we evaluated the effects of benztropine on T-cell activation (1995)). This model is most commonly used to evaluate the and proliferation. Benztropine had no effect on T-cell activa potential efficacy of immunomodulatory agents, but can also tion or proliferation in vitro, as determined by evaluating be used to determine the effectiveness of promyelinating CD4"/CD69" and CD4"/CD25" populations and using a agents that function by enhancing OPC differentiation (M. CFSE assay, respectively (FIG. 27). We evaluated the effect Fernandez et al., Proc Natl Acad Sci U.S.A., 101, 16363 of benztropine on the immune system in vivo in SJL mice in (2004); X. Lee et al., JNeurosci, 27, 220 (2007); reviewed in which EAE had or had not been induced with PLP. In either R. J. Franklin, C. Ffrench-Constant, Nat Rev Neurosci, 9,839 diseased or healthy animals, benztropine had no effect on the (2008)). Benztropine (10 mg/kg) was dosed prophylactically number of circulating splenocytes, CD4 cells, CD8 cells, using a daily intraperitoneal (IP) injection regimen initiated at CD/CD44Hi cells and CD8"/CD44Hi cells (FIGS. 28 and the onset of immunization of 8 week old SJL mice with PLP. 29). A minor but significant decrease in B-cell numbers was Benztropine dramatically decreased the severity of the acute observed following treatment with benztropine (FIGS. 28 and phase of disease and virtually eliminated the observation of a 29). Treatment with benztropine had no effect on cytokine relapse phase as compared to vehicle treated controls (FIGS. production, as determined by evaluating CD4"/IL2", CD4"/ 8A and 25). We next evaluated efficacy when the drug is IFN-y", CD4"/IL-10" and CD4"/TNF-C." T-cell populations dosed therapeutically, by starting daily injections at the first isolated from drug or vehicle treated normal or diseased mice sign of disease onset. Treatment with benztropine in this (FIGS. 28 and 29). We also tested the effects of benztropine mode again led to functional recovery, with significant on T-cell dependent responses induced by TNP-LPS and decreases in clinical severity in remission phases observed TNP-Ficolland on T-cell independent responses produced by and the occurrence of relapse again virtually eliminated (FIG. TNP-KLH. Benztropine had no effect on IgG and IgM pro 8A). In fact, treatment with benztopine in this mode resulted duction in response to any of these antigens (FIG. 30). in decreases in observed clinical severity that exceeded those 0183) We further evaluated the ability of benztropine to observed for the immunomodulating MS drugs FTY720 or induce OPC differentiation and enhance remyelination in interferon-?3 (dosed at near maximally tolerated doses) (FIG. Vivo using the T-cell independent cuprizone-induced model 8A). of demyelination. In this model, mice are fed the copper 0181. In parallel experiments, during relapse phases (day chelatorbis (cyclohexylidenehydrazide) (cuprizone), which 23-25), we isolated spinal cords from benztropine or vehicle results in oxidative/nitrative stress that causes mitochondrial treated mice and stained sections from multiple regions of dysfunction and leads to CNS demyelination (P. Mana et al., each spinal cord with luxol fast blue (LFB) (to visualize Am J Pathol, 168, 1464 (2006); M. Lindner et al., Glia, 56, myelin) or H&E (to visualize infiltrating immune cells). Sec 1104 (2008); P. Mana et al., JNeuroimmunol, 210, 13 (2009): tions from both vehicle and benztropine treated mice showed L. Liu et al., Nat Neurosci, 13,319 (2010); A. J. Steelman, J. significant infiltration by H&E reactive immune cells (FIG. P. Thompson, J. Li, Neurosci Res, 72, 32 (2012)). The demy 26). In vehicle treated mice, areas of infiltration corresponded elinated lesions observed in this model are reminiscent of to areas with significant demyelination (FIG. 26). In contrast, pattern III MS lesions and involve minimal contribution from in benztropine treated mice, a large number of immune cell hematogenous immune cells (L. Liu et al., Nat Neurosci, 13. infiltrated areas stained positive for LFB, consistent with a 319 (2010); C. Lucchinetti et al., Ann Neurol 47,707 (2000); stem cell versus immunomodulatory mechanism (FIG. 26). L. T. Remington et al., Am J Pathol, 170, 1713 (2007)). We further evaluated drug-enhanced remyelination using Inclusion of 0.2% (w/v) cuprizone in the diet of C57BL/6 confocal microscopy, by examining regions of T-cell infiltra mice induces a demyelination program that proceeds with a tion in spinal cord sections stained with markers of mature defined series of events over a characteristic time course, oligodendrocytes (GST-t) and immature OPCs (NG2) (FIG. wherein the corpus callosum shows peak demyelination fol 8B). Quantitative image analysis of multiple random fields lowing 6-7 weeks offeeding (T. Skripuletzetal. Am J Pathol, per group indicates that benztropine treatment causes a sig 172, 1053 (2008)). Spontaneous remyelination is observed nificant increase in the number of GST-II positive mature 2-4 weeks following cuprizone withdrawal (T. Skripuletz et oligodendrocytes from ~500 to -1100 per field (FIG. 8C), al., Am J Pathol, 172, 1053 (2008)). By administering drugs at whereas the number of NG2 positive cells did not differ the time when a cuprizone-free diet is reintroduced, the effi significantly with treatment (FIG. 8C). The observed increase cacy of promyelinating agents can be examined by evaluating in mature oligodendrocyte numbers at sites of T-cell infiltra the relative kinetics of OPC dependent remyelination (T. tion is consistent with a mechanism of benztropine-induced Skripuletzet al., Am J Pathol, 172, 1053 (2008)). Following 7 clinical recovery that involves the stimulation of OPC differ weeks of exposure, upon withdrawal of cuprizone, we admin entiation, leading to enhanced remyelination, in the context istered benztropine (10 mg/kg) intraperitoneally to 15 week of an inflammatory environment. Notably, general toxicity old C57BL/6 mice. Drug or vehicle treated mice were sacri was not observed in drug treated mice, nor was it observed in ficed in groups of 5 weekly for 5 weeks, following drug this histological analysis (i.e., drug induced demyelination treatment, and remyelination was quantitatively evaluated by was not observed) following 4 weeks of daily injections at 10 staining the corpus callosum regions of harvested brains with mg/kg. LFB (FIG. 9A, B). Significant demyelination was clearly 0182. The primary immunological processes involved in observed following seven weeks of treatment with cuprizone, MS and EAE are T-cell mediated and the primary treatment as compared to control animals. Consistent with an enhance paradigms are based on immunomodulatory drugs (T. ment of OPC differentiation and accelerated remyelination, a Kopadze et al., Arch Neurol, 63, 1572 (2006); J. M. Greer et significant increase in myelin staining in the corpus callosum US 2014/0038949 A1 Feb. 6, 2014

was observed at week 2 following treatment with benz 0185. We have identified a centrally acting FDA approved tropine, as compared to the spontaneous remyelination drug that, when dosed alone in the most clinically relevant observed in vehicle controls (FIG. 9A, B). As expected, at model of MS, significantly decreases disease severity. Benz later time points spontaneous remyelination was relatively tropine enhances remyelination, leading to functional recov complete and significant differences were not observed ery, by directly stimulating the differentiation of OPCs by a between drug and vehicle treated animals. A lack of differ mechanism involving antagonism of M/M muscarinic ence at these later time points indicates that, even following 5 receptors expressed on immature OPCs. Evidence that this weeks of treatment at efficacious doses, benztropine is not drug functions by directly stimulating remyelination, and not toxic to mature oligodendrocytes. These data again suggest by inhibiting demyelination, is provided by the observed lack of effect of benztropine on in vitro and in vivo T-cell biology that benztropine enhances the process of in vivo remyelina and by the observed promyelinating activity of benzotropine tion, by directly inducing OPC differentiation. Benztropine in the T-cell independent cuprizone-induced model of demy could also enhance remyelination by exerting a protective elination. Inclusion of benztropine in treatment regimens effect on a “pre-oligodendrocyte' intermediate cell type or by involving existing approved immunomodulatory drugs a process involving a combination of both effects. Caspase-3 resulted in enhanced functional recovery and significantly activation results from both intrinsic and extrinsic apoptotic decreases the dosages of the latter that are required to achieve pathways and serves as a general indicator of stress induced an equivalent level of efficacy. To our knowledge, these cell death. Benztropine treatment was not found inhibit results provide the first in vivo evidence Supporting the notion caspase-3 activation on days 2, 4, and 6 of differentiation, as that a benefit can beachieved by treating MS-like symptoms determined by western blot and immunofluorescence analy using the combination of an immunomodulator with a remy sis (FIG. 32). However, cleaved caspase-3 was barely detect elination enhancer. able in OPCs at any time point in DMSO treated controls, 0186. It is understood that the examples and embodiments indicating that failed OPC differentiation does not likely described herein are for illustrative purposes only and that result from stress induced cell death. various modifications or changes in light thereofwill be Sug gested to persons skilled in the art and are to be included 0184 For the treatment of MS, an OPC differentiation within the spirit and purview of this application and scope of inducing drug would most likely be introduced clinically as the appended claims. All publications, patents, and patent part of a combination therapy with an immunosuppressive applications cited herein are hereby incorporated by refer drug. Using the PLP induced EAE model, we therefore evalu ence in their entirety for all purposes. ated the clinical efficacy of benztropine when combined with 1. A method of stimulating increased myelination of nerves either of two immunomodulating drugs approved for the in a human Subject having a demyelinating disease, the treatment of MS, interferon-?3 and FTY720 (L. Kappos et al., method comprising: NEngl J Med, 355, 1124 (2006); M. Fujino et al., J Pharma administering to the subject a therapeutically effective col Exp Ther,305, 70 (2003); L. Jacobset al., Arch Neurol, 39, dose of a neurotransmitter receptor modulating agent 609 (1982); M. Huber et al., J Neurol, 235, 171 (1988)). The Selected from a muscarinic receptor antagonist, a former reduces T-cell proliferation and alters cytokine dopamine receptor antagonist, a histamine receptor expression (A. Noronha, A. Toscas, M. A. Jensen, J Neuroim antagonist, a beta adrenergic receptor modulator, and an munol, 46, 145 (1993); A. Noronha, A. Toscas, M. A. Jensen, opioid receptor modulator, thereby stimulating Ann Neurol, 27,207 (1990)), while the latterisan S1 Pagonist increased myelination of nerves in the Subject. that regulates T-cell trafficking (V. Brinkmann et al., J Biol 2. The method of claim 1, wherein the neurotransmitter Chem, 277,21453 (2002); V. Brinkmann et al., Nat Rev Drug receptor modulating agent is a muscarinic receptor antago Discov, 9,883 (2010)). We determined whether the combina tion of an OPC inducing drug with either interferon-B or nist. FTY720 would improve efficacy and/or decrease the dose of 3. (canceled) the immunosuppressive agent required to achieve maximal 4. The method of claim 2, wherein the muscarinic receptor benefit. Initially, all three drugs were dosed individually over antagonist is selected from benztropine, carbetapentane, a range of concentrations, to determine Suboptimal and maxi clemastine, ipratropium, atropine, and salts thereof. mal effective/tolerated doses of each in the models (FIGS. 25 5. The method of claim 1, wherein the neurotransmitter and 33 A.B). Benztropine was then administered with either receptor modulating agent is a dopamine receptor antagonist. of two existing immune modulating treatments for MS, and as 6. (canceled) described below, these combinations resulted in significant 7. The method of claim 5, wherein the dopamine receptor benefits in the EAE model. Addition of 2.5 mg/kg benz antagonist is selected from benztropine, GBR12935, triflu tropine to 1 mg/kg FTY720 (FIG. 10A) or 10,000 U/mouse operazine, and salts thereof. interferon-?3 (FIG. 10B) resulted in a significant decrease in 8. The method of claim 1, wherein the neurotransmitter observed clinical severity, as compared to that observed when receptor modulating agent is a histamine receptor antagonist. either FTY720 or interferon-B was dosed alone. Further, the 9. (canceled) combination of 2.5 mg/kg benztropine with a Suboptimal 10. The method of claim 8, wherein the histamine receptor dose of FTY720 (0.1 mg/kg) resulted in a significant decrease antagonist is clemastine or a salt thereof. in clinical severity that was greater than that observed when 11. The method of claim 1, wherein the neurotransmitter either drug was dosed alone and was comparable to the clini receptor modulating agent is a beta adrenergic receptor cal efficacy observed when FTY720 was dosed alone at near antagonist. the maximal observed tolerated dose of 1 mg/kg (FIG. 10C). 12. (canceled) This observation may prove clinically relevant as FTY720 13. The method of claim 11, wherein the beta adrenergic treatment is associated with bradycardia which is dose depen receptor antagonist is selected from pindolol, salmeterol, dent. salbutamol, albuterol, salbutamol, and salts thereof. US 2014/0038949 A1 Feb. 6, 2014 46

14. The method of claim 1, wherein the neurotransmitter 29. The method of claim 23, wherein: receptor modulating agent is an opioid receptor modulator. (i) each of the immunomodulatory agent and the neu 15. (canceled) rotransmitter receptor modulating agent is administered 16. The method of claim 14, wherein the opioid receptor at a therapeutically effective dose; or modulator is selected from carbetapentane, Snc-80, (ii) the neurotransmitter receptor modulating agent is BD-1047, and salts thereof. administered at a therapeutically effective dose and the 17. The method of claim 1, wherein the neurotransmitter immunomodulatory agent is administered at a subthera receptor modulating agent is selected from benztropine, peutic dose; or clemastine, salmeterol, Salbutamol, trifluoperazine, and salts (iii) each of the immunomodulatory agent and the neu thereof. rotransmitter receptor modulating agent is administered 18. (canceled) at a subtherapeutic dose. 19. The method of claim 1, wherein the demyelinating 30-31. (canceled) disease is multiple Sclerosis, idiopathic inflammatory demy 32. The method of claim 23, wherein the immunomodula elinating disease, transverse myelitis, Devic's disease, pro tory agent and the neurotransmitter receptor modulating gressive multifocal leukoencephalopathy, optic neuritis, leu agent are administered systemically. kodystrophy, Guillain-Barre syndrome, chronic 33. The method of claim 23, wherein the immunomodula inflammatory demyelinating polyneuropathy, autoimmune tory agent and the neurotransmitter receptor modulating peripheral neuropathy, Charcot-Marie-Tooth disease, acute agent are administered sequentially. disseminated encephalomyelitis, adrenoleukodystrophy, 34. The method of claim 23, wherein the immunomodula adrenomyeloneuropathy, Leber's hereditary optic neuropa tory agent and the neurotransmitter receptor modulating thy, or HTLV-associated myelopathy. agent are administered concurrently. 20. (canceled) 35. A composition for use in treating a subject having a 21. The method of claim 1, wherein the method further demyelinating disease, the composition comprising: comprises administering to the Subject an immunomodula a neurotransmitter receptor modulating agent; and tory agent. an immunomodulatory agent. 22. The method of claim 21, wherein the immunomodula 36-37. (canceled) tory agent is fingolimod (FTY720), interferon beta-1a, inter 38. The composition of claim 35, wherein the neurotrans feron beta-1b, glatiramer acetate, mitoxantrone, or natali mitter receptor modulating agent is selected from benz Zumab. tropine, clemastine, Salmeterol, Salbutamol, trifluoperazine, 23. A method of enhancing the therapeutic effect of an and salts thereof. immunomodulatory agent in the treatmentofademyelinating 39. (canceled) disease, the method comprising: 40. The composition of claim 35, wherein the immuno administering to a human Subject having the demyelinating modulatory agent is fingolimod (FTY720), interferon beta disease the immunomodulatory agent and a neurotrans 1a, interferon beta-1b, glatiramer acetate, mitoxantrone, or mitter receptor modulating agent; thereby enhancing the natalizumab. therapeutic effect of the immunomodulatory agent in the 41-43. (canceled) treatment of the demyelinating disease. 44. A kit for use in treating a subject having a demyelinat 24. The method of claim 23, wherein the demyelinating ing disease, the kit comprising: disease is multiple Sclerosis, idiopathic inflammatory demy a neurotransmitter receptor modulating agent; and elinating disease, transverse myelitis, Devic's disease, pro an immunomodulatory agent. gressive multifocal leukoencephalopathy, optic neuritis, leu 45-46. (canceled) kodystrophy, Guillain-Barre syndrome, chronic 47. The kit of claim 44, wherein the neurotransmitter inflammatory demyelinating polyneuropathy, auto immune receptor modulating agent is selected from benztropine, peripheral neuropathy, Charcot-Marie-Tooth disease, acute clemastine, Salmeterol, Salbutamol, trifluoperazine, and salts disseminated encephalomyelitis, adrenoleukodystrophy, thereof. adrenomyeloneuropathy, Leber's hereditary optic neuropa 48. (canceled) thy, or HTLV-associated myelopathy. 49. The kit of claim 44, wherein the immunomodulatory 25. (canceled) agent is fingolimod (FTY720), interferon beta-1a, interferon 26. The method of claim 23, beta-1b, glatiramer acetate, mitoxantrone, or natalizumab. wherein the neurotransmitter receptor modulating agent is 50-54. (canceled) Selected from benztropine, clemastine, Salmeterol, Salb 55. The method of claim 1, wherein the neurotransmitter utamol, trifluoperazine, and salts thereof. receptor modulating agent is a muscarinic receptor modulator 27. (canceled) compound, a dopamine receptor modulator compound, a his 28. The method of claim 23, wherein the immunomodula tamine receptor modulating compound, a beta adrenergic tory agent is fingolimod (FTY720), interferon beta-1a, inter receptor modulator compound, or an opioid receptor modu feron beta-1b, glatiramer acetate, mitoxantrone, or natali lator compound listed in Table 1. Zumab. k k k k k