US010493080B2

(12 ) United States Patent (10 ) Patent No.: US 10,493,080 B2 Schultz et al. (45 ) Date of Patent : Dec. 3 , 2019

( 54 ) DIRECTED DIFFERENTIATION OF (56 ) References Cited OLIGODENDROCYTE PRECURSOR CELLS TO A MYELINATING CELL FATE U.S. PATENT DOCUMENTS 7,301,071 B2 11/2007 Zheng (71 ) Applicants : The Scripps Research Institute , La 7,304,071 B2 12/2007 Cochran et al. Jolla , CA (US ) ; Novartis AG , Basel 9,592,288 B2 3/2017 Schultz et al. 2003/0225072 A1 12/2003 Welsh et al. ( CH ) 2006/0258735 Al 11/2006 Meng et al. 2009/0155207 Al 6/2009 Hariri et al . (72 ) Inventors : Peter Schultz , La Jolla , CA (US ) ; Luke 2010/0189698 A1 7/2010 Willis Lairson , San Diego , CA (US ) ; Vishal 2012/0264719 Al 10/2012 Boulton Deshmukh , La Jolla , CA (US ) ; Costas 2016/0166687 Al 6/2016 Schultz et al. Lyssiotis , Boston , MA (US ) FOREIGN PATENT DOCUMENTS (73 ) Assignees : The Scripps Research Institute, La JP 10-218867 8/1998 Jolla , CA (US ) ; Novartis AG , Basel JP 2008-518896 5/2008 (CH ) JP 2010-533656 A 10/2010 WO 2008/143913 A1 11/2008 WO 2009/068668 Al 6/2009 ( * ) Notice : Subject to any disclaimer , the term of this WO 2009/153291 A1 12/2009 patent is extended or adjusted under 35 WO 2010/075239 Al 7/2010 U.S.C. 154 ( b ) by 0 days. (21 ) Appl. No .: 15 /418,572 OTHER PUBLICATIONS Molin - Holgado et al . “ Regulation of muscarinic receptor function in ( 22 ) Filed : Jan. 27 , 2017 developing oligodendrocytes by agonist exposure ” British Journal of Pharmacology, 2003 , 138 , pp . 47-56 . (Year : 2003 ). * (65 ) Prior Publication Data Yong et al. “ Interferon beta in the treatment of multiple sclerosis mechanisms of action ” Neurology , 1998, vol . 51( 3 ), pp . 1-38. ( Year : US 2017/0136029 A1 May 18 , 2017 1998 ). * Kubo et al. “ Antimuscarinic Effects of Antihistamines : Quantitative Evaluation by Receptor- Binding Assay Japan J. Pharmacol. , 1987 , vol . 43, pp . 277-282 . ( Year: 1987 ). * Related U.S. Application Data Cui et al. “Muscarinic acetylcholine receptors mediate oligodendrocyte progenitor survival through Src- like tyrosine kinases and P13K / Akt (62 ) Division of application No. 13 /985,342 , filed as pathways ” Neurochemistry International, 2006 , vol. 48 , pp . 383 application No. PCT/ US2012 /025712 on Feb. 17 , 393. ( Year: 2006 ). * 2012 , now Pat. No. 9,592,288 . Chang et al. , “ Premyelinating Oligodendrocytes in Chronic Lesions of Multple Sclerosis ," N Engl J Med , 2002 , vol . 346 , 165 . Chari et al. , " Efficient Recolonisation of Progenitor- Depleted Areas (60 ) Provisional application No. 61/ 444,666 , filed on Feb. of the CNS by Adult Oligodendrocyte Progenitor Cells ,” Glia , 2002, 37 : 307-313 . 18 , 2011 . Chari et al. , “ Dysfunctional Oligodendrocyte Progenitor Cell (OPC ) Populations May Inhibit Repopulation of OPC Depleted Tissue , ” J. (51 ) Int. Ci. Neurosci Res, 2003 , 73 : 787-793 . A61K 31/5415 ( 2006.01) Chong et al. , “ Tapping into the glial reservoir : cells committed to A61K 31/439 remaining uncommitted ,” J Cell Biol 2010 , 188 ( 3 ), pp . 305-312 . ( 2006.01 ) De Angelis et al ., “ Muscarinic Receptor Subtypes as Potential A61K 31/138 ( 2006.01) Targets to Modulate Oligodendrocyte Progenitor Survival, Prolif A61K 31/40 ( 2006.01 ) eration , and Differentiation , ” Dev Neurobiol, 2012 , 72: 713-728 . A61K 31/135 ( 2006.01) Gobert et al. , “ Convergent functional genomics of oligodendrocyte A61K 31/137 ( 2006.01 ) differentiation identifies multiple autoinhibitory signaling circuits, " A61K 38/21 (2006.01 ) Molecular and Cellular Biology, 2009, 29 : 1538. A61K 39/395 ( 2006.01 ) (Continued ) A61K 31/216 (2006.01 ) Primary Examiner Svetlana M Ivanova A61K 31/46 (2006.01 ) ( 74 ) Attorney , Agent, or Firm — Kilpatrick Townsend & A61K 31/495 ( 2006.01 ) Stockton LLP (52 ) U.S. CI. ( 57 ) ABSTRACT CPC A61K 31/5415 ( 2013.01 ) ; A61K 31/135 The present invention provides methods of inducing differ (2013.01 ); A61K 31/137 (2013.01 ) ; A61K entiation of oligodendrocyte progenitor cells to a mature 31/138 (2013.01 ); A61K 31/216 ( 2013.01 ); myelinating cell fate with a neurotransmitter receptor modu A61K 31/40 ( 2013.01) ; A61K 31/439 lating agent. The present invention also provides methods of ( 2013.01) ; A6IK 31/46 (2013.01 ) ; A61K stimulating increased myelination in a subject in need 31/495 (2013.01 ); A61K 38/215 ( 2013.01 ); thereof by administering said neurotransmitter receptor A61K 39/3955 (2013.01 ) modulating agent. Methods of treating a subject having a (58 ) Field of Classification Search demyelinating disease using a neurotransmitter receptor None modulating agent are also provided . See application file for complete search history . 16 Claims, 52 Drawing Sheets US 10,493,080 B2 Page 2

( 56 ) References Cited Rangachari and Kuchroo , Using EAE to better understand prin ciples of immune function and autoimmune pathology . J.Autoim mun . , 45 :31-39 , 2013 . OTHER PUBLICATIONS Reeves and Swenson , Disorders of the Nervous System . Chapter 23 , “ Demyelinating diseases of the nervous system .” Spalice et al. , Clinical and pharmacological aspects of inflammatory Joubert et al. , “ Chemical inducers and transcriptional markers of demyelinating diseases in childhood : An update . Current oligodendrocyte differentiation ,” Journal of Neuroscience Research , Neuropharmacology , 8 :135-148 , 2010 . 2010 , 88 : 2546-2557 . Steinman , Assessment of animal models for MS and demyelinating Kremer et al. , “ The Complex World of Oligodendroglial Differen disease in the design of rational therapy . Neuron , 24 :511-514 , 1999 . tiation Inhibitors ,” Ann Neurol, 2011 , 69 : 602-618 . Swanborg , Short Analytical Review . Animal models of human Kuhlmann et al ., “ Differentiation block of oligodendroglia progeni disease. Experimental Autoimmune encephalomyelitis in rodents as tor cells as a cause for remyelination failure in chronic multiple a model for human demyelinating disease . Clinical Immunology and Immunopathology, 77 ( 1 ): 4-13 , 1995 . sclerosis ,” Brain , 2008, 131, 1749-1758 . The TMA , “ Animal Models to Study NMO and TM " , Nov. 23 , Patel et al. , “Mediators of oligodendrocyte differentiation during 2015 , https://myelitis.org/animal-models-to-study-nmo-andtm/, printed remyelination ,” FEBS Lett, 2011 , 585 , 3730-3737 . Jul. 14 , 2016 , pp . 1-7 . Stevens et al ., " Adenosine: A Neuron -Glial Transmitter Promoting Tian et al. “ Neuropathic Pain in AnimalModels ofNervous System Myelination in the CNS in Response to Action Potentials ,” Neuron , Autoimmune Diseases ” Mediators of Inflammation , 2013, pp . 1-13 . Dec. 2002 , vol . 36 (5 ) , pp . 855-868 . Kraker et al . “ Autoimmune Neuromuscular Disorder ” , Current Wolswijk , “ Chronic Stage Multiple Sclerosis Lesions Contain a Neuropharmacology , 2011, vol . 9 , pp . 400-408 . Relatively Quiescent Population of Oligodendrocyte Precursor Cells ,” Lehmann et al. “ Pathogenesisi and treatment of immune -mediated neuropathies” Therapeutic Advances in Neurological Disorders , J. Neurosci, 1998 , 18 ( 2 ) , pp . 601-609 . 2009 , vol. 2 , issue 4 , pp . 261-281 . PCT application No. PCT/ US12 /25712 , International Search Report Walgaard et al. “ Emerging drugs for Guillain - Barre syndrome” and Written Opinion , 6 pages. Expert Opinion of Emerging Drugs, Feb. 25 , 2011 , vol. 16 , issue 1 , Deshmukh et al. , “ A regenerative approach to the treatment of pp. 105-120 . multiple sclerosis, ” Nature , Oct. 2013 , vol. 502 ( 7471) , pp . 327-332, Collongues et al. “ Current and future treatment approaches for DOI: 10.1038 / nature12647 . neuromyelitis optica” Therapeutic Advances in Neurological Dis Stangel et al. , “ Remyelination Strategies: New Advancements Toward orders , 2011 , vol. 4 , issue 2 , pp . 111-121 . a Regenerative Treatment in Multiple Sclerosis ,” Current Neurol Oh et al. “ Treatment of HTLV - 1- Associated Myelopathy / Tropical ogy and Neuroscience Reports , May 2006 , vol . 6 ( 3 ) , pp . 229-235 . Spastic Paraparesis : Towards Rational Targeted Therapy ,” Neurol EP Patent Application No. 12 74 7134 , Supplementary Search Clin ., 2008 , vol. 26 , issue 3 , pp . 781-796 ( printed as 1-15 ) . Report , dated Jun . 18 , 2014 , 3 pages. Alexander et al. “ Acute disseminating encephalomyeitis : Treatment Magnaghi et al. , “Novel Pharmacological Approaches to Schwann guidelines” Ann Indian Acad Neurol, 2011, vol. 14, suppli , S60 cells As Neuroprotective Agents for Peripheral Nerve Regenera S64 (printed as pp . 1-12 ) . tion ” , International Review of Neurobiology, 2009, vol. 07 , pp . Khoury et al. , " A Randomized Controlled Double -Masked Trial of 295-312 , DOI: 10.1016 . Albuterol Add - on Therapy in Patients With Multiple Sclerosis ,' Butt et al. , “ Neurotransmitter -Mediated Calcium Signalling in Arch Neurol. 2010 ;67 (9 ): 1055-1061. Oligodendrocyte Physiology and Pathology, ” Sep. 2006 , Wiley Almeida et al . “Methylphenidate -induced Akathisia in a Patient InterScience , pp . 665-675. with Multiple Sclerosis” , Prim Care Companion J Clin Psychiatry , Mendes et al. “ Classical immunomodulatory therapy in multiple 2006 , vol . 8 (6 ), pp . 379-380 . sclerosis” Arq Neuropsiquiatr, 2011, vol. 69 , No. 3 , pp . 536-543 . Ragheb et al ., “ Pharmacological and functional characterization of Bangham et al. , HTLV - 1 -associated myelopathy /tropical spastic muscarinic receptor subtypes in developing oligodendrocytes ,” Jour paraparesis. Nature Reviews, Article No. 15012, Published online : nal of Neurochemistry , 2001, vol . 77 , pp . 1396-1406 . doi: 10_1038 / nrpd.2015.12 , 16 pages , Jun . 18 , 2015 . Smith et al ., “ Recent developments in drug therapy for multiple Guerreiro et al. , Levels of serum chemokines discriminate clinical sclerosis ,” Multiple Sclerosis , 1999 , vol . 5 , 110-120 . myelopathy associated with human T lymphotropic virus type 1 Nakano et al. , “ Antagonizing dopamine D1- like receptor inhibits (HTLV -1 )/ tropical spastic paraparesis (HAM / TSP) disease from Th17 cell differentiation : Preventive and therapeutic effects on HTLV - 1 carrier state . Clinical and Experimental Immunology , 145: 296 experimental autoimmune encephalomyelitis ,” Biochemical and 301, 2006 . Biophysical Research Communications, 2008 , vol. 373 , pp . 286 Linker and Lee, Models of autoimmune demyelination in the central 291 . nervous system : on the way to translational medicine . Experimental Waggoner et al. , “ Presynaptic Regulation of Tyrosine Hydroxylase & Translational Stroke Medicine , 1 ( 5 ): 10 pages , 2009 . Activity in Rat Striatal Synaptosomes by Dopamine Analogs ," Mayo et al. , The Innate immune system in demyelinating disease . Molecular Pharmacology, 1980 , vol . 18 , pp . 91-99 . Immunol Review , 248 ( 1 ) : 170-187 , 2012 . European Patent Application No. 18208362.6 , “ Extended European Merrill, In Vitro and In Vivo pharmacological models to access Search Report ” , dated Apr. 26 , 2019 , 5 pages . demyelination and remyelination . Neuropsychopharmacology Reviews , 34 :55-73 , 2009 . * cited by examiner U.S. Patent Dec. 3 , 2019 Sheet 1 of 52 US 10,493,080 B2

Fig . IA

A. ma Carbetapentane :ECQ- 260 nM Clemastine : ECsa . 780 OM

W

Benztropine : ECso ~ 250 nM Trifluoperazine: ECso- 30 nM

. Salmeterol: EC50-770 nM

GBR12935 : EC50 80 M U.S. Patent Dec. 3 , 2019 Sheet 2 of 52 US 10,493,080 B2

Fig . 1B - C B. 20 GBR 12935 Trifluroperazine sode Carbetapentane - Benztropine Salmeterol

1 (compound ) ( UM )

C.

TiryroidHormone Beratropino Clantestine U.S. Patent Dec. 3 , 2019 Sheet 3 of 52 US 10,493,080 B2

Fig . 2A

3 4 5 6 7 1 : DMSO 2 : T3 3 : Benzatropine MBP 4 : Carbapentane 5 : Clemastine 6 : GBR12935 DOW 7 : Salmeterol Actin U.S. Patent Dec. 3 , 2019 Sheet 4 of 52 US 10,493,080 B2

Fig . 2B - C B. MBP ART PCR voissaidxal

BarvatropineCiemastine Cardupood Simooro

C. MOG ART PCR Expression

ThyroidHormone Bonatropur Carbapoane U.S. Patent Dec. 3 , 2019 Sheet 5 of 52 US 10,493,080 B2

GBR12 Salmeterol Trifluoperazine 3.Fig Carbapentane Clemastine Benzatropine DMSO

DAPI / MBP U.S. Patent Dec. 3 , 2019 Sheet 6 of 52 US 10,493,080 B2

GBR12 Salmeterol Trifluoperazine

4.Fig Carbapentane Clemastine Benzatropin

DMSO

Idvo /bieg DAPI/ 04 U.S. Patent Dec. 3 , 2019 Sheet 7 of 52 US 10,493,080 B2

Fig . 5A Mycophenolate

MeanEAESeverityScore

** Days U.S. Patent Dec. 3 , 2019 Sheet 8 of 52 US 10,493,080 B2

Fig . 5B - C B. Benzatropine

Berzatropine

MeanEAESeverityScore

er

7 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 Days Benzatropine * Mycophenolate C.

# Benzatropine +Mycophenoli

MeanEAESeverityScore

10111213141536 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 Days U.S. Patent Dec. 3 , 2019 Sheet 9 of 52 US 10,493,080 B2

Fig . 5D - E Trifluoperazine

2.0 MeanEAESeverityScore

8 $ 30 H1733 14 15 36 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

E. Trifluoperazine Mycophenolate

- Trifluoperazine + Mycophenolate

MeanSeverityEAEScore 5.5 .

8 9 10 11 12 13 $415 16 17 18 1920222232425 28 27 28 29 30 31 Days U.S. Patent Dec. 3 , 2019 Sheet 10 of 52 US 10,493,080 B2

Fig . 6A - B

A. 25 CASICS GBR12935 * to the Carbapentane Benzaropine Salmeterol

Conc, Carbachol (UM )

B.

Salmetrol, GBR12935 Trifluoperazine , 73 *

Benztropine , Carbapentane, Clemastine U.S. Patent Dec. 3 , 2019 Sheet 11 of 52 US 10,493,080 B2

Fig. 7A - C

A. DMSO

Benztropine (1.5 uM ) Benztropine

B. C. 2009 15 180 Carbachot (OUM ) 160 MOG I Carbachol (0.6uM ) 140 O Carbachol ( 4.74M ) 120 100 % T FoldChange 20 15 10 5

DMSO Benztropine (2.3uM ) U.S. Patent Dec. 3 , 2019 Sheet 12 of 52 US 10,493,080 B2

Fig. 8A - B A. 5 Vehicle Benztropine Therapeutic Benztropine Prophylactic * FTY720 Therapeutic meiten Interferon- ß Therapeutic MeanClinicalEAEScore

0 kr 20 25 Days

Vehicle B. 2000 , Benztropine p < 0.05 1500 CellNumber

NG2 GST- IT U.S. Patent Dec. 3 , 2019 Sheet 13 of 52 US 10,493,080 B2

Fig . 8C

C. Vehicle Benztropine

10X

63X DAPI GST- 11 NG2 U.S. Patent Dec. 3 , 2019 Sheet 14 of 52 US 10,493,080 B2

Fig . 9A - B A. Cuprizone Post Cuprizone Withdrawal Week O Week 1 Week 2 Wehicle vehicle

Benztropine Depooine

No Cuprizone * Control

B.

%ofTotalObjects 20

Grey Scale (0-50 ) O Vehicle Benztropine Untreated U.S. Patent Dec. 3 , 2019 Sheet 15 of 52 US 10,493,080 B2 Fig . 10A - B

A. 4.5 * FTY720 1mg/ kg * BA 2.5mg/ kg wym FTY720 1mg/ kg + 3.5 BA 25mg/ kg

2.0 LILI 1.5

0 5 25 30 Days

B. ne botene Vehicle IFN - B 10000U BA 2.5mg/ kg anonyms IFN - B 100000 + BA 2.5mg/ kg

2.0 LILI

0 20 25 Days 30 U.S. Patent Dec. 3 , 2019 Sheet 16 of 52 US 10,493,080 B2

Fig . 10C - D C. FTY720 0.1mg /kg M FTY720 1mg/ kg talaganaman emotions BA 2.5mg/ kg 3.0 I

0.5 0.0 **** 25 30

D. 4.5 4.0 Vehicle IFN - B 30000 3.5 IFN -B 100000 -BA 2.5mg/ kg

2.5

0 an 20 25 Days U.S. Patent Dec. 3 , 2019 Sheet 17 of 52 US 10,493,080 B2

Fig . 10E - F

E. NU in FTY720 1mg/ kg FTY720 0.1mg /kg 3.5 BA 2.5mg/ kg 3.0 2.5 ???

20 30 Days

F. 4.5 Vehicle my then IFN - B 10000U 3.5 onthos IFN - B 30000U BA 2.5mg/ kg 3.0

2.0 1.5

0 5 Days U.S. Patent Dec. 3 , 2019 Sheet 18 of 52 US 10,493,080 B2

Fig . 11A - B A.

- PDGF Thyroid Hormone

B. Day 0 Dita OPC Oligodendrocyte A2B5 # Compounds U.S. Patent Dec. 3 , 2019 Sheet 19 of 52 US 10,493,080 B2

Fig . 12A - B

A. 25 offee Benztropine 20 EC5o = 350 nM %MBPPositive do 10 1 wak Conc. (UM )

B. Benztropine ( 1.6 UM

MBP

Thyroid Hormone ( 10M ) U.S. Patent Dec. 3 , 2019 Sheet 20 of 52 US 10,493,080 B2

Fig . 13

DMSO TH BA

MOG

Actin U.S. Patent Dec. 3 , 2019 Sheet 21 of 52 US 10,493,080 B2 Fig. 14 Thyroid Benztropine MBP

MOG

CNP

04 U.S. Patent Dec. 3 , 2019 Sheet 22 of 52 US 10,493,080 B2

Fig . 15

1 3 6 10 366 -0.999

DMSO Benztropine HormoneThyroid U.S. Patent Dec. 3 , 2019 Sheet 23 of 52 US 10,493,080 B2

Fig. 16A - B A. OPC / DMSO Benztropinel Thyroid Hormonel DMSO DMSO Apod 0.58 10,79 11.21 Enpp6 0.14 11.58 ; 9.06 Mag 0.10 3.70 4.64 Mbp 0.51 3.82 3.55 Mobp 0,46 2.04 67.70 Mog 0.12 7.98 29.23 Olig1 0.96 10.97 10.75 Plp 0.17 1.97 2.33 Polip 0.44 3.34 3.01 Tpd52 0.24 2.28 2.15

B. Benztropinel Thyroid Hormone/ OPC /DMSO DMSO DMSO Ccnd2 1.93 1.00 1.00 Cited2 0.99 1.10 0.98 E2f1 0.96 0.75 0.80 Egr1 1.19 0.04 0.02 Mki67 1.18 0.67 0.81 Nptxr 1.44 0.82 0.84 Scd1 0.92 1.07 0.43 Sox11 0.87 0.36 0.23 Top2a 1.03 0.67 1.00 Uhrf1 1.18 0.51 0.60 U.S. Patent Dec. 3 , 2019 Sheet 24 of 52 US 10,493,080 B2

Fig . 17A - B

A. Day 6 Lay Day 1 Day 2 Day 3 Partially Mature OD Day 4 Day 5

B. Day 3 Day 3 Day 4 Day 5 Day 6 U.S. Patent Dec. 3 , 2019 Sheet 25 of 52 US 10,493,080 B2

Fig . 17C - D

C. 309 Benztropine (1.5uM ] T 204T %MBP•Cells

T Day 0 Day 1 Day 2 Day 3 Day 4 Day 5

D. Benztropine ( 1.5uM ] T 20 CellsMBP%

Day 3 Day 4 Day 5 Day 6 U.S. Patent Dec. 3 , 2019 Sheet 26 of 52 US 10,493,080 B2

Fig . 18

DMSO

Benztropine ( 2.3uM ) Benztropine (2.3uM )+ Carbachol ( 4.6UM U.S. Patent Dec. 3 , 2019 Sheet 27 of 52 US 10,493,080 B2

Fig . 19A - B A.

2

Benztropine 10.8 UM Histamine ( 1.7 UM )

B.

2

Benztropine 10.8 UMI U.S. Patent Dec. 3 , 2019 Sheet 28 of 52 US 10,493,080 B2

Fig . 20A - B

A. 20 T

Benztropine (0.8 UMI Quinpirole (4.7 UM ) Quinpirole ( 0.6 UM ) Quinpirole (O UM )

B. 20 I

0 Benztropine 10.8 UMI Haloperidol ( 4.7 UM ) Haloperidol (OUM ) U.S. Patent Dec. 3 , 2019 Sheet 29 of 52 US 10,493,080 B2

Fig . 21

monthn Oxybutynin ( EC : 0.61 UM ) 25 . vinstri Atropine ( EC 5.7 UM ) vergeten pratropium ( E65 : 5.6 UM secretario Propiverine (mECrai 0.42 UM ) sotto Scopolamine (~ ECO: 5.1 M )

52

Fig . 22 OPC DMSO ThyroidHormone Brain M2 Receptor

M1 Receptor

M3 Receptor

GAPDH U.S. Patent Dec. 3 , 2019 Sheet 30 of 52 US 10,493,080 B2

Fig. 23A

A. minutes 5 10 20 30 60 Phospho p38MAPK Total p38 MAPK A LES Phospho Akt Total Akt Phospho p42 /44 MAPK Total p42 / 44 Phospho CREB Total CREB -- U.S. Patent Dec. 3 , 2019 Sheet 31 of 52 US 10,493,080 B2

Fig . 23B B. Cyclin D1 Cyclin D2

FoldChange ????? FoldChange ?????????

0.2 0.0 OPCDMSO BenztropineTH OPCDMSO BenztropineTH SO0 unro FoldChange FoldChange OPCDMSO BenztropineTH OPCDMSO BenztropineHIL U.S. Patent Dec. 3 , 2019 Sheet 32 of 52 US 10,493,080 B2

Fig . 23C C. wroteM1/M3 :Muscarinic Misscom Receptor G -protein interactions

4664************ PLC - 1P3

Ca2 + Akt PKC MEK *******

CREB Transcription Cell Cycle U.S. Patent Dec. 3 , 2019 Sheet 33 of 52 US 10,493,080 B2

Fig. 24A - B A.

HT Carbachoir UM Carbachot ( 2 M ) RelativeLightUnits

0 150 Time Iseci

B.

Benztropine ( 20uM ) + Carbachol (50uM ] Benztropine 17uM + Carbachol (GOUM ) 300 Benztropine (24M ) + Carbachol (50uM RelativeLightUnits 200 hehehehehehehehehehehehehehehe 0 50 U.S. Patent Dec. 3 , 2019 Sheet 34 of 52 US 10,493,080 B2

Fig . 24C - D C. Atropine ( 200M ] # Carbachol 1500M Atropine 17uM + Carbachol 150uM Atropine ( 20M + Carbachol 1500M RelativeLightUnits ha ha ha ha ha ha ha 50

D.

Ratio590nm/655nm DMSO I Forskolin Benztropine

[40uM ] ( 13.3uM ] U.S. Patent Dec. 3 , 2019 Sheet 35 of 52 US 10,493,080 B2

Fig . 25

+ Vehicle (Saline ) + Benztropine (0.1 mg/ kg ) Benztropine ( 0.5 mg/ kg ) Benztropine (2.5 mg/ kg ) * Benztropine (12.5 mg/ kg )

w Loch HHH Days U.S. Patent Dec. 3 , 2019 Sheet 36 of 52 US 10,493,080 B2

Fig . 26

Luxol Fast Blue + Hematoxylin and Eosin Luxol Fast Blue

Benztropine U.S. Patent Dec. 3 , 2019 Sheet 37 of 52 US 10,493,080 B2

Fig . 274 - D A. B. WALA C.

D. Benztropine . CD4 /CD69 ALACD4 /CD25 U.S. Patent Dec. 3 , 2019 Sheet 38 of 52 US 10,493,080 B2

Fig . 27E - F E.

CFSE Un -stimulated DMSO Mycophenolate

<

CFSE Un - stimulated DMSO Benztropine U.S. Patent Dec. 3 , 2019 Sheet 39 of 52 US 10,493,080 B2

Fig . 284 - F A. B. Total Splenocytes B Cells 1.8 * 1008 7 * 3007 1.5 * 1008 1.2 * 1008 5x3007 1.0x1008 7.8* 1007 CelNumber 3x1007 2007 2.5x1007 0.0 Vehicle Benztropine Vehicle Benztropine C. D. CD4 + Cells CD8 + Cells

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Fig . 28G - K G. CD4 + / L2 + Cells H. CD4 + AL10 + Cells 2.0 * 1007 2.0x1007

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23.9 % 39.5 % CD4 10.5 %

CD44 U.S. Patent Dec. 3 , 2019 Sheet 41 of 52 US 10,493,080 B2

Fig. 29A - F B Cells A. 1.5x1008 Total Splenocytes B. 1.2 * 1008 1.0 * 1008 5 * 1087 CellNumber 7.5 * 1007 jaqunN100 5.0 * 1007 2.5x1007 Vehicle Benztropine Vehicle Benztropine

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Vehicle Benztropine Vehicle Benztropine U.S. Patent Dec. 3 , 2019 Sheet 42 of 52 US 10,493,080 B2

Fig. 29G- )

CD4 + AL2 + Cells CD4 / L10 + Cells G. 2.5 * 1007 H. 3 * 1007 2.0 * 1007 1.5x1007 10laqunN 1.0x1007 CellNumber 1 * 1007

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Fig . 30A - C A. IgM TNP -LPS Benztropine won

2 Days B. IgG TNP -LPS 350r 3501 Benztropine 250+ 2004

100 M

Days IgM TNP -Ficoli C. Benztropine

2000

Days 12.5 U.S. Patent Dec. 3 , 2019 Sheet 44 of 52 US 10,493,080 B2 Fig . 30D - F IgG TNP -Ficoll D.

OM

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20 25 U.S. Patent Dec. 3 , 2019 Sheet 45 of 52 US 10,493,080 B2

Fig. 31A - D A

B

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50

D. U.S. Patent Dec. 3 , 2019 Sheet 46 of 52 US 10,493,080 B2

Fig. 32

OPC DMSO BenztropineThyroid Hormone

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Actin U.S. Patent Dec. 3 , 2019 Sheet 47 of 52 US 10,493,080 B2

Fig . 33A - B A. 4.57 Vehicle ( Saline ) - BA 10mg /kg 3.5 BA 2.5mg/ kg MeanClinicalEAEScore 2.0 Toutes 1.0 ILI

0 5 1 20 25 Days

B. Vehicle (Saline ) *** IFN - 8 10000U umanghy * IFN - B 3000U olan IFN - B 1000U ------and ClinicalEAEScoreMean 20 1.5 II

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10 15 20 U.S. Patent Dec. 3 , 2019 Sheet 48 of 52 US 10,493,080 B2

Fig . 33C - D

C. Vehicle (Saline ) with BA 2.5mg/ kg + FTY 1mg/ kg * BA 2.5mg/ kg + FTY 0.1mg/ kg 93.5 01 * BA 2.5mg/ kg + FTY 0.01mg/ kg 3.0 MeanClinicalFAEScore 2.5 2.0 I DELLE

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1.54

10 U.S. Patent Dec. 3 , 2019 Sheet 49 of 52 US 10,493,080 B2

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MeanClinicalEAEScore 1.5 1.0 will 0.5 I .... 0.0 0 5 10 15 20 25 30 Days US 10,493,080 B2 1 2 DIRECTED DIFFERENTIATION OF inhibition of OPC differentiation at sites of injury that OLIGODENDROCYTE PRECURSOR CELLS contributes to disease progression ( D. M. Chari, W. F. TO A MYELINATING CELL FATE Blakemore , Glia , 37 , 307 ( 2002 ); D. M. Chari et al ., J Neurosci Res, 73 , 787 (2003 ) ; G.Wolswijk , J Neurosci , 18 , CROSS -REFERENCES TO RELATED 5 601 (1998 ); A. Chang et al. , N Engl J Med, 346 , 165 (2002 ) ; APPLICATIONS T. Kuhlmann et al. , Brain , 131 , 1749 ( 2008 )) . There is no known cure for MS. For treating acute The present application is a divisional of U.S. patent inflammatory attacks, intravenous corticosteroids are typi application Ser. No. 13 / 985,342 , filed Oct. 18 , 2013, which is a national stage application under 35 U.S.C. § 371 of 10 cally administered . Other treatments for MS involve the International Application No. PCT/ US2012 / 025712 , filed administration of an immunomodulator. Although immuno Feb. 17 , 2012 , which claims priority to U.S. Provisional modulators are able to reduce the frequency and severity of Application No. 61/ 444,666 , filed Feb. 18 , 2011, the entire attacks or accumulation of lesions, they do not promote content of each of which is incorporated by reference in its remyelination of damaged axons . entirety . 15 BRIEF SUMMARY OF THE INVENTION BACKGROUND OF THE INVENTION In one aspect , the present invention provides for methods Multiple sclerosis (MS ) afflicts approximately 400,000 of stimulating increased myelination of nerves in a subject people in the United States and 2.5 million worldwide . MS 20 in need thereof. In some embodiments , the method com is an inflammatory disease in which myelin sheaths around prises administering to the subject a therapeutically effective the axons of the brain and spinal cord are damaged . In MS dose of a neurotransmitter receptor modulating agent as well as other demyelinating diseases, autoimmune selected from a muscarinic receptor antagonist, a dopamine inflammatory attack against myelin and oligodendrocytes receptor antagonist , a histamine receptor antagonist , beta causes demyelination . The thinning or loss of myelin sur- 25 adrenergic receptor modulator, and an opioid receptor rounding axons impairs the ability of the axons to effectively modulator; thereby stimulating increased myelination of conduct signals and results in progressive neuronal damage . nerves in the subject. Remyelination is the process by which new myelin In another aspect, the present invention provides for sheaths are generated around axons. Remyelination persists methods of treating a subject having a demyelinating dis throughout adulthood in the CNS and involves the genera- 30 ease . In some embodiments , the method comprises admin tion of new myelinating oligodendrocytes (C. Ffrench istering to the subject a therapeutically effective dose of a Constant, M. C. Raff , Nature , 319 , 499 ( 1986 ) ) . Despite neurotransmitter receptor modulating agent selected from a controversy regarding their intrinsic in vitro and in vivo muscarinic receptor antagonist , a dopamine receptor antago lineage potential (M. C. Nunes et al. , Nat Med , 9 , 439 nist , a histamine receptor antagonist , a beta adrenergic (2003 ) ; S. Belachew et al. , J Cell Biol, 161, 169 ( 2003) ; T. 35 receptor modulator, and an opioid receptor modulator ; Kondo , M.Raff , Science, 289 , 1754 ( 2000 ); Jackson , 2006 ; thereby treating the demyelinating disease. Zhu , 2011 ; Richardson , 2011 ; R. J. Franklin , C. Ffrench In yet another aspect, the present invention provides for Constant, Nat Rev Neurosci, 9 , 839 (2008 ) ), compelling methods of enhancing the therapeutic effect of an immuno evidence indicates that a widespread proliferating popula modulatory agent in a subject in need thereof. In some tion of nerve/ glial antigen -2 (NG2 ) , platelet -derived growth 40 embodiments, the method comprises administering to the factor receptor (alpha subunit, PDGFRa ) positive cells , subject the immunomodulatory agent and a neurotransmitter termed NG2- glia or oligodendrocyte precursor cells (OPCs ), receptor modulating agent; thereby enhancing the therapeu are the major source of newly formed mature oligodendro tic effect of the immunomodulatory agent. cytes required for remyelination ( P. J. Homer et al. , J In some embodiments , the neurotransmitter receptor Neurosci, 20 , 2218 (2000 ); M.C. Nunes et al. , Nat Med , 9, 45 modulating agent is a muscarinic receptor antagonist. In 439 (2003 ) ; J. M.Gensert , J. E. Goldman , Neuron , 19 , 197 some embodiments , the muscarinic receptor antagonist is a ( 1997 ) ; M.S. Windrem et al. , Nat Med , 10 , 93 (2004 ) ; R. J. muscarinic receptor modulator compound listed in Table 1 . Franklin , C. Ffrench -Constant , Nat Rev Neurosci, 9, 839 In some embodiments , the muscarinic receptor is selected ( 2008) ; Richarson , 2011) . from benztropine , carbetapentane, clemastine , ipratropium , Remyelination can occur following the loss of myelin in 50 atropine , and salts thereof . diseases such as MS, thus restoring neurological function to In some embodiments , the neurotransmitter receptor axons . However , although remyelination can occur in the modulating agent is a dopamine receptor antagonist. In some early stages of MS, oligodendrocytes are unable to com embodiments , the dopamine receptor antagonist is a dop pletely rebuild the myelin sheath , and repeated inflammatory amine receptor modulator compound listed in Table 1. In attacks ultimately lead to fewer effective remyelinations 55 some embodiments , the dopamine receptor antagonist is until plaques build up around the damaged axons. A primary selected from benztropine, GBR12935 , trifluoperazine , and cause of remyelination failure is the progressive inability of salts thereof . somatic oligodendrocyte precursor cells to differentiate at In some embodiments , the neurotransmitter receptor the sites of injury . Thus, remission in MS is largely depen modulating agent is a histamine receptor antagonist . In some dent upon OPCs migrating to sites of injury , and subse- 60 embodiments , the histamine receptor antagonist is a hista quently differentiating to a mature cell fate capable of repair mine receptor modulator compound listed in Table 1. In ( J. R. Patel , R. S. Klein , FEBS Lett, 585 , 3730 ( 2011 ); D. some embodiments , the histamine receptor antagonist is Kremer et al. , Ann Neurol, 69 , 602 (2011 ) ; A. Chang et al. , clemastine or a salt thereof. N Engl J Med , 346 , 165 ( 2002 ) ). Studies aimed at evaluating In some embodiments , the neurotransmitter receptor the presence and relative densities of OPCs at sites of 65 modulating agent is a beta adrenergic receptor modulator. In chronically demyelinated MS lesions indicate that it is not a some embodiments , the beta adrenergic receptor modulator failure of repopulation or migration of OPCs, but rather is a beta adrenergic receptor modulator compound listed in US 10,493,080 B2 3 4 Table 1. In some embodiments , the beta adrenergic receptor some embodiments , the method comprises administering the modulator is selected from pindolol , salmeterol, salbutamol, neurotransmitter receptor modulating agent and the immu albuterol, and salts thereof. nomodulatory agent sequentially . In some embodiments , the In some embodiments , the neurotransmitter receptor method comprises administering the neurotransmitter recep modulating agent is an opioid receptor modulator. In some 5 tor modulating agent concurrently . embodiments , the opioid receptor modulator is an opioid 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 demy embodiments , the opioid receptor modulator is carbetapen elinating disease . In some embodiments , the composition tane , Snc - 80 , BD - 1047 , or salts thereof. comprises : In some embodiments , the neurotransmitter receptor 10 a neurotransmitter receptor modulating agent ; and modulating agent is benztropine , carbetapentane, clemas an immunomodulatory agent. tine , pindolol , ipratropium , atropine , GBR12935, Snc - 80 , In yet another aspect , the present invention provides for BD - 1047 , salmeterol, albuterol, trifluoperazine , or a salt kits for use in treating a subject having a demyelinating thereof. In some embodiments , the neurotransmitter receptor disease . In some embodiments , the composition comprises : modulating agent is benztropine , clemastine, salmeterol, 15 a neurotransmitter receptor modulating agent; and salbutamol , trifluoperazine, or a salt thereof. In some an immunomodulatory agent. embodiments , the neurotransmitter receptor modulating In some embodiments , the composition or kit comprises agent is benztropine or a salt thereof ( e.g. , benztropine a neurotransmitter receptor modulating agent as described mesylate ) herein and an immunomodulatory agent as described herein . In some embodiments , the subject has a demyelinating 20 In some embodiments , the neurotransmitter receptor modu disease . In some embodiments , the demyelinating disease is lating agent is selected from a muscarinic receptor antago multiple sclerosis, idiopathic inflammatory demyelinating nist, a dopamine receptor antagonist , a histamine receptor disease, transverse myelitis , Devic's disease, progressive antagonist , a beta adrenergic receptor ator, and an multifocal leukoencephalopathy, optic neuritis , leukoystro opioid receptor modulator. In some embodiments , the neu phy, Guillain -Barre syndrome, chronic inflammatory demy- 25 rotransmitter receptor modulating agent is benztropine , cle elinating polyneuropathy , autoimmune peripheral neuropa mastine , salmeterol, salbutamol, trifluoperazine , or a salt thy, Charcot- Marie - Tooth disease , acute disseminated thereof. In some embodiments, the neurotransmitter receptor encephalomyelitis, adrenoleukodystrophy, adrenomy modulating agent is benztropine or a salt thereof. In some eloneuropathy, Leber's hereditary optic neuropathy, or embodiments , the immunomodulatory agent is fingolimod human T -cell lymphotropic virus (HTLV ) -associated 30 (FTY720 ), interferon beta - la , interferon beta -lb , glatiramer myelopathy. In some embodiments , the demyelinating dis acetate , mitoxantrone, or natalizumab . In some embodi ease is multiple sclerosis . In some embodiments , the demy ments , the neurotransmitter receptor modulating agent is elinating disease is relapsing -remitting multiple sclerosis benztropine, clemastine , salmeterol, salbutamol, trifluopera (RRMS ). In some embodiments , the demyelinating disease zine, or a salt thereof and the immunomodulatory agent is is secondary progressive multiple sclerosis (SPMS ) . In some 35 fingolimod (FTY720 ), interferon beta - la , or interferon beta embodiments , the demyelinating disease is primary progres 1b . sive multiple sclerosis (PPMS ) . In some embodiments , the In some embodiments , the neurotransmitter receptor demyelinating disease is progressive relapsingmultiple scle modulating agent is formulated as a therapeutically effective rosis ( PRMS ). or optimal dose and the immunomodulatory agent is formu In some embodiments , the subject is a human . In some 40 lated as a therapeutically effective or optimal dose . In some embodiments , the subject is a non -human mammal. embodiments , the neurotransmitter receptor modulating In some embodiments , the method further comprises agent is formulated as a therapeutically effective or optimal administering to the subject an immunomodulatory agent. In dose and the immunomodulatory agent is formulated as a some embodiments , the immunomodulatory agent is fingoli subtherapeutic dose . In some embodiments , the immuno mod (FTY720 ) , interferon beta -la , interferon beta -lb , glati- 45 modulatory agent is formulated as a therapeutically effective ramer acetate , mitoxantrone , or natalizumab . In some or optimal dose and the neurotransmitter receptor modulat embodiments , the immunomodulatory agent is fingolimod ing agent is formulated as a subtherapeutic dose . In some (FTY720 ), interferon beta -la , or interferon beta - lb . embodiments , the neurotransmitter receptor modulating In some embodiments , the method comprises administer agent is formulated as a subtherapeutic dose and the immu ing to the subject a therapeutically effective or optimal dose 50 nomodulatory agent is formulated as a subtherapeutic dose . of one or both of the neurotransmitter receptor modulating In yet another aspect, the present invention also provides agent and the immunomodulatory agent. In some embodi for use of a composition as described herein for the manu ments , the method comprises administering to the subject a facture of a medicament for the treatment of a demyelinating subtherapeutic dose of one or both of the neurotransmitter disease . receptor modulating agent and the immunomodulatory 55 agent . In some embodiments , the method comprises admin DEFINITIONS istering to the subject a therapeutically effective dose or optimal dose of the neurotransmitter receptor modulating As used herein , the term “ neurotransmitter receptor agent and a subtherapeutic dose of the immunomodulatory modulating agent” refers to an agent that inhibits or activates agent. In some embodiments , the method comprises admin- 60 the activity of a neurotransmitter receptor. In some embodi istering to the subject a therapeutically effective dose or ments , the term refers to a compound that modulates the optimal dose of the immunomodulatory agent and a sub activity of a muscarinic receptor ( e.g., a muscarinic receptor therapeutic dose of the neurotransmitter receptor modulating antagonist ), a dopamine receptor ( e.g. , a dopamine receptor agent . antagonist ) , a histamine receptor ( e.g., a histamine receptor In some embodiments , the method comprises administer- 65 antagonist ), a beta adrenergic receptor ( e.g. , a beta adren ing one or both of the neurotransmitter receptor modulating ergic receptor antagonist ) , or an opioid receptor ( e.g., an agent and the immunomodulatory agent systemically . In opioid receptor modulator ). For any compound that is iden US 10,493,080 B2 5 6 tified as a neurotransmitter receptor modulating agent ( e.g., leukoystrophy , Guillain - Barre syndrome, chronic inflamma a compound described in Table 1 herein ), it is also contem tory demyelinating polyneuropathy, autoimmune peripheral plated that any pharmaceutically acceptable salts , prodrugs, neuropathy, Charcot- Marie - Tooth disease, acute dissemi racemic mixtures , conformational and /or optical isomers , nated encephalomyelitis , adrenoleukodystrophy, adrenomy crystalline polymorphs and isotopic variants of the com- 5 eloneuropathy, Leber's hereditary optic neuropathy, or pound may also be used . In some embodiments , the neu HTLV - associated myelopathy. In some embodiments , the rotransmitter receptor modulating agent is a small molecule , demyelinating disease is multiple sclerosis . e.g., a molecule having a molecular weight of less than 800 As used herein , the term “ subject” refers to animals such kDa. In some embodiments , the neurotransmitter receptor as mammals , including, but not limited to , primates ( e.g., modulating agent is a small molecule that is able to cross the 10 humans) , cows, sheep , goats , horses , dogs , cats , rabbits , rats , blood - brain barrier . mice and the like . In some embodiments , the subject is a As used herein , the term “ oligodendrocyte precursor cell” human . or “OPC ” refers to an undifferentiated progenitor cell with As used herein , the term " compound ” refers to any the capacity to self - renew and differentiate into a myelinat molecule, either naturally occurring or synthetic , e.g., pep ing oligodendrocyte . A “mature myelinating cell fate ” refers 15 tide , protein , oligopeptide ( e.g., from about 5 to about 50 to cell that is capable of forming myelin , e.g., a myelinating amino acids in length ) , small organic molecule , polysaccha oligodendrocyte . “ Differentiation ” refers to the process by ride , peptide , circular peptide , peptidomimetic , lipid , fatty which a specialized cell type is formed from a less special acid , siRNA , polynucleotide , oligonucleotide, etc., to be ized cell type, for example , a myelinating oligodendrocyte tested for the capacity to induce OPC differentiation . The from an OPC . In some embodiments , an OPC is identified by 20 compound to be tested can be in the form of a library of test morphology and /or by the presence of a biomarker , e.g. , compounds, such as a combinatorial or randomized library PDGFR - a or NG2. In some embodiments , a myelinating that provides a sufficient range of diversity . Test compounds oligodendrocyte is identified by morphology and /or by the are optionally linked to a fusion partner , e.g., targeting presence of a marker, e.g. , myelin basic protein (MBP ), compounds, rescue compounds, dimerization compounds, myelin oligodendrocyte glycoprotein (MOG ), 2'3 '- cyclic- 25 stabilizing compounds, addressable compounds, and other nucleotide 3 ' phosphodiesterase (CNP ), galactocebroside functional moieties. Conventionally , compounds are (GalC ), 01 antigen (01 ) , or 04 antigen (04 ). screened by identifying a test compound ( called a “ screening As used herein , the terms “ stimulating increased myeli hit” ) with some desirable property or activity , e.g., inducing nation ” or “ stimulate increased myelination ” refer to induc activity , and screening hits are confirmed and validated ing an increased amount of myelin surrounding an axon , 30 using in vitro and in vivo assays. Often , high throughput e.g. , by administering an agent that induces the differentia screening (HTS ) methods are employed for such an analysis . tion of oligodendrocyte precursor cells to a mature myeli An “ agonist ” refers to an agent that stimulates , increases , nating cell fate , as compared to the amount of myelin activates , or enhances activation of a neurotransmitter recep surrounding the axon in the absence of the agent being tor ( e.g., muscarinic receptor, dopamine receptor, histamine administered . In some embodiments , an agent stimulates 35 receptor, beta adrenergic receptor, and / or opioid receptor ) of " increased ” myelination when the amount of myelin sur the invention . rounding the axon in a sample ( e.g. , a brain tissue sample An “ antagonist” refers to an agent that partially or totally from a subject having a demyelinating disease ) subsequent blocks stimulation , decreases, prevents , inactivates , or to administration of an agent that induces the differentiation delays activation of a neurotransmitter receptor ( e.g., mus of OPCS to a mature myelinating cell fate is at least about 40 carinic receptor, dopamine receptor, histamine receptor, beta 5 % , 10 % , 15 % , 20 % , 30 % , 40 % , 50 % , 60 % , 70 % , 80 % , adrenergic receptor, and /or opioid receptor) of the invention . 90 % , or more as compared to the amount of myelin sur As used herein , the terms “ therapeutically effective rounding the axon in the sample prior to administration of amount or dose ” or “ therapeutically sufficient amount or the agent. The amountof myelin surrounding an axon can be dose " or " effective or sufficient amount or dose ” refer to a measured by any method known in the art , e.g. , using 45 dose that produces therapeutic effects for which it is admin magnetic resonance imaging (MRI ) . In some embodiments , istered when it is administered on its own . The exact dose an agent stimulates increased myelination when one ormore will depend on the purpose of the treatment, and will be characteristics of a demyelinating disease ( e.g., multiple ascertainable by one skilled in the art using known tech sclerosis ) improves subsequent to administration of an agent niques ( see , e.g., Lieberman , Pharmaceutical Dosage Forms that induces differentiation ofOPCS to a mature myelinating 50 ( vols . 1-3 , 1992 ); Lloyd , The Art , Science and Technology of cell fate as compared to the characteristic of the diseases Pharmaceutical Compounding ( 1999) ; Pickar , Dosage Cal prior to administration of the agent. As a non -limiting culations (1999 ) ; and Remington : The Science and Practice example , an agent is said to stimulate increased myelination of Pharmacy, 20th Edition , 2003 , Gennaro , Ed ., Lippincott, subject having multiple sclerosis when the frequency Williams & Wilkins ) . and / or severity of inflammatory attacks decreases subse- 55 As used herein , the terms “ administer ” or “ administering ” quent to administration of an agent as compared to the refer to any type of administration , including but not limited frequency and /or severity of inflammatory attacks prior to to oral administration , administration as a suppository , topi administration of the agent. cal contact, parenteral, intravenous, intraperitoneal , intra As used herein , the term “ demyelinating disease ” refers to muscular , intralesional, intranasal or subcutaneous admin a disease or condition of the nervous system characterized 60 istration , intrathecal administration , or the implantation of a by damage to or loss of the myelin sheath of neurons. A slow -release device e.g., a mini- osmotic pump , to the sub demyelinating disease can be a disease affecting the central ject. nervous system or a disease affecting the peripheral nervous As used herein , the terms “ treat” or “ treating ” or “ treat system . Examples of demyelinating diseases include , but are ment” refer to any indicia of success in the treatment or not limited to , multiple sclerosis , idiopathic inflammatory 65 amelioration of an injury, pathology, condition , or symptom demyelinating disease, transverse myelitis , Devic's disease , ( e.g., pain ) , including any objective or subjective parameter progressive multifocal leukoencephalopathy, optic neuritis , such as abatement; remission ; diminishing of symptoms or US 10,493,080 B2 7 8 making the symptom , injury, pathology or condition more ers . Immunofluorescence staining using specific antibodies tolerable to the patient; decreasing the frequency or duration for MBP, MOG , and 2 ', 3 ' - cyclic nucleotide 3 ' phosphodies of the symptom or condition ; or, in some situations , pre terase (CNP ) . Nuclei were identified using DAPI. Com venting the onset of the symptom or condition . The treat pound treatment of OPCs at EC90 concentrations for 6 days ment or amelioration of symptoms can be based on any 5 induces the robust expression of mature oligodendrocyte objective or subjective parameter; including, e.g. , the result markers . of a physical examination . FIG . 4. Immunofluorescence analysis of compound As used herein , the term " subtherapeutic dose” refers to treated OPCs using mature oligodendrocyte specific mark a dose of a pharmacologically active agent( s) , either as an ers . Immunofluorescence staining using specific antibodies administered dose of pharmacologically active agent , or 10 for galactocereberoside (Gal ), oligodendrocyte marker 01 actual level of pharmacologically active agent in a subject , (01 ) and oligodendrocyte marker 04 (04 ) . Nuclei were that functionally is insufficient to elicit the intended phar identified using DAPI. Compound treatment of OPCs at macological effect in itself , or that quantitatively is less than EC90 concentrations for 6 days induces the robust expression the established therapeutic dose for that particular pharma of mature oligodendrocyte markers . cological agent ( e.g., as published in a reference consulted 15 FIG . 5A - E . Representative neurotransmitter receptor by a person of skill, for example , doses for a pharmacologi modulating agents benzatropine and trifluoperazine amelio cal agent published in the Physicians ' Desk Reference , 66th rate symptoms in a PLP induced relapsing EAE mouse Ed ., 2012 , PDR Network , LLC ; or Brunton , et al . , Goodman model . SJL mice were immunized with proteolipid peptide & Gilman's The Pharmacological Basis of Therapeutics , (PLP ) and pertussis toxin and monitored daily with scoring 12th edition , 2011, McGraw -Hill Professional ) when admin- 20 on the standard clinical EAE scale ( 0-5 ). Compounds were istered on its own . A “ subtherapeutic dose ” can be defined administered daily for the duration of the study in saline at in relative terms ( i.e., as a percentage amount ( less than 10 mg/ kg via intra- peritoneal injection (0.1 ml) , starting on 100 % ) of the amount of pharmacologically active agent the day of the appearance of clinical EAE symptoms (day conventionally administered ) . For example , a subtherapeutic 10 ) . Sub -optimal dosing of mycophenolate mofetil at 20 dose amount can be about 1 % to about 75 % of the amount 25 mg/ kg in sterile saline (pH 5 ) was administered in combi of pharmacologically active agent conventionally adminis nation with benzatropine and trifluoperazine respectively , tered . In some embodiments , a subtherapeutic dose can be starting on the day of peak clinical EAE symptoms (day 14 ) about 75 % , 50 % , 30 % , 25 % , 20 % , 10 % or less, of the post PLP injection . Mean clinical EAE score and standard amount of pharmacologically active agent conventionally error ofmean (SEM ) are shown for each study group . Mice administered . 30 in the vehicle control group, n = 6 (FIG . 5A - E , open boxes ) show appearance of acute phase (mean maximal clinical BRIEF DESCRIPTION OF THE DRAWINGS EAE score of 2 : 0.8 on day 11) followed by remission (mean maximal clinical EAE score of 0.3-0.3 on day 19 ) and FIG . 1A -C . Neurotransmitter receptor modulating agents relapse of EAE symptoms (mean maximal clinical EAE dose dependently induce OPC differentiation . (A ) Chemical 35 score of 1.5 =0.4 on day 25 ). Mycophenolate mofetil, n = 6 structures of representative identified OPC differentiating (FIG . 5A closed boxes ) shows partial reduction in the molecules. The calculated EC50 values were determined severity of relapse (mean maximal EAE score 0.7 + 0.3 on using appropriate Graphpad Prism 5.0 curve fitting software . day 25 , p value < 0.05 ) . Benzatropine , n = 5 (FIG . 5B closed ( B ) Dose dependent compound induced OPC differentiation . boxes ) and trifluoperazine, n = 6 (FIG . 5D closed boxes ) OPCs were plated in 384 well plates at 1000 cells per well 40 show a significant reduction in the severity of relapse (mean in OPC media containing basal PDGF- aa (2 ng /mL ) and maximal clinical EAE score 0.2 +0.2 ( day 25 ) and 0.4 + 0.4 treated with serial dilutions of compounds. Following six ( day 26 ) respectively , p values< 0.001 ) . Benzatropine and days of compound treatment, cells were fixed and subjected trifluoperazine in combination with mycophenolate mofetil , to immunofluorescence analysis using anti -myelin basic n = 6 for both ( FIG . 5C and FIG . 5E respectively , closed protein (MBP ) antibody . Image acquisition and quantifica- 45 boxes ) show complete suppression of relapse (mean maxi tion of MBP staining was performed using the OPERA mal clinical EAE score 0 : 0 for both on day 25, p val imaging system . ( C ) Maximal OPC differentiation inducing ues < 0.001) . activity of identified compounds compared to DMSO con FIG .6A - B . Muscarinic receptor antagonism induces OPC trol. differentiation but is not the sole pharmacological mecha FIG . 2A - C . Neurotransmitter receptor modulating agents 50 nism of all identified differentiation inducing agents . ( A ) induce the differentiation of OPCs to a myelinating oligo Themuscarinic receptor agonist carbachol antagonizes com dendrocyte cell fate . ( A ) Western blot analysis of OPCs pound induced OPC differentiation in some cases. OPCs treated with compounds at EC90 concentrations for 6 days. were plated in 384 -well plates at 1000 cells per well in Total protein was isolated from cell pellets and probed for media containing compounds at EC90 concentrations and myelin basic protein (MBP ) and myelin oligodendrocyte 55 treated with 1 : 3 serial dilutions of carbachol . Following 6 glycoprotein (MOG ) using specific antibodies. ( B - C ) Quan days of treatment, cells were fixed and stained for MBP . titative RT- PCR (QRT - PCR ) analysis of OPCs treated with Plates were imaged using the OPERA high content screen compounds at EC70 concentrations for 6 days. Total RNA ing system and MBP staining was quantified as described . was isolated from cell pellets and reverse transcribed .MBP Carbachol treatment results in dose dependent inhibition of and MOG expression was quantified using specific probes 60 the differentiation activity of benzatropine, carbapentane and Taqman -based qRT- PCR . Expression was normalized to and clemastine , while no effect is observed on the differen the internal controls ß -actin and GAPDH . Fold change in tiation activity of salmeterol, GBR12935 and trifluopera gene expression over DMSO treated control cells is plotted zine . (B ) Carbachol- induced calcium ( Ca2 + ) influx is for MBP ( B ) and MOG ( C ) . Results are displayed as blocked by the muscarinic receptor antagonist activity of mean +/- standard deviation , n = 3 . 65 some compounds. OPCs were plated in 384 -well plates at FIG . 3. Immunofluorescence analysis of compound 5000 cells per well in media containing basal PDGF -aa . treated OPCs using mature oligodendrocyte specific mark Cells were equilibrated with Ca2 + sensitive Fluo -3 AM® dye US 10,493,080 B2 9 10 in HBSS for 30 min . Multiple concentrations of OPC mice treated with FTY720 (0.1 mg/ kg ) or FTY720 (0.1 differentiation - inducing compounds ( 3 times the EC90 , EC90 mg/ kg ) or benztropine (BA ; 2.5 mg/ kg ). ( D ) Clinical EAE and EC50 ) were added followed by addition of 1 : 3 serial scores for mice treated with interferon -ß ( IFN ; 3000 dilutions of the muscarinic receptor agonist Carbachol. Ca2 + U /mouse ) or interferon - ß (IFN ; 10,000 U /mouse ) or benz influx was immediately measured using the FLIPR 5 tropine (BA ; 2.5 mg/ kg ) . ( E ) Clinical EAE scores for mice TETRA® system . Shown are representative data for carba treated with FTY720 (0.1 mg/ kg ) in combination with chol treatment at 100 uM and compound treatment at EC90 . benztropine (BA ; 2.5 mg/ kg ) show a comparable decrease in Relative light units (RLU ) indicating Ca2 + influx are plotted clinical severity scores as compared to FTY720 ( 1 mg/ kg ) , on the y -axis and time ( in sec ) after addition of carbachol facilitating the reduction in the dose ofFTY720 . (F ) Clinical plotted on the x -axis . GBR12935 , trifluoperazine, and sal- 10 EAE scores for mice treated with interferon - B (IFN ; 3000 meterol ( top ) show no effect on carbachol induced Ca2 + U /mouse ) in combination with benztropine (BA ; 2.5 mg/ kg ) influx while clemastine , benzatropine, and carbapentane do not show a comparable decrease in clinical severity (bottom ) inhibit Ca2 + influx induced by carbachol . scores as interferon - ß ( IFN ; 10,000 U /mouse ). Error bars FIG . 7A - C . A high throughput screen identified muscar indicate standard deviation of the mean within each group of inic receptor antagonist benztropine as an inducer of OPC 15 8 mice. differentiation . ( A ) Benztropine [ 1.5 uM ] and positive con FIG . 11A - B . Screen to identify inducers of OPC differ trol thyroid hormone [ 1 uM ] treated rat OPCs were cultured entiation . ( A ) OPCs were maintained as proliferating A2B5 under basal differentiation conditions ( 2 ng /ml PDGF) for 6 positive cells under basal growth conditions (Neurobasal days and stained for MBP ( green ). The structure of benz medium , B27 supplement without Vitamin A , non -essential tropine is also shown . ( B ) Benztropine [ 1.5 uM ] treated 20 amino acids, L -Glutamine , 30 ng /ml PDGF ). OPCs were OPCs were analyzed for MBP and MOG expression after 6 plated in basal differentiation media (Neurobasal medium , days in culture using qRT- PCR . ( C ) OPCs were co -treated B27 supplement without Vitamin A , non - essential amino with benztropine [2.3 uM ] and carbachol [ O UM , 0.6 uM and acids, L -Glutamine , 2 ng/ ml PDGF) , treated with DMSO 4.7 uM ] for 6 days under basal differentiation conditions and ( < 0.1 % ) or thyroid hormone ( 1 uM ), fixed after 6 days in stained for MBP. 25 culture , and stained using antibodies for CNP, 04 , or MBP. FIG . 8A - C . Benztropine decreases the clinical severity of A2B5- positive OPCs differentiate into immature oligoden disease in the PLP induced EAE model for MS. ( A ) Benz drocytes that express CNP and 04 , but not MBP, upon tropine decreased the clinical severity of disease in the PLP withdrawal of PDGF. Addition of thyroid hormone induces induced EAE model when dosed prophylactically ( starting the differentiation of OPCs into mature oligodendrocytes on the day of PLP injection ) as well as therapeutically (at the 30 that express MBP. ( B ) Screen strategy using A2B5 positive start of EAE symptoms) and showed efficacy comparable to OPCs cultured under basal differentiation conditions and FTY720 ( 1 mg/ kg ) and interferon - B ( 10,000 U /mouse ) . ( B ) treated with compounds for 6 days to identify small mol Quantification of confocal images of spinal cord sections ecules that induce the differentiation of OPCs to mature , from EAE mice treated with benztropine and stained with MBP expressing oligodendrocytes . specific antibodies for GST -1 ( a marker of mature oligo- 35 FIG . 12A - B . Primary hit confirmation . (A ) Dose response dendrocytes ) and NG2 (a marker of OPCs) showed assay used to confirm primary screening hits and determine increased GST -1 positive cells as compared to vehicle potency ( EC50 ) . (EC50 ) is defined as the concentration that treated mice , with no change in the number of NG2 positive results in a half maximal increase in the percentage of total cells . (C ) Representative confocal images of spinal cord cells that express MBP as detected by immunostaining . sections from EAE mice treated with benztropine and 40 OPCs were cultured in differentiation medium and treated stained with specific antibodies for GST- 1 (mature oligo with benztropine or control (DMSO < 0.01 % ) for 6 days . dendrocytes ) and NG2 ( OPCs) . Cells were fixed and immunostained using antibodies for FIG . 9A - B . Benztropine treatment induces remyelination MBP . Error bars represent standard deviations from rep in vivo in the cuprizone model. (A ) Representative images licate experiments. ( B ) Representative images show dose from the corpus callosum region of the brain at various time 45 dependent activity of benztropine . points show increased remyelination observed in benzatro FIG . 13. Benztropine induces differentiation of OPCs to pine treated mice as compared to vehicle treated mice 2 mature oligodendrocytes . OPCs were plated in differentia weeks after cuprizone withdrawal and commencement of tion medium (Neurobasal medium , B27 supplement without drug administration . (B ) Quantification of the myelinated Vitamin A , non - essential amino acids, L -Glutamine , 2 ng/ ml areas in the corpus callosum region shows a significant 50 PDGF) and treated with DMSO ( < 0.01 % ) , benztropine [ 1.5 ( ~ 2 - fold ) increase in myelin staining in benztropine treated UM ] or thyroid hormone [ 1 uM ] . After 6 days in culture , mice as compared vehicle controls 2 weeks after drug cells were analyzed for MBP and MOG expression by treatment. Data is represented in terms of threshold bins on Western blot. the grey scale (0-50 ) as described . Error bars represent FIG . 14. Compound treatment induces the differentiation standard deviations of at least 6 corpus callosum regions . 55 of OPCs to mature oligodendrocytes . OPCs were plated in FIG . 10A - F . Combination with benztropine improves differentiation medium (Neurobasal medium , B27 supple efficacy and allows for a reduction in the dose of FTY720 ment without Vitamin A , non - essential amino acids, L -Glu and interferon - ß . (A ) Clinical EAE scores for mice treated tamine, 2 ng /ml PDGF ) and treated with DMSO ( < 0.01 % ) , with FTY720 ( 1 mg/ kg ) in combination with benztropine benztropine [ 1.5 uM ) or thyroid hormone [ 1 uM ] for 6 days. (BA ; 2.5 mg/ kg ) show a significantly decreased clinical 60 Cells were fixed and immunostained for MBP , MOG , CNP , severity as compared to mice treated with FTY720 ( 1 Galc , 01, or 04. Representative images of DMSO , benz mg/ kg ) or benztropine ( 2.5 mg/ kg ) alone . ( B ) Clinical EAE tropine and thyroid hormone treated cells show expression scores for mice treated with Interferon - ß (IFN ; 10,000 of mature oligodendrocyte markers in benztropine and thy U /mouse ) in combination with benztropine (BA , 2.5 mg/ kg ) roid hormone treated cells , but not in DMSO treated cells. show a significantly decreased clinical severity as compared 65 FIG . 15.Gene expression profile of OPCs differentiated to to mice treated with Interferon - y (IFN ; 10,000 U /mouse ) or oligodendrocytes by benztropine treatment. OPCs were Benztropine (2.5 mg/ kg ) alone. (C ) Clinical EAE scores for plated in differentiation medium and treated with DMSO US 10,493,080 B2 11 12 ( < 0.1 % ), benztropine [2.3 uM ] or thyroid hormone [ 1 uM ] FIG . 22. OPCs express muscarinic receptors and choline for 6 days. OPCs from the same passage were also pelleted acetyl transferase. Total RNA was isolated from OPCs and frozen . Total RNA was isolated from the cells and gene treated with DMSO (< 0.1 % ) or thyroid hormone [ 1 uM ] for expression analysis was performed using rat genome arrays 6 days, or from whole rat brain . RNA was reverse tran from Affymetrix . Global clustering analysis of mRNA 5 scribed to cDNA and gene expression ofmuscarinic recep expression probe sets that displayed a > 2 - fold change in tors M1, M2, or M3 was detected by PCR using gene specific expression across samples shows clustering of benztropine primers. treated samples with thyroid hormone treated samples while FIG . 23A - C . Antagonism of M2/ M3 muscarinic signaling gene expression profiles of DMSO treated cells cluster with pathway by benztropine primes OPCs for differentiation . OPCs. Data are represented as fold change over DMSO 10 ( A ) OPCs were treated with benztropine [ 10 UM ] for the treated controls . Increased expression of mature oligoden indicated times and pelleted for Western blot analysis of drocyte genes was seen in compound treated cells as com total protein . Benztropine inhibits signaling proteins down pared to DMSO treated cells . stream of M2/M3 muscarinic receptors by down -regulating FIG . 16A - B . Gene expression profiles of benztropine 15 phosphorylation of Akt, p42MAP Kinase, and increasing treated cells show a downregulation of OPC genes and an phosphorylation of p38MAP Kinase and CREB . ( B ) OPCs upregulation of mature oligodendrocyte genes. ( A ) were plated in basal differentiation conditions and treated Increased expression ( fold change ) of mature oligodendro with benztropine [ 1.5 uM ] , thyroid hormone [ 1 uM ] or cyte genes in compound treated cells as compared to DMSO DMSO (< 0.1 % ). OPCs from the same passage were also treated cells . ( B ) Decreased expression (fold change ) of 20 pelleted and frozen . Total RNA was isolated from the cells , OPC genes in compound treated cells as compared to reverse transcribed to first strand cDNA and used as a DMSO treated cells . template for qRT- PCR . Gene specific FAM labeled probes FIG . 17A -D . Timing of compound treatment determines were used to detect expression levels of various genes, with the efficiency of differentiation . ( A ) OPCs were plated in probes for beta -actin and GAPDH as internal controls . Gene differentiation medium on day 0 and treated with com- 25 expression shows a downregulation in cell cycle genes such pounds on various days. Cells were fixed and immunos as Cyclin D1, Cyclin D2 , C - Fos, C - Jun indicating an exit tained for MBP on day 6 after plating . (B ) OPCs were plated from cell cycle . ( C ) Signaling pathway downstream of the in differentiation medium on day 0 and treated with com pounds 12 hours later. Cells were fixed on various days M2/M3 muscarinic receptors. following compound treatment and immunostained for 30 rinicFIG receptors. 24A - D . Benztropinebut not the antagonizesM2/M4 muscarinic the M2/M3 receptors musca. MBP. (C ) Treatment with benztropine within 48 hours of OPCs were plated in differentiation media for 12 hours . plating in differentiation medium induced efficient differen Media was changed to Hank's Balanced Salt Solution tiation of OPCs to ma ire oligodendrocytes. Compound (HBSS ) with 4-( 2 -hydroxyethyl ) -1 - piperazineethanesulfo mediumtreatment reduced 72 hours the or efficiency more after of platingdifferentiation in differentiation of OPCs. 35 nic acid (HEPES ) and cells were treated with benztropine at ( D ) Benztropine treatment for a minimum of 5 days was various concentrations for 1 hour. Carbachol was added and necessary to induce efficient differentiation of OPCs to calcium flux was measured for 186 seconds using the FLIPR mature oligodendrocytes. TETRA® system . ( A ) Carbachol induced a dose dependent FIG . 18. Carbachol antagonizes benztropine induced OPC increase in intracellular Ca2 + levels . ( B ) Benztropine dose differentiation . OPCs were plated in basal differentiation 40 dependently blocked carbachol induced calcium influx media and co - treated with benztropine [ 2.3 UM ] and carba through antagonism of M./M2 muscarinic receptors . ( C ) chol [ O UM , 0.6 uM or 4.7 uM ] for 6 days and stained for Atropine , a muscarinic antagonist, serves as a positive MBP (green ). control. ( D ) OPCs were plated in differentiation medium for FIG . 19A - B . Benztropine has no effect on histamine 12 hours . A CAMP- HTRF assay was performed using the receptor signaling. OPCswere plated in basal differentiation 45 CAMP dynamic 2 kit . Benztropine had no effect on the levels medium (Neurobasal medium , B27 supplement without of CAMP. IBMX was added as a cAMP stabilizer, and Vitamin A , non -essential amino acids, L -Glutamine , 2 ng /ml forskolin was a positive control. PDGF) and co - treated with various concentrations of ben FIG . 25. Dose dependent activity of prophylactic benz ztropine and ( A ) histamine or ( B ) the histamine receptor tropine in the EAE model. EAE was induced in mice using agonist histamine trifluoromethyltoluidide (HTMT ) . 50 PLP and pertussis toxin . Benztropine dissolved in saline at FIG . 20A - B . Benztropine has no effect on dopamine D2 various doses was injected via I.P. injection daily using the and D3 receptor signaling . OPCs plated in basal differen prophylactic mode followed by scoring clinical symptoms. tiation medium (Neurobasal medium , B27 supplement with Prophylactic dosing is defined as administration of com out Vitamin A , non - essential amino acids, L -Glutamine , 2 pound commenced on the day of PLP injection . Error bars ng/ ml PDGF ) and co - treated with various concentrations of 55 indicate standard deviation of the mean within each group of benztropine and the dopamine receptor ( A ) agonist quin 8 mice . pirole or ( B ) antagonist haloperidol. Error bars indicate FIG . 26. Benztropine treatment induces remyelination in standard deviations of 3 replicate measurements . vivo in the PLP induced EAE models for MS. Benztropine FIG . 21. Muscarinic antagonists induce differentiation of does not block lymphocyte infiltration in EAE mice, but OPCs to oligodendrocytes . OPCs were plated in differen- 60 leads to significantly increased LFB staining, indicating the tiation medium ( 2 ng/ ml PDGF ) and treated with various presence of myelin in areas infiltrated by lymphocytes as concentrations of compounds for 6 days . Cells were fixed compared to vehicle treated mice . EAE was induced in mice and immunostained for MBP . Selective muscarinic antago by injecting PLP and pertussis toxin and the mice were nists oxybutynin , atropine , ipratropium , propiverine, and treated with benztropine ( 10 mg/ kg ) or vehicle controls at scopolamine induced differentiation of OPCs in a dose 65 the first appearance of EAE symptoms. Spinal cords were dependent manner. EC50 values for compound induced dif isolated from mice representative of the average group ferentiation are indicated . scores during the relapse phase of EAE , sectioned and US 10,493,080 B2 13 14 stained with Luxol Fast Blue and H & E (left panel ) and as serum IgM and IgG levels . ( C , D ) Benztropine showed no Luxol Fast Blue only ( right panel) . Arrows point to regions effect on TNP -Ficoll induced T - cell independent B -cell of lymphocyte infiltration . responses measured as serum IgM and IgG levels . ( E , F ) FIG . 27A - F . Benztropine has no effect on T - cell activation Benztropine showed no effect on TNP - KLH induced T -cell and proliferation in vitro . (A - F ) Total splenocytes were 5 dependent B -cell responses measured as serum IgM and IgG isolated from mice and stimulated with CD3 and analyzed levels . Error bars represent standard deviations from 3 by flow cytometry for expression of T - cell activation mark replicate ELISAs performed on samples from 5 mice in each ers CD69 and CD25 and T -cell proliferation using CFSE . treatment group . ( A ) Unstimulated cells . ( B ) DMSO treated cells . (C ) Myco FIG . 31A - D . Quantification ofmyelination staining in the phenolate suppresses the activation and proliferation of 10 cuprizone model. (A ) Luxol Fast Blue staining was per T- cells as compared to DMSO . (D ) Benztropine has no effect formed on sections from the corpus callosum region of the on T -cell activation . ( E ) Mycophenolate suppresses T -cell brains isolated from mice treated either with benztropine ( 10 proliferation . ( F ) Benztropine has no effect on T - cell prolif mg/ kg ) or vehicle control after 7 weeks of exposure to eration . The numbers represent percentage gated popula cuprizone. ( B ) Images were converted to a 256 shade grey tions positive for the given marker . 15 scale . (C ) The 256 shades of grey were divided into 5 bins FIG . 28A - K . Benztropine shows no immunosuppressive of 50 shades each . Number of objects in the corpus callosum effects in vivo after induction of EAE in mice. ( A - K ) EAE region in each bin were counted using Image -Pro plus. ( D ) was induced in mice by injecting PLP and pertussis toxin . Representative images of Image - Pro rendering of the Benztropine (10 mg/ kg ) and saline ( vehicle control) were tification of objects in each bin . injected intraperitoneally in the therapeutic mode for 14 20 FIG . 32. Western blot analysis of cleaved caspase activity days . Total splenocytes were isolated from the mice , stimu in differentiated OPCs. OPCs were plated in basal differen lated with PMA and ionomycin , and analyzed for various tiation media and treated with benztropine [ 1.5 uM ], thyroid populations of immune cells and cytokine secretion after 48 hormone [ 1 uM ) or DMSO [0.1 % ] for 6 days . Total protein hours . Protein transport was blocked in cells used for was isolated and analyzed by Western blot using a specific cytokine analysis using Monoeiosin . Benztropine treatment 25 antibody for the expression of caspase 3 and cleaved caspase had no effect on the number of total splenocytes ( A ), number 3. No expression of cleaved caspase 3 was detected in the of B cells ( B ) , number of CD4 + T -cells ( C ) , number of CD8 + compound treated cells or in untreated OPCs . T- cells (D ), number of CD4+ CD44Hi T -cells (E ), and FIG . 33A - F. Combination with benztropine improves number of CD8 +/ CD44Hi T- cells (F ). Benztropine also had efficacy and allows for a reduction in the dose of FTY720 no effect on cytokine production measured as the number of 30 and interferon -ß . (A - F ) EAE was induced in mice using PLP IL2 producing CD4 + T - cells (G ), number of IL10 producing and pertussis toxin . Benztropine (2.5 mg/ kg ) and FTY720 CD4 + T -cells ( H ) , number of TNF - a producing CD4 + T -cells ( various doses) and interferon ( various does ) were injected and number of IFN - y producing CD4 + T - cells . Error bars via intraperitoneal injections in the therapeutic mode at the indicate standard deviation of the mean within each group of start of EAE symptoms. ( A ) Clinical EAE scores for mice 5 mice. Representative flow cytometry scatter plots ( K ) 35 treated with FTY720 at doses of 1 mg/ kg , 0.1 mg/ kg and show similar numbers of CD4 * , CD8 + , and CD44 * cells in 0.01 mg/ kg show a dose dependent activity for FTY720 . ( B ) spleens isolated from vehicle treated mice and benztropine Clinical EAE scores for mice treated with interferon - ß at treated mice . doses of 10,000 U , 3000 U and 1000 U per mouse shows a FIG . 29A -J . Benztropine shows no immunosuppressive dose dependent activity for interferon - ß . ( C ) Combinations effects in vivo in normalmice . ( A -J ) Benztropine (10 mg/kg ) 40 of benztropine ( 2.5 mg/ kg ) with FTY720 (1 mg/ kg , 0.1 and saline (vehicle control) were injected intraperitoneally mg/ kg and 0.01 mg/ kg ). (D ) Combinations of benztropine in normal mice for 14 days . Total splenocytes were isolated ( 2.5 mg/kg ) with interferon - B ( 10,000 U , 3000 U and 1000 from the mice , stimulated with PMA and ionomycin and U per mouse ). ( E ) Clinical EAE scores for mice treated with analyzed for various populations of immune cells and FTY720 (0.01 mg/ kg ) in combination with benztropine cytokine secretion after 48hrs. Protein transport was blocked 45 (BA ; 2.5 mg/ kg ) does not show a significantly decreased in cells used for cytokine analysis using monoeiosin . Ben clinical severity as compared to mice treated with FTY720 ztropine treatment had no effect on the number of total (0.01 mg/ kg ) or benztropine (2.5 mg/ kg ) alone . (F ) Clinical splenocytes (A ), number of B cells ( B ), number of CD4 + EAE scores for mice treated with interferon - B (IFN ; 1000 T -cells (C ), number of CD8 + T -cells (D ), number of CD4 +/ U /mouse ) in combination with benztropine (BA , 2.5 mg/ kg ) CD44Hi T -cells ( E ) and number of CD8 + / CD44Hi T - cells 50 does not show a significantly decreased clinical severity as ( F ) . Benztropine also had no effect on cytokine production compared to mice treated with interferon - ß (IFN ; 1000 measured as the number of IL2 producing CD4 + T - cells ( G ), U /mouse ) or benztropine ( 2.5 mg/ kg ) alone. Error bars number of IL10 producing CD4 + T -cells ( H ) , number of indicate standard deviation of the mean within each group of TNF - y producing CD4 + T- cells (1 ) and number of IFN -Y 8 mice . producing CD4 + T- cells ( I ). Error bars indicate standard 55 FIG . 34A - F . Identified compounds decrease clinical deviation of the mean within each group of 5 mice . severity in the EAE model . ( A - F ) EAE was induced in mice FIG . 30A -F . Benztropine does not suppress T -cell depen by injecting PLP in complete Freund's adjuvant (CFA ) and dent and independent immune responses. ( A - F ) Mice were pertussis toxin , and the animals were scored daily for injected with Keyhole Limpet Hemocyanin protein conju clinical severity of disease on a scale of 0-5 . ( A ) Clinical gated to 2,4,6 , trinitrophenylhapten ( TNP- KLH ), lipopoly- 60 EAE scores for mice treated with benztropine ( 10 mg/ kg ) in saccharide conjugated to 2,4,6 , trinitrophenylhapten ( TNP the therapeutic mode (injections starting at the first appear LPS ), or TNP (2,4,6 - Trinitrophenyl) -FICOLL conjugate ance of EAE symptomson day 8-10 ) showed a significantly ( TNP- Ficoll) in appropriate adjuvants and treated with decreased clinical severity in the relapse phase of the disease vehicle or benztropine ( 10 mg/ kg ). Serum was isolated at as compared to vehicle treated mice . (B ) Clinical EAE various time points and IgG and IgM levels were measured 65 scores for mice treated with benztropine ( 10 mg/ kg ) in the by ELISA . ( A , B ) Benztropine showed no effect on TNP prophylactic mode ( injections starting on day 0 ) showed a LPS induced T- cell independent B -cell responses measured significantly decreased clinical severity in both acute and US 10,493,080 B2 15 16 relapse phase of the disease as compared to vehicle treated receptor modulator . The present invention also provides mice . ( C ) Benztropine showed a dose dependent efficacy in methods of treating a subject having a demyelinating disease decreasing clinical severity scores in the EAE model , with by administering to a subject a neurotransmitter receptor 12.5 mg/ kg being the most effective dose and 0.1 mg/kg modulating agent. showing no effect. Clinical EAE scores for mice treated with 5 II. Agents that Stimulate Increased Myelination of Nerves ( D ) trifluoperazine ( 10 mg/ kg ), ( E ) clemastine (10 mg/ kg ) , A. Neurotransmitter Receptor Modulating Agents or ( E ) salbutamol (10 mg/ kg ) in the therapeutic mode A neurotransmitter receptor modulating agent is an agent showed a significantly decreased clinical severity in the that induces oligodendrocyte precursor cell (OPC ) differen relapse phase of the disease as compared to vehicle treated tiation to a mature myelinating cell fate ( e.g., myelinating mice . Error bars indicate standard deviation of the mean 10 oligodendrocytes ) and /or stimulates increased myelination . within each group of 8-10 mice . In some embodiments , a neurotransmitter receptor modu DETAILED DESCRIPTION OF THE lating agent is selected from a muscarinic receptor antago INVENTION nist , a dopamine receptor antagonist , a histamine receptor 15 antagonist, a beta adrenergic receptor modulator , and an I. Introduction opioid receptor modulator. As shown in the Examples sec The present invention is based , in part, on the discovery tion below , exemplary members of each of these classes of that compounds that modulate various classes of neurotrans compounds has been shown to induce OPC differentiation to mitter receptors, such as muscarinic receptor antagonists , a myelinating oligodendrocyte cell fate , and thus stimulate dopamine receptor antagonists , histamine receptor antago- 20 increased myelination . Based on the data showing this nists , beta adrenergic receptor antagonists, and opioid recep activity of exemplary muscarinic receptor antagonists , dop tor modulators , promote the differentiation of oligodendro amine receptor antagonists , histamine receptor antagonists , cyte precursor cells (OPCs ) into a mature myelinating cell beta adrenergic receptor antagonists , and opioid receptor fate (e.g. , myelinating oligodendrocytes ). Accordingly , in modulators , other compounds in each of these classes and one aspect , the present invention provides for methods of 25 having similar pharmacological mechanisms to the exem inducing OPC differentiation to myelinating oligodendro plified compounds, such as the compounds listed in Table 1, cytes . are also predicted to be useful for inducing OPC differen Without being bound by a particular theory , it is believed tiation to a mature myelinating cell fate ( e.g., myelinating that in demyelinating diseases such as multiple sclerosis , oligodendrocytes ) and /or stimulating increased myelination . OPCs are present and able to migrate to demyelinated 30 In some embodiments , a compound that is identified as an regions, suggesting that the progressive decrease in remy agent that stimulates increased myelination has " selective” elination in these diseases is not due to defects in OPC activity for one of these classes ofneurotransmitter receptors population or recruitment, but rather is due to impaired ( i.e., has an agonistic or antagonistic effect against one of a differentiation of OPCs (reviewed in Chong and Chan , J Cell muscarinic receptor, a dopamine receptor , a histamine recep Biol. 188 : 305-312 ( 2010 )) . Accordingly , the present inven- 35 tor , a beta adrenergic receptor, or an opioid receptor , or a tion further provides for methods of stimulating increased subtype of any of these receptors , and has a weaker effect or myelination of nerves in a subject in need thereof by substantially no effect against the other receptors) . In some administering to a subject a neurotransmitter receptor modu embodiments , a compound that is identified as an agent that lating agent, such as a muscarinic receptor antagonist , a stimulates increased myelination has activity against two or dopamine receptor antagonist, a histamine receptor antago more of these classes of neurotransmitter receptors or sub nist , a beta adrenergic receptor antagonist, or an opioid types of neurotransmitter receptors . TABLE 1 Neurotransmitter receptor modulating agents Neurotransmitter Receptor( s ) Modulated Low Molecular Weight Compound Muscarinic Dopaminergic Histaminergic Adrenergic Opioid Antitussive Serotonergic ( - ) -Norephedrine ( - ) -Quinpirole hydrochloride - Terbuclomine ( + )- Butaclamol ( + ) - BUTACLAMOL HYDROCHLORIDE ( + - ) - Ethylketazocine ( + )- Lappaconitine ( + ) - Scopolamine ( + - ) - Trimethoquinol ( +/- ) - Epibatidine ( 1r, 2r )-cyclohexane - 1,2 - dicarboxylic acid (3,4 -dihydroxyphenylamino ) -2 - imidazoline .DELTA.9 - Tetrahydrocannabinol [ 11C ]MNPA 1 - ( (6,7 -Dimethoxy - 3 -methyl - 2 benzofuranyl) carbonyl ) -4 -methylpiperazine monohydrochloride 1- ( 3 -bromo - 5 - isoxazolyl) -2- (tert butylamino )ethanol hydrochloride 1-( 3 - Chlorophenyl) piperazine 1-( 3 - Chlorophenyl) piperazine dihydrochloride 1-( 3 - Chlorophenyl) piperazine hydrochloride US 10,493,080 B2 17 18 TABLE 1 -continued Neurotransmitter receptor modulating agents Neurotransmitter Receptor ( s ) Modulated Low Molecular Weight Compound Muscarinic Dopaminergic Histaminergic Adrenergic Opioid Antitussive Serotonergic 1- (4 - Hydroxyphenyl) -2 - aminoethanol 1,2,9,10 - tetramethoxyaporphine 1,4 -bis { 2 - [ ( 4 -methoxynaphthalen - 1 yl) methylidene ]hydrazinyl } phthalazine 1,5 - Trimethylenetetrazole 1-[ 3- ( Trifluoromethyl) phenyl ] piperazine 121524-08-1 125-71-3 (Parent ) 127-35-5 17- Hydroxy - 2,3 - cyclopropanoandrostane 19 -Propylorvinol 1 - Benzylimidazole 1 - M - Thiq 1 - PHENYLPIPERAZINE 1 -Propanamine , 3 -dibenzo [ b , e ]thiepin - 11 (6H ) ylidene - N , N - dimethyl 2 - ( ( 2 - Ethoxyphenoxy )phenylmethyl )morpholine methanesulfonate 2-( 1 -Piperazinyl ) pyrimidine 2-( 2 - Aminoethyl )pyridine 2-( 2 - Aminoethyl) pyridine dihydrochloride 2 - Detpq 2 -Methoxyidazoxan 2 -METHYL - 1,3 - DIOXOLANE 2 - Thiazoleethanamine 3 -iodopindolol 3 - Quinuclidinyl benzilate 4 - DAMP 4 -Damp methiodide 4 - DOI 4 -NMPB 5,6 - Dihydroxytryptamine 5,7 - DIHYDROXYTRYPTAMINE 5 - Carboxamidotryptamine 5 - Fhdpat 5 -Hmdptme 5 - Hydroxypropafenone 5 -Methylfurmethide 5 -Methylurapidil 6 - Dtaf 6 -Hydroxydopamine hydrobromide 6 -Hydroxydopamine hydrochloride 6 -nitroquipazine 76-57-3 7 - Ohdpat 87-00-3 8 - Artp 8 -OH -Dpat A - 38503 A -77636 hydrochloride acebutolol Acebutolol hydrochloride aceclidine Aceclidine hydrochloride Acepromazine maleate Aceroxatidine acetaminophen Acetylpromazine Acide tolfenamique [INN - French ] ACRIVASTINE Actifed ACTINOQUINOL SODIUM Adobiol ADRAFINIL Adrenaline bitartrate ADTN Aerolone Aerovent Afdx 384 AJ 76 Aktamin hydrochloride Alaproclate Alaproclate hydrochloride Alesion US 10,493,080 B2 19 20 TABLE 1 -continued Neurotransmitter receptor modulating agents Neurotransmitter Receptor ( s ) Modulated Low Molecular Weight Compound Muscarinic Dopaminergic Histaminergic Adrenergic Opioid Antitussive Serotonergic Aleudrin Alfenta ALFENTANIL Alfentanil hydrochloride alfuzosin Alginor Algolysin Alimezine ( TN ) Allegra Allergival Allococaine Almotriptan Alnespiron Alnespirone [ INN ] Alomide ( TN ) ALOSETRON HYDROCHLORIDE Aloxi Aloxi ( TN ) alpha -Ergocryptine alpha -Methyldopa alpha -Methylhistamine alpha- Methyl - L - dopa alpha -Methylserotonin Alphaprodin alprenolol Alprenolol hydrochloride alrestatin Altat amantadine Amantadine hydrochloride Ambenonium chloride Amerge Amfebutamone amfebutamonum Amibegron hydrochloride amidephrine amisulpride amitriptyline Amitriptyline hydrochloride Amosulalol amosulalol hydrochloride amoxapine Amperozide Amperozide hydrochloride AMPHETAMINE AMPHETAMINE SULFATE Amsulosin Anatran Andantol Anemet anisodamine anisodine Anplag antazoline Antazoline hydrochloride Antazoline phosphate Antergan Antussan Anzemet apomorphine Apomorphine HCl apraclonidine APRACLONIDINE HYDROCHLORIDE a -prodine Aprofen hydrochloride Aprofene Ara -putp Arbutamina ARBUTAMINE Arbutamine hydrochloride Arc 239 arecoline Arformoterol Arotinolol US 10,493,080 B2 21 22 TABLE 1 -continued Neurotransmitter receptor modulating agents Neurotransmitter Receptor ( s ) Modulated Low Molecular Weight Compound Muscarinic Dopaminergic Histaminergic Adrenergic Opioid Antitussive Serotonergic arotinolol hydrochloride Artane Arterenol bitartrate Artex Astelin astemizole Astomin Astramorph Atarax atenolol Atipamezole Atomoxetine ATROPINE Atropine iodomethylate Atropine methyl nitrate Atropine sulfate Atrovent Auteral Avacan Avapyrazone Avinza Axert Azaperone AZATADINE azelastine Azepexole Azepexole hydrochloride Bamipine Banistyl Banthine Batebulast Batebulast hydrochloride BD1047 BE 2254 Beforal Befunolol Beldavrin Bemesetron BENACTYZINE Benactyzine hydrochloride benalfocin Benextramine Benfuran Benoxathian Benoxathian hydrochloride BENPERIDOL benserazide Bentanidol Benzetimide BENZETIMIDE HYDROCHLORIDE Benzfetamine Benzhexol Benzonatate benzphetamine benztropine Benztropinum Berachin beta - Cft beta - ENDORPHIN beta - Endorphin ( 1-31 ) beta -Flupenthixol betahistine Betahistine dihydrochloride Betahistine mesilate betaxolol Betaxolol hydrochloride BETAZOLE Betazole hydrochloride bethanechol Bethanechol chloride Bethanidine BINOSPIRONE MESYLATE biperiden Biperiden hydrochloride US 10,493,080 B2 23 24 TABLE 1 -continued Neurotransmitter receptor modulating agents Neurotransmitter Receptor ( s ) Modulated Low Molecular Weight Compound Muscarinic Dopaminergic Histaminergic Adrenergic Opioid Antitussive Serotonergic Bisguanidinium phosphate BISOPROLOL Bisoprolol fumarate Bitolterol Bladderon Bmy- 7378 Bonamine BOPINDOLOL Bornaprine BrAAM Bretylium tosylate brimonidine Brl 15572 Brl 26830 Brl 37344A Brl 48553 Brl - 15572 Br135135A Brocadisipal Brolamfetamine bromocriptine bromopride Bromopride hydrochloride BROMPHENIRAMINE Brompheniramine maleate Broncaspin Bronitin Mist Bronkometer Broxaterol BTCP Buccastem Bucindolol Bucindolol hydrochloride Buclizine Buclodin Budipine Budipine hydrochloride Bufetolol Bufotenine Bufuralol Bunazosin Bunolol Bunolol hydrochloride BUPRANOLOL bupranolol hydrochloride Buprenorfina [ INN -Spanish ] buprenorphine BUPRENORPHINE HYDROCHLORIDE Buprenorphine hydrochloride solution bupropion Bupropion hydrochloride BURIMAMIDE Buscapine buspirone Buspirone hydrochloride BUTACLAMOL HYDROCHLORIDE butanoic acid Butaxamina Butofilolol butorphanol Butorphanol tartrate Butoxamine [ INN ] BUTOXAMINE HYDROCHLORIDE Butylhyoscine Butylscopolamine C11796 C12H19NO2.HCI C16H22CINO C19H25NO.HCI C19H27CIN204.HCI C20H27NO3.HCI C50H81N1509 C5976_SIGMA cabergoline US 10,493,080 B2 25 26 TABLE 1 -continued Neurotransmitter receptor modulating agents Neurotransmitter Receptor ( s ) Modulated Low Molecular Weight Compound Muscarinic Dopaminergic Histaminergic Adrenergic Opioid Antitussive Serotonergic Caffeine benzoate Calcium fusarate Camylofin Carazolol carbachol carbamazepine Carbamazepine dihydrate Carbamylcholine carbetapentane Carbetapentane citrate carbidopa Carbidopa hydrate Carbidopa Monohydrate Carbidopa, ( S )-Isomer Carbidopa levodopa carbinoxamine CARBINOXAMINE MALEATE Carebastine CARFENTANIL CARTEOLOL carteolol hydrochloride carvedilol CCRIS 3490 CEC dihydrochloride celiprolol Centralvet Cerocral cetirizine Cevimeline CGP 20712A CGP 20712A methanesulfonate CGS 12066B CGS 12066B dimaleate CH 38083 CHEBI: 104181 CHEBI: 117275 CHEBI: 124645 CHEBI: 126213 CHEBI: 136626 CHEBI: 142179 CHEBI: 148898 CHEBI: 159721 CHEBI: 161127 CHEBI: 178303 CHEBI: 201827 CHEBI: 238638 CHEBI: 268876 CHEBI: 334862 CHEBI: 350546 CHEBI: 36796 CHEBI: 399928 CHEBI: 40751 CHEBI: 431080 CHEBI: 471632 CHEBI: 48295 CHEBI: 517861 CHEBI: 583615 CHEBI: 584626 CHEBI: 623294 CHEBI: 648957 CHEBI: 702837 CHEMBL446167 CHEMBL93361 CHLORAZINE Chlorethylclonidine Chloropyramine Chloropyramine hydrochloride chlorpheniramine Chlorpheniramine maleate chlorpromazine Chlorpromazine hydrochloride chlorprothixene CIANOPRAMINE Cid 105105 US 10,493,080 B2 27 28 TABLE 1 -continued Neurotransmitter receptor modulating agents Neurotransmitter Receptor ( s ) Modulated Low Molecular Weight Compound Muscarinic Dopaminergic Histaminergic Adrenergic Opioid Antitussive Serotonergic CID5122 CID517557 cimaterol cimetidine Cimetidine hydrochloride CINANSERIN Cinanserin hydrochloride cinnarizine ciproxifan cirazoline cisapride Cisapride monohydrate citalopram citalopram hydrobromide CL 316,243 CL 316243 Cl- Apb Clearnal clemastine Clemastine (USAN ) CLEMASTINE FUMARATE clenbuterol Clenbuterol hydrochloride clidinium CLIDINIUM BROMIDE clobenpropit Clobutinol clomipramine Clomipramine hydrochloride clonidine CLONIDINE HYDROCHLORIDE CLOPENTHIXOL Clopixol Cloranolol Clovoxamine Clovoxamine fumarate clozapine cocaethylene Cocaethyline Cocain - chlorhydrat (German ] cocaine COCAINE HYDROCHLORIDE Cocaine muriate codeine Cogentin Cogentin mesylate Cognex Compazine Concerta Concordin Congesteze Contristamine Corindolan Corlopam Corynanthin corynanthine Corynanthine hydrochloride CP 93129 Crispin Cromakalim CV 705 Cyanopindolol CYCLAZOCINE CYCLIZINE Cyclizine hydrochloride Cyclogyl cyclopentolate CYPROHEPTADINE cyproheptadine hydrochloride Cystospaz d1 -hyoscyamine DAGO Daipin Dalcipran US 10,493,080 B2 29 30 TABLE 1 -continued Neurotransmitter receptor modulating agents Neurotransmitter Receptor ( s ) Modulated Low Molecular Weight Compound Muscarinic Dopaminergic Histaminergic Adrenergic Opioid Antitussive Serotonergic Dalgan Dalmee DAMGO Dapiprazole Dapiprazole hydrochloride Darifenacin Darifenacin hydrobromide Darvon D - Chlorpheniramine D - Dopa Debridat DEBRISOQUIN SULFATE Debrisoquine deisopropyldisopyramide Deltorphin C Deltorphin I Denopamine Deprenalin Deprenil Deptropine Deptropine citrate Deramciclane [ INN ] Deramciclane fumarate Dermorphin desipramine Desipramine hydrochloride Desloratadine Desoxedrine Detrol Dexbrompheniramine DEXBROMPHENIRAMINE MALEATE Dexchlorpheniramine maleate DEXETIMIDE Dexfenfluramine DEXMEDETOMIDINE Dexmedetomidinum [ INN - Latin ] Dexmethylphenidate DEXPROPRANOLOL Dexpropranolol hydrochloride dextroamphetamine dextromethorphan Dextromethorphan hydrobromide monohydrate DEXTROMORAMIDE Dextropropoxyphene Dextrostat DiacetylmonoximeDEZOCINE Diacetylmorphine Diamaprit- 2HCl Dicetel Dicodethal dicyclomine Dicyclomine hydrochloride Difril DIHYDREXIDINE Dihydro - alpha - ergocryptine mesylate DIHYDROALPRENOLOL DIHYDROCODEINE Dihydrocodeine bitartrate Dihydroergocornine Dihydroergocristine DIHYDROERGOCRISTINE MESYLATE Dihydroergocryptine dihydroergotamine Dihydroergotamine mesilate Dihydroetorphine Dihydromorphine Dihydroquinidine Dihydroquinine dihydroxyphenylalanine Dilaudid Dilevalol Dilevalol hydrochloride Dimaprit US 10,493,080 B2 31 32 TABLE 1 -continued Neurotransmitter receptor modulating agents Neurotransmitter Receptor ( s ) Modulated Low Molecular Weight Compound Muscarinic Dopaminergic Histaminergic Adrenergic Opioid Antitussive Serotonergic Dimemorfan Dimemorfan (INN ) Dimepheptanol Dimethindene maleate DIMETHYLTRYPTAMINE Dimetindene Diphemanil diphenhydramine Diphenhydramine citrate DIPHENHYDRAMINE HYDROCHLORIDE DIPHENOXYLATE Diphenoxylate HCl dipivefrin Dipivefrin hydrochloride Dironyl Ditropan Dixyrazine DL - Adrenaline dl- Desoxyephedrine DL - DOPA dl - Narcotine DL -Oxyfedrine DL - threo - 3,4 - Dihydroxyphenylserine DL - threo - DOPS dobutamine Dobutamine hydrochloride docarpamine dolasetron DOLASETRON MESYLATE Dolasetronum [INN - Latin ] Domin domperidone Domperidone Maleate Dopabain dopamine Dopamine hydrochloride dopazinol Dopexamine DOPEXAMINE HYDROCHLORIDE dosulepin hydrochloride Dotarizine [ INN ] Dothiepin doxazosin Doxazosin mesylate doxepin Doxepin Hydrochloride Doxepine DOXOFYLLINE doxylamine Doxylamine succinate DPDPE Dramamine Drixoral Dronabinol droperidol dropropizine drotaverine droxidopa DSP 4 DSP - 4 hydrochloride DU - 29373 duloxetine DULOXETINE HYDROCHLORIDE DuP 734 Duremesin Dynorphin 1-13 ebastine Ebrotidine Ecopipam Edronax EEDO Efaroxan Efaroxan hydrochloride Effexor US 10,493,080 B2 33 34 TABLE 1 -continued Neurotransmitter receptor modulating agents Neurotransmitter Receptor ( s ) Modulated Low Molecular Weight Compound Muscarinic Dopaminergic Histaminergic Adrenergic Opioid Antitussive Serotonergic Effortilvet Eldoral Eletriptan Eletriptan hydrobromide Eltoprazine Eltoprazine hydrochloride Emadine emedastine Emepronum Emergil Enantio - PAF C - 16 Endomorphin 1 Endomorphin 2 Endovalpin ENTACAPONE Epanolol eperisone Ephedrine Ephedrine hydrochloride EPHEDRINE SULFATE Ephetonine Epibatidine epinastine epinephrine Epinephrine hydrochloride Eplivanserin fumarate Ergocryptine Ergocryptine mesylate Ergocryptine - alpha Ergoloid mesylate Ergomar Ergotamin ERGOTAMINE ergotamine tartrate Esbuphon Escitalopram Escitalopram oxalate Eseroline ESMOLOL Esmolol hydrochloride ethaverine Ethaverine hydrochloride ethopropazine Ethopropazine hydrochloride ETHYLKETOCYCLAZOCINE Ethylmorphine Eticlopride Eticlopride hydrochloride Etilefrine etilefrine hydrochloride Etintidine Etintidine hydrochloride Etorphine EU -0100372 Eupaverina Euspirol Evoxac exaprolol Exaprolol hydrochloride Falipamil Famotidina famotidine Famotidine HCI Fananserin Fastin Femoxetine Femoxetinum [INN -Latin ] Fencarbamide hydrochloride Fencarol Fenclonine fenfluramine Fenistil fenoldopam Fenoldopam bromide US 10,493,080 B2 35 36 TABLE 1 -continued Neurotransmitter receptor modulating agents Neurotransmitter Receptor ( s ) Modulated Low Molecular Weight Compound Muscarinic Dopaminergic Histaminergic Adrenergic Opioid Antitussive Serotonergic fenoterol Fenoterol hydrobromide Fenoverine fentanyl Fentora fexofenadine FG 4963 Finaten Finibron flavoxate Flavoxate hydrochloride Flb 457 Flesinoxan Flesinoxan hydrochloride FLESTOLOL FLESTOLOL SULFATE Fluanxol depot ( TN ) flunarizine flunarizine hydrochloride Fluorofen fluoxetine FLUPENTHIXOL DECANOATE Flupentixol FLUPHENAZINE Fluphenazine hydrochloride Fluspirilene fluvoxamine FLUVOXAMINE MALEATE Focalin FOMINOBEN FONAZINE formoterol Frovatriptan Frovatriptan succinate fusaric acid gabapentin Galantamin Galantamine Galantamine hydrobromide Ganglefene Ganglerone Gastrozepin Gbr 12783 GBR12935 Geodon Gepirone GEPIRONE HYDROCHLORIDE Gevatran glafenine Glaucine Glauconex Glaxo Wellcome brand of acrivastine GLYCOPYRROLATE Gnoscopine Gotensin GR 113808 GR - 127935 granisetron Granisetron hydrochloride guanabenz Guanabenz acetate GUANETHIDINE guanethidine sulfate GUANFACINE guanfacine hydrochloride guanidine Guanidine bromide Guanidine hydrochloride Guanidine nitrate GUANIDINIUM Gynergen Hag - PC haloperidol Haymine US 10,493,080 B2 37 38 TABLE 1 -continued Neurotransmitter receptor modulating agents Neurotransmitter Receptor ( s ) Modulated Low Molecular Weight Compound Muscarinic Dopaminergic Histaminergic Adrenergic Opioid Antitussive Serotonergic HEAT Hemicholinium Hemicholinium - 3 Heroin hydrochloride Hhsi -difenidol Higenamine Himbacine histamine Histamine dihydrochloride Histamine diphosphate Histamine hydrochloride Histantin Hoe - 893d HOMATROPINE Homatropine hydrobromide (RS ) Homocodeine Hycodan Hydergine Hydriatine Hydroaminacrine HYDROCODONE hydromorphone Hydromorphone hydrochloride hydroquinidine Hydroquinidine hydrochloride HYDROQUININE hydroxyzine Hydroxyzine pamoate Hyoscine hydrobromide Hyoscine Methobromide hyoscyamine Hyoscyamine ( D ) Hyoscyamine sulfate Hyoscyamine sulfate (USP ) Hypostamine Hysco Ibopamine ibuprofen IBZM Icatibant Icatibant acetate Ici 118551 ICI- 89406 IDAZOXAN IDAZOXAN HYDROCHLORIDE Ifenprodil ifenprodil tartrate IHEAT Ildamen imetit Imetit dihydrobromide Imidacloprid imipramine Imipramine hydrochloride IMPROMIDINE Impromidine hydrochloride Inapetyl INDALPINE indanidine Indenolol Inderal Indolophenanthridine INDORAMIN Indorenate Hydrochloride Inopamil Insidon intropin Iodocyanopindolol ipratropium ipratropium bromide Ipratropium bromide monohydrate Iprazochrome Ips -339 IPSAPIRONE US 10,493,080 B2 39 40 TABLE 1 -continued Neurotransmitter receptor modulating agents Neurotransmitter Receptor ( s ) Modulated Low Molecular Weight Compound Muscarinic Dopaminergic Histaminergic Adrenergic Opioid Antitussive Serotonergic Ipsapirone hydrochloride Ismelin Isoaminile Isocodeine isoetharine isoproterenol ISOPROTERENOL HYDROCHLORIDE Isoproterenol sulfate Isospaglumic acid Isothipendyl isoxsuprine Isoxsuprine hydrochloride Itrop Janimine Jetrium tartrate Kadian Kerlone Ketanserin Ketanserin tartrate Ketobemidone Ketogan ketotifen KETOTIFEN FUMARATE Kinichron Kytril L 657743 L - 741,626 labetalol Labetalol hydrochloride lafutidine L - alpha - Acetyl- N -normethadol Landiolol lappaconitine Lazabemide Legatrin Leoplexamin l - Ephedrine Lethidrone Levacetylmethadol Levallorphan Levamfetamine Levcromakalim Levetimide LEVOBUNOLOL Levobunolol • HCI levocabastine LEVOCABASTINE HYDROCHLORIDE Levocetirizine levodopa Levodropropizine Levomeprazine Levomepromazine Levomethorphan Levomethorphan hydrobromide levorphanol Levosalbutamol Levospasme Levsinex LIDAMIDINE LIDAMIDINE HYDROCHLORIDE Lilly 53857 1 - Isoprenaline chloride LISURIDE lisuride maleate L -Noradrenaline bitartrate LODOXAMIDE TROMETHAMINE Lofentanil Lofentanil oxalate Longifene Lopaco_000714 Lopac - A - 164 Lopac - C - 130 Lopressor loratadine US 10,493,080 B2 41 42 TABLE 1 -continued Neurotransmitter receptor modulating agents Neurotransmitter Receptor ( s ) Modulated Low Molecular Weight Compound Muscarinic Dopaminergic Histaminergic Adrenergic Opioid Antitussive Serotonergic Lorcet Lotronex loxapine Loxapine hydrochloride Loxapine succinate Loxtidine L -pentazocine LSD tartrate LUPITIDINE HYDROCHLORIDE LY 235959 LY 277359 maleate Ly- 165163 Lysergide Lysivane Mabuterol madopar Malexil maprotiline Maprotiline hydrochloride Marzine Maxolon mazindol Mci 9042 Men 5652 McN - A - 343 mCPBG m - CPBG hydrochloride MDL - 100907 MDMA mebeverine MEBEVERINE HYDROCHLORIDE Meclastine meclizine Meclizine hydrochloride Meclizine Mixture With Niacin Mecloprodine Medetomidine Medetomidine hydrochloride MEDROXALOL Medroxalol hydrochloride Melevodopa memantine MEMANTINE HYDROCHLORIDE meperidine Mepindolol Meptazinol MEPTAZINOL HYDROCHLORIDE mequitazine Merital mescaline Mescomine mesoridazine Mesulergine Mesulergine hydrochloride Metabolites ( street ) metaproterenol Metaproterenol hemisulfate Metaraminol bitartrate Metatsin metergoline methacholine Methacholine chloride methadone Methadyl acetate METHAMPHETAMINE Methamphetamine hydrochloride methantheline methapyrilene METHAPYRILENE HYDROCHLORIDE Metharsinat methiothepin Methiothepin maleate Methoctramine Metholes US 10,493,080 B2 43 44 TABLE 1 -continued Neurotransmitter receptor modulating agents Neurotransmitter Receptor ( s ) Modulated Low Molecular Weight Compound Muscarinic Dopaminergic Histaminergic Adrenergic Opioid Antitussive Serotonergic Methorphan methoxamine METHOXAMINE HYDROCHLORIDE Methylatropine methyldopa METHYLDOPA SESQUIHYDRATE Methylfurmetide methyloctatropine bromide methylphenidate Methylscopolamine methysergide Methysergide maleate METIAMIDE Metipranolol metoclopramide Metoclopramide dihydrochloride metoprolol Metoprolol fumarate mianserin Mianserin hydrochloride Mictonorm Midaglizole Midaglizole hydrochloride midodrine MIDODRINE HYDROCHLORIDE Mifentidine Milnacipran Minipress Mintussin Minusine Mirapex mirtazapine Mivazerol Mizolastine MK - 212 MK - 912 M -Mptp Mofegiline Mofegiline hydrochloride MONATEPIL MALEATE morphine Morphine hydrochloride MORPHINE SULFATE morphinesulfate Mosapride Mosapride citrate Moxaverine Moxisylyte moxisylyte hydrochloride MPTP Muscarin Myonal Myophedrine N , 3 -Dimethylmorphinan Naaxia N -acetylaspartylglutamate N - Acetyl- Asp -Glu nadolol Nafadotride nafronyl Nafronyl oxalate naftopidil nalbuphine Nalorphine Nalorphine hydrochloride naloxone Naloxone hydrochloride NAN - 190 hydrobromide naphazoline NAPHAZOLINE HYDROCHLORIDE Naphazoline nitrate Naphthisen naratriptan Nargoline US 10,493,080 B2 45 46 TABLE 1 -continued Neurotransmitter receptor modulating agents Neurotransmitter Receptor ( s ) Modulated Low Molecular Weight Compound Muscarinic Dopaminergic Histaminergic Adrenergic Opioid Antitussive Serotonergic Narphen Navaron Nazasetron NCGC00015261-01 nchembio873 -comp43 nchembio873 - comp53 nchembio873 -comp67 Ncq 298 Nebivolol Nebracetam Nebracetam fumarate Nedeltran nefopam Nefopam hydrochloride nemonapride NEOSTIGMINE neostigmine bromide Neostigmine methyl sulfate nicergoline NIH - 8805 Nipradilol Nisentil nitrous oxide nizatidine N -Methylspiroperidol Noleptan Nomifensine Norephedrine norepinephrine Norfenefrine Norfenfloramine Norflex Norprolac nortriptyline Nortriptyline hydrochloride Norzine Ampuls Noscapalin noscapine Novopropoxyn n -Propylapomorphine NSC10004 NSC114335 NSC289336 NSC61391 NSC61806 NSC69886 NSC79303 Nubain nylidrin Nylidrin hydrochloride Octatropine octopamine Oils , peppermint olanzapine Olopatadine ondansetron ONDANSETRON HYDROCHLORIDE Opana Opcon Opipramol Opreal_021650 Optimine Orlaam orphenadrine Orphenadrine hydrochloride Otenzepad Otilonium Bromide oxatomide Oxeladin oxidopamine Oxifedrinum oxitropium bromide OXMETIDINE Oxmetidine hydrochloride US 10,493,080 B2 47 48 TABLE 1 -continued Neurotransmitter receptor modulating agents Neurotransmitter Receptor ( s ) Modulated Low Molecular Weight Compound Muscarinic Dopaminergic Histaminergic Adrenergic Opioid Antitussive Serotonergic Oxolamine Oxolamine citrate Oxotremorine oxotremorine methiodide oxprenolol Oxprenolol hydrochloride oxybutynin Oxybutynin chloride oxycodone Oxycodone hydrochloride oxymetazoline oxymorphone oxyphenonium OXYPHENONIUM BROMIDE ozagrel ozagrel hydrochloride Palfadonna Palladone Palonosetron Palonosetron hydrochloride Papaveretum PAPP Paracodin Paracymethadol Parasan Parlodel paroxetine Paroxetine hydrochloride Paroxetine maleate Pataday Paxil PBPO p - Chloramphetamine Pemilaston Pemirolast penbutolol PENFLURIDOL Pentalgine pentazocine Peracon Perazine maleate pergolide PerhydrohistrionicotoxinPERGOLIDE MESYLATE Periactin . Pernazine Pernovine perphenazine Pethidine hydrochloride PFHHSID phenacetin PHENAZOCINE Phencarbamide Phenindamine pheniramine PHENIRAMINE MALEATE PHENOPERIDINE Phenoperidine hydrochloride Phenopropamine Phenoxene phenoxybenzamine Phenoxybenzamine hydrochloride phentermine phentolamine Phentolamine mesylate phenylbiguanide phenylephrine PHENYLEPHRINE HYDROCHLORIDE phenylpropanolamine PHENYLPROPANOLAMINE HYDROCHLORIDE PHOLCODINE Phospholine iodide Picumast US 10,493,080 B2 49 50 TABLE 1 -continued Neurotransmitter receptor modulating agents Neurotransmitter Receptor ( s ) Modulated Low Molecular Weight Compound Muscarinic Dopaminergic Histaminergic Adrenergic Opioid Antitussive Serotonergic Picumast dihydrochloride pilocarpine Pilocarpine Pilocarpine hydrochloride Pilocarpine nitrate Pilocarpine nitrate salt pimozide Pinaverium pindolol Pipamperone Pipazethate Piperoxan PIRBUTEROL pirenzepine Pirenzepine dihydrochloride piribedil Piribedil hydrochloride Piribedil mesylate Piritramide PIZOTYLINE Plegine p -MPPI Pnu 99194A Pondimin practolol Pramipexol [Spanish ] pramipexole prazosin Prazosin hydrochloride PRBCM Precedex Preclamol Preclamolum [Latin ] Prednisolone 21 -pivalate Prenalterol PRENYLAMINE Prestwick 144 Prialt Privine Prizidilol PROCATEROL procaterol hydrochloride prochlorperazine Prochlorperazine dimaleate Prochlorperazine edisylate Prochlorperazine maleate procyclidine Procyclidine hydrochloride Progabide Prolixin promazine PROMAZINE HYDROCHLORIDE Promedol promethazine PROMETHAZINE HYDROCHLORIDE propantheline PROPANTHELINE BROMIDE Propitan Propiverine propiverine hydrochloride propranolol Propranolol hydrochloride Proroxan [ INN ] Proscomide Prothiaden protopine PROTOPINE HYDROCHLORIDE protriptyline Protriptyline hydrochloride Proxicromil Prozac Psxeladine Psychostyl pyrilamine US 10,493,080 B2 51 52 TABLE 1 -continued Neurotransmitter receptor modulating agents Neurotransmitter Receptor ( s ) Modulated Low Molecular Weight Compound Muscarinic Dopaminergic Histaminergic Adrenergic Opioid Antitussive Serotonergic PYRILAMINE MALEATE Pyrroxane Quadramet QuifenadineQuinagolide Quinelorane Quinidex quinidine Quinidine sulfate Quinine Quinine hydrochloride quinine sulfate QUININE SULFATE DIHYDRATE QUINPIROLE quipazine QUIPAZINE MALEATE R ( - ) -Denopamine R - 50547 RACLOPRIDE Raclopride C11 Raclopridum [Latin ] RACTOPAMINE Ractopamine hydrochloride Ramosetron Ramosetron hydrochloride ranitidine Ranitidine bismuth citrate ranitidine hydrochloride Rapimine Rauwolscine Reboxetine Reboxetine mesylate Reboxetine mesylate hydrate Redux Reglan Relaspium REMIFENTANIL REMIFENTANIL HYDROCHLORIDE Remoxipride REMOXIPRIDE HYDROCHLORIDE Renzapride Renzapride hydrochloride repirinast Reproterol REPROTEROL HYDROCHLORIDE reserpine Respilene Restenacht Rexigen Rexolate Rilmenidine Rilmenidine phosphate Rimiterol Rimiterol Hydrobromide risperidone ritanserin ritodrine Ritodrine hydrochloride rizatriptan Rizatriptan benzoate Ro 363 Robinal Robinul Rociverine ropinirole Ropinirole hydrochloride Rotenolone Rotigotine RS 86 , hydrobromide RS 86HB RS - 25259-197 RU 24969 RU - 24213 S ( - ) Eticlopride hydrochloride US 10,493,080 B2 53 54 TABLE 1 -continued Neurotransmitter receptor modulating agents Neurotransmitter Receptor ( s ) Modulated Low Molecular Weight Compound Muscarinic Dopaminergic Histaminergic Adrenergic Opioid Antitussive Serotonergic S - 20500 Salbutamol Saligren Salmeterol salmeterol xinafoate Samarium Sm 153 lexidronam Sarpogrelate Savella SB 206553 Sch 23982 Sch - 23982 scopolamine scopolamine butylbromide Scopolamine hydrobromide Sdz 205,557 Secoverine SECOVERINE HYDROCHLORIDE Selecal selegiline Selegiline hydrochloride Selozok Serc Serotone serotonin sertraline Sertraline hydrochloride Setoperone Sgd 101-75 Silomat Sinemet SKF 38393 SKF 38393 hydrochloride SKF 81297 Skf 83566 SKF 91488 SKF 91488 dihydrochloride Sm - Edtmp SMR000449272 Snc80 Solifenacin Solifenacin succinate Sordinol sotalol Sotalol hydrochloride Soventol Soventol ( TN ) Soventol hydrochloride Spasril Spectrum_001815 spiperone Spiriva Spiriva Handihaler Spiroxatrine SQ 10643 SR 59230A ST 91 Stadol Stelazine stepholidine STP (hallucinogen ) Strattera subecholine Suberyldicholine SUFENTANIL Sufentanil citrate sulpiride Sultopride sultopride hydrochloride sumatriptan SUMATRIPTAN SUCCINATE Suplatast Tosilate Sympatholytin Synephrine T123_SIGMA US 10,493,080 B2 55 56 TABLE 1 -continued Neurotransmitter receptor modulating agents Neurotransmitter Receptor ( s ) Modulated Low Molecular Weight Compound Muscarinic Dopaminergic Histaminergic Adrenergic Opioid Antitussive Serotonergic Tacrine Tacrine hydrochloride Tacrine hydrochloride hydrate tageflar Talacen Talinolol Talipexole Talwin Talwin 50 TAMSULOSIN tandospirone citrate Tbhpbo TCMDC- 125509 Tegaserod maleate Telenzepine Teletux TEMELASTINE Tempium Tenamfetamine terazosin Terazosin hydrochloride terbutaline TERBUTALINE HEMISULFATE Terbutaline sulfate terfenadine terguride Terodiline TERODILINE HYDROCHLORIDE Tersigat TERTATOLOL Tesmilifene TETRABENAZINE TETRAHYDROCANNABINOL tetrahydropalmatine Tetraspasmin - Lefa TFMPP Thecodinum Thephorin hydrochloride Theratuss thiethylperazine Thiethylperazine maleate Thioperamide thioridazine Thioridazine hydrochloride thiothixene thonzylamine THONZYLAMINE HYDROCHLORIDE tiapride TILIDINE Tilisolol timolol Timolol hemihydrate Timolol maleate TIOTIDINE Tiotixene Tiotropium TIOTROPIUM BROMIDE TIPP Tiropramide tizanidine Tizanidine hydrochloride Tobanum Tocodilydrin tolazoline Tolazoline hydrochloride Tolcapone tolfenamic acid tolterodine Tolterodine tartrate Torecan Tramadol tramadol hydrochloride tranilast Traxanox US 10,493,080 B2 57 58 TABLE 1 -continued Neurotransmitter receptor modulating agents Neurotransmitter Receptor ( s ) Modulated Low Molecular Weight Compound Muscarinic Dopaminergic Histaminergic Adrenergic Opioid Antitussive Serotonergic Traxanox sodium trazodone Trazodone hydrochloride Tremblex Tretoquinol Tricodein trifluoperazine Trifluoperazine dihydrochloride Trifluperidol triflupromazine Triflupromazine hydrochloride TRIMAZOSIN trimebutine Trimebutine maleate Trimeperidine Trimethoquinol trimipramine Trimipramine maleate tripelennamine Tripelennamine citrate Tripelennamine hydrochloride triprolidine Triprolidine hydrochloride tropicamide Tropine tropate tropisetron TROPISETRON HCI Tropyl 3,5 -dichlorobenzoate trospium chloride tulobuterol Tulobuterol hydrochloride Tuscodin tyramine Tyr - D - Ala -Gly - N -Methyl - Phe -Gly -ol Tyr - tic -phe - phe - OH UNII - 7LG286J8GV urapidil Uroxatral Valoron Vanoxeamine Vanoxerine venlafaxine Ventipulmin Ventolin Verton viloxazine Viloxazine hydrochloride Vistaril Volinanserin Vuf 9153 Way 100635 Way - 100135 Win - 35428 wortmannin . Wyamine sulfate Xamoterol Xamoterol hemifumarate XANOMELINE Xanomeline tartrate xylamidine Xylamidine tosylate xylazine Xylazine hydrochloride Xyzall Yohimbine YOHIMBINE HYDROCHLORIDE Zacopride ZACOPRIDE HYDROCHLORIDE ZATOSETRON ZATOSETRON MALEATE Zebeta Zelmac Zelnorm Zeneca ZD7114 US 10,493,080 B2 59 60 TABLE 1 -continued Neurotransmitter receptor modulating agents Neurotransmitter Receptor ( s ) Modulated Low Molecular Weight Compound Muscarinic Dopaminergic Histaminergic Adrenergic Opioid Antitussive Serotonergic Zetidoline Zimeldine Zimeldine hydrochloride Zimelidine Zimelidine dihydrochloride ZINTEROL ZINTEROL HYDROCHLORIDE Zipeprol Ziprasidone Ziprasidone hydrochloride Ziprasidone mesylate Zofran Zolantidine zolmitriptan Zuclopenthixol Zyrtec

1. Muscarinic Receptor Antagonists dopamine receptor's function . As shown in the Examples , A muscarinic receptor is a G -protein coupled acetylcho benztropine , GBR12935 , and trifluoperazine have been line receptor . There are five known subtypes of muscarinic 25 shown to antagonize the function of a dopamine receptor receptors: My receptors , M2 receptors , Mz receptors , M4 and / or are known antagonists of a dopamine receptor. There receptors , and M , receptors . A muscarinic receptor antago fore, in some embodiments , the dopamine receptor antago nist is an agent able to inhibit one or more characteristic nist is a compound selected from benztropine , GBR12935 , responses of a muscarinic receptor or receptor subtype . As trifluoperazine , and salts , prodrugs , racemic mixtures , con a non - limiting example , an antagonist may competitively or 30 formational and / or optical isomers , crystalline polymorphs, non - competitively bind to (1 ) a muscarinic receptor , (2 ) an and isotopic variants thereof. Alternatively , any of the dop agonist or partial agonist ( or other ligand ) of the muscarinic amine receptor modulators listed in Table 1 can be used to receptor, and / or ( 3 ) a downstream signaling molecule to antagonize a dopamine receptor. in some embodi inhibit the muscarinic receptor's function . As shown in the ments , the dopamine receptor antagonist is a dopamine Examples, benztropine, carbetapentane , clemastine , ipratro- 35 receptor modulator compound listed in Table 1. The com pium , and atropine have been shown to antagonize the pounds described in Table 1 are readily available . function of a muscarinic receptor and /or are known antago In some embodiments , the neurotransmitter receptor nists of a muscarinic receptor . Therefore , in some embodi modulating agent is benztropine or a salt thereof ( e.g. , ments , the muscarinic receptor antagonist is a compound benztropine mesylate ). In some embodiments , the neu selected from benztropine , carbetapentane, clemastine , ipra- 40 rotransmitter receptor modulating agent is trifluoperazine or tropium , atropine, and salts , prodrugs, racemic mixtures , a salt thereof ( e.g., trifluoperazine hydrochloride) . conformational and /or optical isomers , crystalline poly 3. Histamine Receptor Antagonists morphs, and isotopic variants thereof. Alternatively , any of A histamine receptor is a G -protein coupled receptor , for the muscarinic receptor modulators listed in Table 1 can be which the neurotransmitter histamine is the primary endog used to antagonize a muscarinic receptor. Thus, in some 45 enous ligand . There are four known subtypes of histamine embodiments , the muscarinic receptor antagonist is a mus receptors : H , receptors , H , receptors , H2 receptors , and H4 carinic receptor modulator compound listed in Table 1. The receptors . A histamine receptor antagonist is an agent able to compounds described in Table 1 are readily available . inhibit one or more characteristic responses of a histamine In some embodiments , the neurotransmitter receptor receptor or receptor subtype . As a non - limiting example , an modulating agent is benztropine or a salt thereof ( e.g., 50 antagonist may competitively or non -competitively bind to benztropine mesylate ). In some embodiments , the neu ( 1 ) a histamine receptor , ( 2 ) an agonist or partial agonist (or rotransmitter receptor modulating agent is clemastine or a other ligand ) of the histamine receptor , and / or ( 3 ) a down salt thereof ( e.g. , clemastine fumarate ). stream signaling molecule to inhibit the histamine receptor's 2. Dopamine Receptor Antagonists function . As shown in the Examples, clemastine has been A dopamine receptor is a G -protein coupled receptor , for 55 shown to antagonize the function of a histamine receptor . which the neurotransmitter dopamine is the primary endog Therefore , in some embodiments , the histamine receptor enous ligand . There are five known subtypes of dopamine antagonist is clemastine or a salt , prodrug , racemic mixture , receptors : D , and D , receptors , the D -like receptors , acti conformational and /or optical isomer, crystalline poly vate adenylyl cyclase, while the D2, D3, and D4 receptors , morph , or isotopic variant thereof. Alternatively , any of the the D2- like receptors , inhibit adenylyl cyclase and activate 60 histamine receptor modulators listed in Table 1 can be used K + channels. A dopamine receptor antagonist is an agent to antagonize a histamine receptor. Thus, in some embodi able to inhibit one or more characteristic responses of a ments , the histamine receptor antagonist is a histamine dopamine receptor or receptor subtype . As a non - limiting receptor modulator compound listed in Table 1. The com example , an antagonist may competitively or non -competi pounds described in Table 1 are readily available . tively bind to ( 1 ) a dopamine receptor, ( 2 ) an agonist or 65 In some embodiments , the neurotransmitter receptor partial agonist (or other ligand ) of the dopamine receptor , modulating agent is clemastine or a salt thereof ( e.g. , cle and /or ( 3 ) a downstream signaling molecule to inhibit the mastine fumarate ). US 10,493,080 B2 61 62 4. Beta Adrenergic Receptor Modulators screening a plurality of agents to identify an agent that A beta adrenergic receptor is a subtype of the adrenergic increases the number of cells in a sample having a differ receptor, a G -protein coupled receptor, for which cat entiated , myelinating cell fate ( e.g., mature myelinating echolamines (e.g. , epinephrine and norepinephrine ) are the oligodendrocyte ) . In some embodiments , an agent promotes primary endogenous ligand . There are three known subtypes 5 or increases OPC differentiation when it increases the per of beta adrenergic receptors : B , receptors , B2 receptors , and centage of OPCs ( e.g. , in a sample comprising a plurality of Bz receptors . A beta adrenergic receptor antagonist is an OPCs) that differentiate to a mature myelinating cell fate by agent able to inhibit one or more characteristic responses of at least about 5 % , 10 % , 15 % , 20 % , 25 % , 30 % , 40 % , 50 % , a beta adrenergic receptor or receptor subtype. As a non or more as compared to the percentage of OPCs that limiting example , an antagonist may competitively or non- 10 differentiate to a mature myelinating cell fate in the absence competitively bind to (1 ) a beta adrenergic receptor, (2 ) an of the agent. agonist or partial agonist ( or other ligand ) of the beta 1. Marker Assays adrenergic receptor, and /or ( 3 ) a downstream signaling mol In some embodiments , agents that stimulate increased ecule to inhibit the beta adrenergic receptor's function . As a myelination of nerves are identified by screening for induc non -limiting example , pindolol is able to antagonize the 15 tion of markers of mature myelinating oligodendrocytes . In function of a beta adrenergic receptor. A beta adrenergic some embodiments , samples comprising a plurality of OPCs receptor agonist is an agent able to induce or stimulate one are contacted with a candidate agent, incubated under con or more characteristic responses of a beta adrenergic recep ditions suitable for the differentiation of OPCs, and evalu tor or receptor subtype . As shown in the Examples , pindolol, ated for the presence or absence of one or more markers of salmeterol , salbutamol, and albuterol have been shown to 20 mature myelinating oligodendrocytes . Examples of markers agonize the function of a beta adrenergic receptor and / or are of mature myelinating oligodendrocytes include, but are not known agonists of a beta adrenergic receptor. Therefore, in limited to , myelin basic protein (MBP ) , myelin oligoden some embodiments , the beta adrenergic receptor modulator drocyte glycoprotein (MOG ), 2'3 - cyclic- nucleotide 3 phos is a compound selected from pindolol, salmeterol , salbuta phodiesterase ( CNP ) , GalC , 01 , or 04 . mol, albuterol, and salts , prodrugs, racemic mixtures, con- 25 Markers of mature myelinating oligodendrocytes can be formational and /or optical isomers, crystalline polymorphs , detected using any number of established analytical tech and isotopic variants thereof. Alternatively , any of the beta niques. For example , detection can be accomplished by adrenergic receptor modulators listed in Table 1 can be used detecting nucleic acid ( e.g., by in situ hybridization or to modulate a beta adrenergic receptor . Thus, in some RT- PCR ) or protein (e.g. , by immunoassay or Western blot embodiments , the beta adrenergic receptor modulator is a 30 analysis ) levels , followed by visualization and/ or quanitifi beta adrenergic receptor modulator compound listed in cation using any one of a variety of methods known in the Table 1. The compounds described in Table 1 are readily art . In some embodiments , a marker ofmature myelinating available . oligodendrocytes is detected by in situ hybridization . In situ In some embodiments , the neurotransmitter receptor hybridization techniques are generally described in In Situ modulating agent is salmeterol or a salt thereof ( e.g., sal- 35 Hybridization : A Practical Approach (Wilkinson , D. G., meterol xinfoate ) . In some embodiments , the neurotransmit ed .) , Oxford University Press , 1992. In some embodiments , ter receptor modulating agent is salbutamol or a salt thereof a marker ofmature myelinating oligodendrocytes is detected ( e.g., salbutamol hemi sulfate ) . by immunoassay. Immunoassay techniques and protocols 5. Opioid Receptor Modulators are generally described in Price and Newman , “ Principles An opioid receptor is a G - protein coupled receptor , for 40 and Practice of Immunoassay ,” 2nd Edition , Grove's Dic which opioids are the primary endogenous ligand . An opioid tionaries , 1997 ; and Gosling , “ Immunoassays: A Practical receptor antagonist is an agent able to inhibit one or more Approach , ” Oxford University Press , 2000. In some characteristic responses of an opioid receptor or receptor embodiments , the immunoassay is an immunofluorescence subtype . As a non - limiting example, an antagonist may assay . competitively or non -competitively bind to ( 1 ) an opioid 45 A detectable moiety can be used in the assays described receptor , ( 2 ) an agonist or partial agonist (or other ligand ) of herein . A wide variety of detectable moieties can be used , a receptor, and/ or ( 3 ) a downstream signaling molecule to with the choice of label depending on the sensitivity inhibit a receptor's function . An opioid receptor agonist is an required , ease of conjugation with the antibody, stability agent able to induce or stimulate one or more characteristic requirements , and available instrumentation and disposal responses of an opioid receptor or receptor subtype. For 50 provisions. Suitable detectable moieties include, but are not example , an agonist may activate an opioid receptor. As limited to , radionuclides , fluorescent dyes ( e.g., fluorescein , shown the Examples, carbetapentane , Snc - 80 , and fluorescein isothiocyanate (FITC ) , Oregon GreenTM rhod BD - 1047 have been shown to modulate the function of an amine, Texas red, tetrarhodimine isothiocynate ( TRITC ), opioid receptor. Therefore, in some embodiments , the opioid Cy3, Cy5, etc.) , fluorescent markers (e.g. , green fluorescent receptor antagonist is a compound selected from carbeta- 55 protein (GFP ) , phycoerythrin , etc. ), autoquenched fluores pentane, Snc -80 , BD -1047 , and salts , prodrugs , racemic cent compounds that are activated by tumor - associated mixtures , conformational and/ or optical isomers , crystalline proteases, enzymes (e.g. , luciferase , horseradish peroxidase , polymorphs, and isotopic variants thereof. Alternatively , any alkaline phosphatase, etc.) , nanoparticles, biotin , digoxi of the opioid receptor modulators listed in Table 1 can be genin , and the like . used to modulate an opioid receptor. Thus, in some embodi- 60 2. Cells and Reagents ments , the opioid receptor modulator is an opioid receptor The primary screens for identifying agents that induce modulator compound listed in Table 1. The compounds OPC differentiation and /or stimulate increased myelination described in Table 1 are readily available . of nerves can be performed in cell- based assays using B. Identification of Modulating Agents cultured OPC cell lines or OPCs derived from a subject ( e.g. , A number of different screening protocols can be utilized 65 from a mammal) . to identify agents that stimulate increased myelination of OPCs can be derived from any of a variety of sources. In nerves. In general terms, the screening methods involve some embodiments , OPCs are harvested from a tissue , for US 10,493,080 B2 63 64 example , brain tissue , spinal cord tissue , or optic nerve ies ” or “ ligand libraries” are then screened in one or more tissue . The tissue can be from a rodent ( e.g., rat or mouse ) , assays , as described herein , to identify those library mem chicken , dog , cat, rabbit , cow , sheep , goat, or primate ( e.g., bers (particular chemical species or subclasses) that display a monkey , a chimpanzee , or a human ). In some embodi a desired characteristic activity . The compounds thus iden ments , OPCs are derived from fetal tissue. In some embodi- 5 tified can serve as conventional “ lead compounds ” or can ments , OPCs are derived from adult tissue . Alternatively, themselves be used as potential or actual therapeutics. OPCs can be derived from culturing stem cells ( e.g., neural A combinatorial chemical library is a collection of diverse stem cells or embryonic stem cells ) or from other cells that chemical compounds generated by either chemical synthesis can be induced to give rise to OPCs ( e.g., bone marrow or biological synthesis , by combining a number of chemical stromal cells) . 10 " building blocks. ” For example , a linear combinatorial Examples of conditions suitable for OPC differentiation chemical library such as a polypeptide library is formed by are described in the Examples section below . Cell culture combining a set of chemical building blocks (amino acids ) conditions are described in more detail , e.g. , in Picot, in every possible way for a given compound length ( i.e., the Human Cell Culture Protocols (Methods in Molecular Medi number of amino acids in a polypeptide compound ) . Mil cine) 2010 ed ., and in Davis , Basic Cell Culture 2002 ed . 15 lions of chemical compounds can be synthesized through OPCs are cultured with growth factor, for example , such combinatorial mixing of chemical building blocks. PDGFaa . As a non -limiting example , OPCs are proliferated Preparation and screening of combinatorial chemical in culture on poly - D - Lysine coated cell culture dishes using libraries is well known to those of skill in the art. Such OPC media (Neurobasal media , B27 supplement without combinatorial chemical libraries include , but are not limited vitamin A , non - essential amino acids ) containing 30 ng/ mL 20 to , peptide libraries (see , e.g., U.S. Pat . No. 5,010,175 , PDGFaa . For differentiation , OPCs are seeded on poly - D Furka, Int. J. Pept. Prot. Res. 37 : 487-493 ( 1991) and Hough Lysine coated cell culture dishes using OPC media contain ton et al. , Nature 354 : 84-88 ( 1991) ) . Other chemistries for ing 2 ng /mL PDGFaa and treated with compounds dis generating chemical diversity libraries can also be used . solved in DMSO ( < 1 % final concentration ). Differentiating Such chemistries include, but are not limited to : peptoids OPCs are incubated at 37 ° C., 5 % CO2 for 6 days . At the end 25 (e.g. , PCT Publication No. WO 91/19735) , encoded peptides of 6 days, cells are fixed with 4 % paraformaldehyde for ( e.g. , PCT Publication WO 93/20242) , random bio -oligom immunofluorescence analysis or are harvested for biochemi ers ( e.g., PCT Publication No. WO 92/00091) , benzodiaz cal analysis . epines ( e.g., U.S. Pat . No. 5,288,514 ), diversomers such as 3. Candidate Agents hydantoins, benzodiazepines and dipeptides (Hobbs et al. , The agents that are screened for the ability to promote 30 Proc . Nat. Acad. Sci. USA 90 :6909-6913 ( 1993 )) , vinylo OPC differentiation can be any small chemical compound , gous polypeptides (Hagihara et al ., J. Amer. Chem . Soc . or a biological entity , such as a polypeptide, sugar, nucleic 114 :6568 (1992 )) , nonpeptidal peptidomimetics with glu acid or lipid . Typically , test compounds will be small chemi cose scaffolding (Hirschmann et al ., J. Amer. Chem . Soc . cal molecules and peptides . Essentially any chemical com 114 :9217-9218 (1992 ) ) , analogous organic syntheses of pound can be used as a potential modulator or ligand in the 35 small compound libraries (Chen et al. , J. Amer. Chem . Soc. assays of the invention , although most often compounds that 116 : 2661 ( 1994 ) ), oligocarbamates (Cho et al ., Science can be dissolved in aqueous or organic ( especially DMSO 261: 1303 (1993 ) ) , and / or peptidyl phosphonates (Campbell based ) solutions are used . The assays are designed to screen et al. , J. Org. Chem . 59 :658 (1994 )) , nucleic acid libraries large chemical libraries by automating the assay steps and ( see Ausubel, Berger and Sambrook , all supra ), peptide providing compounds from any convenient source to assays , 40 nucleic acid libraries ( see , e.g., U.S. Pat . No. 5,539,083 ) , which are typically run in parallel ( e.g., in microtiter formats antibody libraries (see , e.g. , Vaughn et al. , Nature Biotech on microtiter plates in robotic assays ). It will be appreciated nology , 14 ( 3 ) : 309-314 1522 ( 1996 ) and U.S. Pat . No. 5,593, that there are many suppliers of chemical compounds , 853 ), small organic molecule libraries ( see , e.g., benzodiaz including Sigma (St. Louis , Mo.) , Aldrich ( St. Louis , Mo.) , epines , Baum C & EN , January 18 , page 33 ( 1993 ) ; Sigma- Aldrich ( St. Louis , Mo. ), Fluka Chemika -Bio- 45 isoprenoids , U.S. Pat . No. 5,569,588 ; thiazolidinones and chemica Analytika (Buchs , Switzerland ) and the like. metathiazanones , U.S. Pat. No. 5,549,974 ; pyrrolidines , In some embodiments , the agents have a molecular U.S. Pat. Nos. 5,525,735 and 5,519,134 ; morpholino com weight of less than 1,500 daltons , and in some cases less pounds , U.S. Pat. No. 5,506,337 ; benzodiazepines, U.S. Pat . than 1,000 , 800 , 600 , 500 , or 400 daltons. The relatively No. 5,288,514 , and the like ). small size of the agents can be desirable because smaller 50 Devices for the preparation of combinatorial libraries are molecules have a higher likelihood of having physiochemi commercially available ( see , e.g., 357 MPS , 390 MPS , cal properties compatible with good pharmacokinetic char Advanced Chem Tech , Louisville Ky. , Symphony, Rainin , acteristics, including oral absorption than agents with higher Woburn , Mass ., 433A Applied Biosystems, Foster City , molecular weight. For example , agents less likely to be Calif ., 9050 Plus, Millipore , Bedford , Mass. ). In addition , successful as drugs based on permeability and solubility 55 numerous combinatorial libraries are themselves commer were described by Lipinski et al . as follows : having more cially available ( see , e.g., ComGenex , Princeton , N.J., Tri than 5 H -bond donors (expressed as the sum of OHs and pos , Inc., St. Louis , Mo. , 3D Pharmaceuticals , Exton , Pa. , NHs) ; having a molecular weight over 500 ; having a LogP Martek Biosciences , Columbia , Md ., etc. ). over 5 ( or MLogP over 4.15 ) ; and /or having more than 10 In some embodiments , candidate agents are able to pen H -bond acceptors (expressed as the sum of Ns and Os ). See , 60 etrate the blood -brain barrier. In some embodiments , candi e.g., Lipinski et al ., Adv Drug Delivery Res 23 :3-25 ( 1997) . date agents have a low molecular weight ( i.e., a molecular Compound classes that are substrates for biological trans weight of no more than 800 kDa ). In some embodiments , porters are typically exceptions to the rule . candidate agents are screened for one or more other criteria , In some embodiments , the agents are from a combinato such as toxicity or brain pharmacokinetics . rial chemical or peptide library containing a large number of 65 4. Validation potential therapeutic compounds (potential modulator or Agents that are initially identified by any of the foregoing ligand compounds ). Such " combinatorial chemical librar screening methods can be further tested to validate the US 10,493,080 B2 65 66 apparent activity . In some embodiments , validation assays dopamine receptor modulator compound , histamine receptor are in vitro assays . In some embodiments , such studies are modulator compound , beta adrenergic receptor modulator conducted with suitable animal models. The basic format of compound , or opioid receptor modulator compound listed in such methods involves administering a lead compound Table 1 ) . In some embodiments , the neurotransmitter recep identified during an initial screen to an animal that serves as 5 tor modulating agent is benztropine , cerbetapentane , clem a disease model for humans and then determining if the astine , pindolol, ipratropium , atropine , GBR12935 , Snc -80 , disease ( e.g., a demyelinating disease ) is in fact modulated BD - 1047, salmeterol, albuterol, or trifluoperazine, or a salt and / or the disease or condition is ameliorated . The animal thereof. In some embodiments , the neurotransmitter receptor models utilized in validation studies generally are mammals modulating agent is benztropine, clemastine, salmeterol, of any kind . Specific examples of suitable animals include , 10 salbutamol, trifluoperazine , or a salt thereof. In some but are not limited to, primates , mice , rats and zebrafish . embodiments , the neurotransmitter receptor modulating III . Methods using Neurotransmitter Receptor Modulating agent is benztropine or a salt thereof (e.g. , benztropine Agents mesylate ) The neurotransmitter receptor modulating agents In some embodiments , the demyelinating disease is mul described herein can be used in various therapeutic and / or 15 tiple sclerosis , idiopathic inflammatory demyelinating dis prophylactic methods . In one aspect , the present invention ease, transverse myelitis , Devic's disease , progressive mul provides methods of inducing oligodendrocyte precursor tifocal leukoencephalopathy , optic neuritis , leukodystrophy, cell (OPC ) differentiation to a mature myelinating cell fate Guillain - Barre syndrome, chronic inflammatory demyelinat ( e.g., myelinating oligodendrocytes ). In some embodiments , ing polyneuropathy, autoimmune peripheral neuropathy, the method comprises contacting the OPC with a neurotrans- 20 Charcot- Marie - Tooth disease , acute disseminated encepha mitter receptor modulating agent as described herein and lomyelitis , adrenoleukodystrophy, adrenomyeloneuropathy , culturing the OPC under conditions suitable for OPC dif Leber's hereditary optic neuropathy, or human T- cell lym ferentiation . In some embodiments , the neurotransmitter photropic virus (HTLV ) -associated myelopathy . receptor modulating agent is selected from a muscarinic In some embodiments , the demyelinating disease is mul receptor antagonist, a dopamine receptor antagonist, a his- 25 tiple sclerosis (MS ) . There are several subtypes of MS , tamine receptor antagonist , a beta adrenergic receptor modu including relapsing - remitting multiple sclerosis (RRMS ), lator , and an opioid receptor modulator. In some embodi secondary progressive multiple sclerosis (SPMS ) , primary ments , the OPC is cultured in the presence of the progressive multiple sclerosis (PPMS ), and progressive neurotransmitter receptor modulating agent for at least 2 relapsing multiple sclerosis (PRMS ) . In some embodiments , days , at least 3 days, at least 4 days, at least 5 days , at least 30 the subject has RRMS . In some embodiments , the subject 6 days or longer under conditions suitable for OPC differ has SPMS . In some embodiments , the subject has PPMS. In entiation . Differentiation to a mature myelinating cell fate some embodiments , the subject has PRMS. A subject may can be determined by detecting the presence of one ormore initially be diagnosed as having one subtype of MS (e.g. , biologicalmarkers ofmature myelinating oligodendrocytes . RRMS) , and subsequently the subtype ofMS afflicting the Marker assays for detecting the presence or level ofmyeli- 35 subject may convert to another subtype of MS ( e.g., from nating oligodendrocytes are described herein , for example in RRMS to SPMS ). It is contemplated that the methods of the Section II (B ) above . present invention can be applied to treat a subject whose In another aspect, the present invention provides methods subtype of MS converts to another subtype of MS. of stimulating increased myelination of nerves in a subject In some embodiments , a subject in need thereof ( e.g., a in need thereof. In some embodiments , the method com- 40 subject having a demyelinating disease or at risk for having prises administering to the subject a neurotransmitter recep a demyelinating disease ) is administered a neurotransmitter tor modulating agent as described herein ; thereby stimulat receptor modulating agent in combination with at least one ing increased myelination of nerves in the subject . In some other therapy. In some embodiments , the at least one other embodiments , the neurotransmitter receptor modulating therapy is an immunomodulatory agent. As used herein , an agent is a compound selected from a muscarinic receptor 45 “ immunomodulatory agent” refers to a disease -modifying antagonist, a dopamine receptor antagonist , a histamine drug which alters the course of a demyelinating disease ( e.g. , receptor antagonist , a beta adrenergic receptor modulator, multiple sclerosis , idiopathic inflammatory demyelinating and an opioid receptor modulator. disease , transverse myelitis , Devic's disease , progressive In some embodiments , a subject in need of a method of multifocal leukoencephalopathy, optic neuritis , leukodystro stimulating increased myelination of nerves is a subject 50 phy , Guillain -Barre syndrome, chronic inflammatory demy having a demyelinating disease . Thus, in yet another aspect , elinating polyneuropathy, autoimmune peripheral neuropa the present invention provides methods of treating and / or thy, Charcot- Marie - Tooth disease, acute disseminated ameliorating a subject having a demyelinating disease . In encephalomyelitis , adrenoleukodystrophy , adrenomy some embodiments , a subject in need of a method of eloneuropathy, Leber's hereditary optic neuropathy, or stimulating increased myelination of nerves is a subject at 55 HTLV -associated myelopathy ) . risk of having a demyelinating disease. Thus, in yet another In some embodiments, an immunomodulatory agent is a aspect , the present invention provides methods of preventing disease -modifying drug that alters the course of multiple a demyelinating disease or delaying the occurrence of a sclerosis ( e.g. , RRMS, SPMS, PPMS , or PRMS ). For demyelinating disease . example , a disease -modifying drug can reduce the frequency In some embodiments , the method comprises administer- 60 or severity of an MS relapse and / or reduce development of ing to the subject a neurotransmitter receptor modulating lesions or scars at regions of demyelination . In some agent selected from a muscarinic receptor antagonist , a embodiments , an immunomodulatory agent is an immuno dopamine receptor antagonist , a histamine receptor antago suppressant ( i.e. , an agent that suppresses or prevents an nist, a beta adrenergic receptor modulator, and an opioid immune response ). In some embodiments , an immuno receptor modulator. In some embodiments , the neurotrans- 65 modulatory agent is an agent that modulates an immune mitter receptor modulating agent is a compound listed in response ( e.g., by stimulating the induction of suppressor T Table 1 ( e.g., a muscarinic receptor modulator compound , cells ). Examples of immunomodulatory agents for the treat US 10,493,080 B2 67 68 ment ofMS include, but are not limited to , interferons (e.g. , and an opioid receptor modulator, and the immunomodula interferon - B , e.g. , interferon beta - la or interferon beta - 1 ), tory agent is selected from fingolimod (FTY720 ) , interferon glatiramer acetate , mitoxantrone, fingolimod (FTY720 ), or beta - la , interferon beta - lb , glatiramer acetate , mitoxan monoclonal antibodies ( e.g. , natalizumab , rituximab , dacli trone , natalizumab , rituximab , daclizumab , and alemtu zumab , or alemtuzumab ). Thus, in some embodiments , the 5 zumab . In some embodiments , the neurotransmitter receptor method of the present invention comprises administering to modulating agent is a muscarinic receptor modulator com a subject having MS a neurotransmitter receptor modulating pound, a dopamine receptor modulator compound , a hista agent ( e.g., a compound selected from a muscarinic receptor mine receptor modulator compound , a beta adrenergic antagonist, a dopamine receptor antagonist , a histamine receptor modulator compound , or an opioid receptor modu receptor antagonist, a beta adrenergic receptor antagonist, 10 lator compound listed in Table 1, and the immunosuppres and an opioid receptor modulator ) in combination with sant is fingolimod (FTY720 ) , interferon beta - la , interferon fingolimod (FTY720 ), interferon beta -la , interferon beta beta - 1b , glatiramer acetate , mitoxantrone, natalizumab , 1b , glatiramer acetate , mitoxantrone, natalizumab , ritux rituximab , daclizumab , or alemtuzumab . In some embodi imab , daclizumab , or alemtuzumab . ments , the neurotransmitter receptor modulating agent is In another aspect , the present invention provides methods 15 benztropine, clemastine, salmeterol, salbutamol, or trifluop of enhancing the therapeutic effect of an immunomodulatory erazine , or a salt thereof , and the immunomodulatory agent agent in a subject in need thereof. It has been surprisingly is fingolimod (FTY720 ) , interferon beta -la , interferon beta found that in mouse models of demyelinating disease , 1b , glatiramer acetate , mitoxantrone, natalizumab , ritux administering a combination of a neurotransmitter receptor imab , daclizumab , or alemtuzumab . In some embodiments , modulating agent and an immunomodulatory agent results 20 the neurotransmitter receptor modulating agent is benz in a significantly larger decrease in the clinical severity of tropine and the immunomodulatory agent is fingolimod the demyelinating disease as compared to the decrease in the (FTY720 ) , interferon beta -la , or interferon beta - 1b . clinical severity of the demyelinating disease that can be Therapeutic doses for the immunomodulatory agents fin achieved with either the neurotransmitter receptor modulat golimod (FTY720 ), interferon beta - la , interferon beta - 1b , ing agent or the immunomodulatory agent alone. Thus , in 25 glatiramer acetate , mitoxantrone , and natalizumab are some embodiments , the method comprises administering to known in the art. See , e.g., Kappos et al ., N Engl J Med a subject an immunomodulatory agent and a neurotransmit 362 :387-401 (2010 ); Cohen et al. , N Engl J Med 362: 402-15 ter receptor modulating agent; thereby enhancing the thera (2010 ) ; Gottesman et al. , Mult Scler 12 :271-80 (2006 ) ; peutic effect of the immunomodulatory agent in the subject. Hurwitz et al ., Clin Ther 30 : 1102-12 ( 2008 ) ; Gaindh et al. , In some embodiments , the subject has a demyelinating 30 Expert Opin Biol Ther 8 : 1823-29 ( 2008 ) ; Koch - Henriksen et disease or is at risk of having a demyelinating disease . al. , Neurology 66 :1056-60 (2006 ); Benatar, Lancet 360 :1428 Furthermore, it has also surprisingly been found that (2008 ) ; Jacobs et al. , Ann Neurol 39 :285-94 ( 1996 ) ; Lancet administering a neurotransmitter receptor modulating agent, 352 :1498-1504 ( 1998 ) ; Johnson et al. , Neurology 45 : 1268 in combination with an immunomodulatory agent at a dose 76 ( 1995) ; Johnson et al. , Neurology 50 : 701-08 ( 1998 ) ; that , on its own , is insufficient to be therapeutic for the 35 Comi et al ., Ann Neurol . 69 : 75-82 ( 2011 ) ; Calabresi Nat treatment of a demyelinating disease , results in a therapeutic Clin Pract Neurol 3 :540-1 ( 2007 ) ; and Polman et al. , N Engl effect that is greater than the therapeutic effect from admin J Med 354 :899-910 (2006 ); the contents of each of which are istering each agent alone. Thus, in another aspect, the incorporated by reference herein in their entirety . present invention provides methods of treating a subject in In some embodiments , the immunomodulatory agent need thereof by administering an immunomodulatory agent 40 ( e.g., fingolimod , interferon beta - la , interferon beta - 1b , and a neurotransmitter receptor modulating agent, wherein glatiramer acetate , mitoxantrone, or natalizumab ) is admin the immunomodulatory agent is administered at a subthera istered at a therapeutically effective dose . In some embodi peutic dose . In some embodiments , the subject has a demy ments , the immunomodulatory agent ( e.g., fingolimod , inter elinating disease or is at risk of having a demyelinating feron beta -la , interferon beta - 1b , glatiramer acetate , disease . 45 mitoxantrone , or natalizumab ) is administered at a subthera In some embodiments , the demyelinating disease is mul peutic dose , e.g., at a dose that is less than about 75 % , less tiple sclerosis , idiopathic inflammatory demyelinating dis than about 70 % , less than about 60 % , less than about 50 % , ease , transverse myelitis, Devic's disease , progressive mul less than about 40 % , less than about 30 % , less than about tifocal leukoencephalopathy, optic neuritis , leukodystrophy, 25 % , less than about 20 % , less than about 15 % , less than Guillain - Barre syndrome, chronic inflammatory demyelinat- 50 about 10 % , or less than about 5 % of the dose that is ing polyneuropathy, autoimmune peripheral neuropathy , conventionally administered for the immunomodulatory Charcot- Marie - Tooth disease , acute disseminated encepha agent. lomyelitis , adrenoleukodystrophy, adrenomyeloneuropathy , Fingolimod (FTY720 ) is conventionally administered at a Leber's hereditary optic neuropathy, or HTLV -associated therapeutically effective dose of from about 0.5 mg per day myelopathy . In some embodiments , the demyelinating dis- 55 to about 1.5 mg per day (e.g. , about 0.5 , about 0.6 , about 0.7 , ease is multiple sclerosis , e.g., relapsing -remitting multiple about 0.8 , about 0.9 , about 1.0 , about 1.1 , about 1.2 , about sclerosis (RRMS ) , secondary progressive multiple sclerosis 1.3 , about 1.4 , or about 1.5 mg per day ) . See , e.g., Kappos (SPMS ) , primary progressive multiple sclerosis ( PPMS) , or et al. , N Engl J Med 362 : 387-401 ( 2010 ). Thus, in some progressive relapsing multiple sclerosis (PRMS ) . In some embodiments , a subtherapeutic dose of fingolimod is from embodiments , the subject is initially diagnosed as having 60 about 0.005 mg per day to about 0.375 mg per day ( e.g., one subtype of MS ( e.g., RRMS) , and subsequently the about 0.005 , about 0.01, about 0.02 , about 0.03 , about 0.04 , subtype of MS afflicting the subject converts to another about 0.05 , about 0.06 , about 0.07 , about 0.08 , about 0.09 , subtype of MS ( e.g., from RRMS to SPMS ) . about 0.1 , about 0.15 , about 0.2 , about 0.25 , about 0.3 , about In some embodiments , the neurotransmitter receptor 0.35 , or about 0.375 mg per day ). modulating agent is selected from a muscarinic receptor 65 Interferon beta - la is conventionally administered at a antagonist, a dopamine receptor antagonist , a histamine therapeutically effective dose of about 30 ug per week . See receptor antagonist , a beta adrenergic receptor modulator, e.g., Jacobs et al ., Ann Neurol 39: 285-94 ( 1996 ). Thus, in US 10,493,080 B2 69 70 some embodiments , a subtherapeutic dose of interferon 1b . In some embodiments , benztropine is administered at a beta - la is from about 0.3 ug per week to about 23 ug per therapeutically effective dose of from about 1 mg per day to week (e.g. , about 0.3 , about 0.5 , about 0.75 , about 1 , about about 10 mg per day ; fingolimod is administered at a 2 , about 3 , about 4 , about 5 , about 6 , about 7 , about 8 , about therapeutically effective dose of from about 0.5 mg per day 9 , about 10 , about 11 , about 12 , about 13 , about 14 , about 15 , 5 to about 1.5 mg per day ; interferon beta - la is administered about 16 , about 17 , about 18 , about 19 , about 20 , about 21 , at a therapeutically effective dose of about 30 ug per week ; about 22 , or about 23 ug per week ) . and /or interferon beta - lb is administered at a therapeutically Interferon beta - lb is conventionally administered at a effective dose of from about 250 ug every other day to about therapeutically effective dose of from about 250 ug every 500 ug every other day . other day to about 500 ug every other day . See , e.g. , 10 In some embodiments , a neurotransmitter receptor modu Gottesman et al. , Mult Scler 12 :271-80 (2006 ). Thus, in lating agent ( e.g., a muscarinic receptor antagonist , a dop some embodiments , a subtherapeutic dose of interferon amine receptor antagonist, a histamine receptor antagonist , beta - 1b is from about 2 ug every other day to about 190 ug a beta adrenergic receptor modulator , or an opioid receptor every other day (e.g. , about 2 , about 5 , about 10 , about 20 , modulator listed in Table 1, e.g. , benztropine, clemastine , about 30 , about 40 , about 50 , about 60 , about 70 , about 80 , 15 salmeterol, salbutamol , trifluoperazine , or a salt thereof) is about 90 , about 100 , about 110 , about 120 , about 130 , about administered at a therapeutically effective dose and an 140 , about 150 , about 160 , about 170 , about 180 , or about immunomodulatory agent ( e.g., fingolimod , interferon beta 190 ug every other day ). 1a, interferon beta - 1b , glatiramer acetate , mitoxantrone, or In some embodiments , the neurotransmitter receptor natalizumab ) is administered at a subtherapeutic dose , e.g. , modulating agent (e.g. , a muscarinic receptor antagonist, a 20 at a dose that is less than about 75 % , less than about 70 % , dopamine receptor antagonist , a histamine receptor antago less than about 60 % , less than about 50 % , less than about nist , a beta adrenergic receptor modulator, or an opioid 40 % , less than about 30 % , less than about 25 % , less than receptor modulator listed in Table 1 ) is administered at a about 20 % , less than about 15 % , less than about 10 % , or less therapeutically effective dose . In some embodiments , the than about 5 % of the dose that is conventionally adminis neurotransmitter receptor modulating agent ( e.g. , muscar- 25 tered for the immunomodulatory agent. In some embodi inic receptor antagonist, dopamine receptor antagonist, his ments , the neurotransmitter receptor modulating agent is tamine receptor antagonist, beta adrenergic receptor modu benztropine or a salt thereof ( e.g., benztropine mesylate ) and lator, or opioid receptor modulator listed in Table 1 ) is the immunomodulatory agent is fingolimod (FTY720 ) , administered at a subtherapeutic dose , e.g., at a dose that is interferon beta - la , or interferon beta - 1b . In some embodi less than about 75 % , less than about 70 % , less than about 30 ments , benztropine is administered at a therapeutically 60 % , less than about 50 % , less than about 40 % , less than effective dose of from about 1 mg per day to about 10 mg about 30 % , less than about 25 % , less than about 20 % , less per day ; fingolimod is administered at a subtherapeutic dose than about % , less than about 10 % , or less than about 5 % of from about 0.005 mg per day to about 0.375 mg per day ; of the dose that is conventionally administered for the interferon beta - la is administered at a subtherapeutic dose of neurotransmitter receptor modulating agent. In some 35 from about 0.3 ug per week to about 23 ug per week ; and/ or embodiments , the neurotransmitter receptor modulating interferon beta - 1b is administered at a subtherapeutic dose agent that is administered at a therapeutically effective dose of from about 2 ug every other day to about 190 ug every or at a subtherapeutic dose is benztropine, clemastine , other day. salmeterol, salbutamol, trifluoperazine , or a salt thereof. In In some embodiments , a neurotransmitter receptor modu some embodiments , the neurotransmitter receptor modulat- 40 lating agent (e.g. , muscarinic receptor antagonist, dopamine ing agent that is administered at a therapeutically effective receptor antagonist, histamine receptor antagonist, beta dose or at a subtherapeutic dose is benztropine or a salt adrenergic receptor modulator, or opioid receptor modulator thereof (e.g. , benztropine mesylate ). As a non - limiting listed in Table 1 , e.g., benztropine , clemastine, salmeterol, example , a therapeutically effective dose of benztropinemay salbutamol, trifluoperazine , or a salt thereof) is administered be from about 1 mg per day to about 10 mg per day ( e.g., 45 at a subtherapeutic dose , e.g., at a dose that is less than about about 1, about 2 , about 3 , about 4 , about 5 , about 6 , about 75 % , less than about 70 % , less than about 60 % , less than 7 , about 8 , about 9 , or about 10 mg per day ) . Thus, in some about 50 % , less than about 40 % , less than about 30 % , less embodiments , a subtherapeutic dose of benztropine may be than about 25 % , less than about 20 % , less than about 15 % , from about 0.01 mg per day to about 0.75 mg per day ( e.g., less than about 10 % , or less than about 5 % of the dose that about 0.01 , about 0.05 , about 0.10 , about 0.015 , about 0.20 , 50 is conventionally administered for the neurotransmitter about 0.25 , about 0.30 , about 0.35 , about 0.40 , about 0.45 , receptor modulating agent, and an immunomodulatory agent about 0.50 , about 0.55 , about 0.60 , about 0.65 , about 0.70 , (e.g. , fingolimod , interferon beta -la , interferon beta - 1b , or about 0.75 mg per day ). glatiramer acetate, mitoxantrone , or natalizumab ) is admin In some embodiments , a neurotransmitter receptor modu istered at a therapeutically effective dose . In some embodi lating agent (e.g. , a muscarinic receptor antagonist , a dop- 55 ments , the neurotransmitter receptor modulating agent is amine receptor antagonist, a histamine receptor antagonist , benztropine or a salt thereof ( e.g., benztropine mesylate ) and a beta adrenergic receptor modulator, or an opioid receptor the immunomodulatory agent is fingolimod (FTY720 ), modulator listed in Table 1 , e.g. , benztropine , clemastine, interferon beta - la , or interferon beta - 1b . In some embodi salmeterol, salbutamol, trifluoperazine , or a salt thereof) is ments, bentropine is administered at a subtherapeutic dose administered at a therapeutically effective dose and an 60 of from about 0.01 mg per day to about 0.75 mg per day ; immunomodulatory agent ( e.g., fingolimod , interferon beta fingolimod is administered at a therapeutically effective dose 1a, interferon beta -1b , glatiramer acetate , mitoxantrone , or of from about 0.5 mg per day to about 1.5 mg per day ; natalizumab ) is administered at a therapeutically effective interferon beta -la is administered at a therapeutically effec dose . In some embodiments , the neurotransmitter receptor tive dose of about 30 ug per week ; and / or interferon beta - 1b modulating agent is benztropine or a salt thereof ( e.g., 65 is administered at a therapeutically effective dose of from benztropine mesylate ) and the immunomodulatory agent is about 250 ug every other day to about 500 ug every other fingolimod (FTY720 ), interferon beta -la , or interferon beta day . US 10,493,080 B2 71 72 In some embodiments , a neurotransmitter receptor modu prevention of a demyelinating disease ( e.g. , multiple scle lating agent ( e.g., muscarinic receptor antagonist, dopamine rosis ) . In some embodiments , a neurotransmitter receptor receptor antagonist , histamine receptor antagonist , beta modulating agent (e.g. , a muscarinic receptor antagonist , a adrenergic receptor modulator, or opioid receptor modulator dopamine receptor antagonist , a histamine receptor antago listed in Table 1 , e.g., benztropine, clemastine , salmeterol, 5 nist , a beta adrenergic receptor modulator , or an opioid salbutamol, trifluoperazine , or a salt thereof) is administered receptor modulator listed in Table 1, e.g., benztropine, at a subtherapeutic dose , e.g., at a dose that is less than about clemastine, salmeterol, salbutamol, trifluoperazine, or a salt 75 % , less than about 70 % , less than about 60 % , less than thereof ) is administered at a subtherapeutic dose and an about 50 % , less than about 40 % , less than about 30 % , less immunomodulatory agent ( e.g., fingolimod , interferon beta than about 25 % , less than about 20 % , less than about 15 % , 10 la , interferon beta - 1b , glatiramer acetate , mitoxantrone, or less than about 10 % , or less than about 5 % of the dose that natalizumab ) is administered at a subtherapeutic dose for the is conventionally administered for the neurotransmitter treatment or prevention of a demyelinating disease ( e.g. , receptor modulating agent, and an immunomodulatory agent multiple sclerosis ) . (e.g. , fingolimod , interferon beta -la , interferon beta - 1b , IV . Pharmaceutical Compositions glatiramer acetate , mitoxantrone, or natalizumab ) is admin- 15 In another aspect , the present invention provides pharma istered at a subtherapeutic dose , e.g. , at a dose that is less ceutical compositions for use in the treatment of a demy than about 75 % , less than about 70 % , less than about 60 % , elinating disease ( e.g. , multiple sclerosis , idiopathic inflam less than about 50 % , less than about 40 % , less than about matory demyelinating disease , transverse myelitis , Devic's 30 % , less than about 25 % , less than about 20 % , less than disease , progressive multifocal leukoencephalopathy, optic about 15 % , less than about 10 % , or less than about 5 % of the 20 neuritis , leukodystrophy, Guillain -Barre syndrome, chronic dose that is conventionally administered for the immuno inflammatory demyelinating polyneuropathy , autoimmune modulatory agent. In some embodiments, the neurotrans peripheral neuropathy, Charcot- Marie - Tooth disease , acute mitter receptor modulating agent is benztropine or a salt disseminated encephalomyelitis , adrenoleukodystrophy, thereof ( e.g. , benztropine mesylate ) and the immunomodu adrenomyeloneuropathy, Leber's hereditary optic neuropa latory agent is fingolimod ( FTY720 ), interferon beta -la , or 25 thy, or human T- cell lymphotropic virus (HTLV ) -associated interferon beta - 1b . In some embodiments , bentropine is myelopathy ) . In some embodiments , the composition com administered at a subtherapeutic dose of from about 0.01mg prises a mixture of a neurotransmitter receptor modulating per day to about 0.75 mg per day ; fingolimod is administered agent and an immunomodulatory agent. In some embodi at a subtherapeutic dose of from about 0.005 mg per day to ments , the pharmaceutical composition comprises a neu about 0.375 mg per day ; interferon beta - la is administered 30 rotransmitter receptor modulating agent selected from a at a subtherapeutic dose of from about 0.3 ug per week to muscarinic receptor antagonist , a dopamine receptor antago about 23 ug per week ; and / or interferon beta -1b is admin nist, a histamine receptor antagonist, a beta adrenergic istered at a subtherapeutic dose of from about 2 ug every receptor modulator, and an opioid receptormodulator and an other day to about 190 ug every other day. immunomodulatory agent selected from interferon beta - la, In some embodiments , a neurotransmitter receptormodu- 35 interferon beta - 1b , glatiramer acetate , mitoxantrone , fingoli lating agent (e.g. , a muscarinic receptor antagonist , a dop mod (FTY720 ), natalizumab , rituximab , daclizumab , and amine receptor antagonist , a histamine receptor antagonist , alemtuzumab . In some embodiments , the pharmaceutical a beta adrenergic receptor modulator, or an opioid receptor composition comprises a neurotransmitter receptor modu modulator listed in Table 1, e.g. , benztropine , clemastine , lating agent selected from benztropine , clemastine , salmet salmeterol, salbutamol, trifluoperazine, or a salt thereof) is 40 erol, salbutamol, trifluoperazine , and salts thereof and an administered at a therapeutically effective dose and an immunomodulatory agent selected from interferon beta - la , immunomodulatory agent ( e.g. , fingolimod, interferon beta interferon beta - 1b , glatiramer acetate ,mitoxantrone , fingoli 1a, interferon beta -1b , glatiramer acetate , mitoxantrone , or mod (FTY720 ) , natalizumab , rituximab , daclizumab , and natalizumab ) is administered at a therapeutically effective alemtuzumab . In some embodiments , the pharmaceutical dose for the treatment or prevention of a demyelinating 45 composition comprises benztropine or a salt thereof and an disease ( e.g. , multiple sclerosis ). In some embodiments , a immunomodulatory agent selected from interferon beta - la, neurotransmitter receptor modulating agent ( e.g. , a musca interferon beta- 1b , and fingolimod (FTY720 ). rinic receptor antagonist, a dopamine receptor antagonist , a In some embodiments , one or both of the neurotransmitter histamine receptor antagonist , a beta adrenergic receptor receptor modulating agent and the immunomodulatory agent modulator, or an opioid receptor modulator listed in Table 1 , 50 are formulated as a therapeutically effective or optimaldose . e.g., benztropine, clemastine, salmeterol, salbutamol, triflu In some embodiments , one or both of the neurotransmitter operazine , or a salt thereof) is administered at a therapeu receptor modulating agent and the immunomodulatory agent tically effective dose and an immunomodulatory agent ( e.g., are formulated as a subtherapeutic dose. In some embodi fingolimod , interferon beta- la , interferon beta - lb , glati ments , the neurotransmitter receptor modulating agent is ramer acetate , mitoxantrone , or natalizumab ) is adminis- 55 formulated as a therapeutically effective or optimal dose and tered at a subtherapeutic dose for the treatment or prevention the immunomodulatory agent is formulated as a subthera of a demyelinating disease ( e.g. ,multiple sclerosis ). In some peutic dose . In some embodiments , the immunomodulatory embodiments , a neurotransmitter receptor modulating agent agent is formulated as a therapeutically effective or optimal ( e.g. , a muscarinic receptor antagonist , a dopamine receptor dose and the neurotransmitter receptor modulating agent is antagonist , a histamine receptor antagonist, a beta adrener- 60 formulated as a subtherapeutic dose . Suitable dosage ranges gic receptor modulator , or an opioid receptor modulator for therapeutically effective and subtherapeutic doses of listed in Table 1 , e.g., benztropine , clemastine , salmeterol, neurotransmitter receptor modulating agents and immuno salbutamol, trifluoperazine , or a salt thereof) is administered modulatory agents are described above . at a subtherapeutic dose and an immunomodulatory agent An agent for use in any of the therapeutic methods of the ( e.g., fingolimod , interferon beta - la, interferon beta - 1b , 65 present invention ( e.g. , an agent that stimulates increased glatiramer acetate, mitoxantrone , or natalizumab ) is admin myelination as described herein , or an immunomodulatory istered at a therapeutically effective dose for the treatment or agent as described herein ) may be in any pharmaceutically US 10,493,080 B2 73 74 acceptable form , including any pharmaceutically acceptable tion include those described in U.S. Pat. Nos . 6,635,680 ; salts , prodrugs, racemic mixtures , conformational and /or 6,624,200 ; 6,613,361; 6,613,358 , 6,596,308 ; 6,589,563 ; optical isomers , crystalline polymorphs and isotopic variants 6,562,375 ; 6,548,084 ; 6,541,020 ; 6,537,579; 6,528,080 and of the neurotransmitter receptor modulating agents . 6,524,621 , each of which is hereby incorporated herein by A combination of a neurotransmitter receptor modulating 5 reference . Controlled release formulations of particular agent and an immunomodulatory agent can be incorporated interest include those described in U.S. Pat. Nos. 6,607,751 ; into a variety of formulations for therapeutic or prophylactic 6,599,529 ; 6,569,463 ; 6,565,883 ; 6,482,440 ; 6,403,597 ; administration . More particularly , a combination of a neu 6,319,919 ; 6,150,354 ; 6,080,736 ; 5,672,356 ; 5,472,704 ; rotransmitter receptor modulating agent and an immuno 5,445,829 ; 5,312,817 and 5,296,483 , each of which is modulatory agent can be formulated into pharmaceutical 10 hereby incorporated herein by reference. Those skilled in the compositions, e.g. , a single composition , by formulation art will readily recognize other applicable sustained release with appropriate pharmaceutically acceptable carriers or formulations . diluents , and can be formulated into preparations in solid , The pharmaceutical formulations of the invention can be semi- solid , liquid or gaseous forms, such as tablets , cap provided as a salt and can be formed with many acids, sules , pills, powders, granules , dragees , gels , slurries, oint- 15 including but not limited to hydrochloric , sulfuric , acetic , ments , solutions , suppositories , injections , inhalants and lactic , tartaric , malic , succinic , etc. Salts tend to be more aerosols . As such , administration of an agent of the present soluble in aqueous or other protonic solvents that are the invention can be achieved in various ways , including oral, corresponding free base forms. In other cases, the prepara buccal , parenteral, intravenous, intradermal ( e.g. , subcuta tion may be a lyophilized powder in 1 mM -50 mM histidine , neous , intramuscular ), transdermal, etc. , administration . 20 0.1 % -2 % sucrose , 2 % -7 % mannitol at a pH range of 4.5 to Moreover , the agent can be administered in a local rather 5.5 , that is combined with buffer prior to use . than systemic manner , for example , in a depot or sustained For oral administration , an agent of the present invention release formulation . can be formulated readily by combining with pharmaceuti Formulations cally acceptable carriers that are well known in the art . Such Suitable formulations for use in the present invention are 25 carriers enable the compounds to be formulated as tablets , found in Remington : The Science and Practice of Pharmacy , pills , dragees , capsules, emulsions, lipophilic and hydro 21st Ed ., Gennaro , Ed ., Lippencott Williams & Wilkins philic suspensions, liquids, gels, syrups, slurries , suspen ( 2003) , which is hereby incorporated herein by reference . sions and the like, for oral ingestion by a patient to be The pharmaceutical compositions described herein can be treated . Pharmaceutical preparations for oral use can be manufactured in a manner that is known to those of skill in 30 obtained by mixing the compounds with a solid excipient, the art, i.e., by means of conventional mixing, dissolving , optionally grinding a resulting mixture , and processing the granulating , dragee- making , levigating , emulsifying, encap mixture of granules, after adding suitable auxiliaries , if sulating , entrapping or lyophilizing processes. The follow desired , to obtain tablets or dragee cores . Suitable excipients ing methods and excipients are merely exemplary and are in are , in particular, fillers such as sugars , including lactose , no way limiting . 35 sucrose, mannitol, or sorbitol; cellulose preparations such In some embodiments , an agent is prepared for delivery in as , for example , maize starch , wheat starch , rice starch , a sustained - release , controlled release , extended -release , potato starch , gelatin , gum tragacanth , methyl cellulose , timed -release or delayed - release formulation , for example , hydroxypropylmethyl- cellulose , sodium carboxymethylcel in semipermeable matrices of solid hydrophobic polymers lulose, and /or polyvinylpyrrolidone (PVP ). If desired , dis containing the therapeutic agent. Various types of sustained- 40 integrating agents can be added , such as a cross - linked release materials have been established and are well known polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof by those skilled in the art . Current extended -release formu such as sodium alginate . lations include film -coated tablets , multiparticulate or pellet Pharmaceutical preparations which can be used orally systems, matrix technologies using hydrophilic or lipophilic include push - fit capsules made of gelatin , as well as soft, materials and wax - based tablets with pore - forming excipi- 45 sealed capsules made of gelatin and a plasticizer , such as ents (see , for example , Huang, et al. Drug Dev. Ind . Pharm . glycerol or sorbitol. The push - fit capsules can contain the 29:79 (2003 ) ; Pearnchob , et al . Drug Dev. Ind . Pharm . active ingredients in admixture with filler such as lactose , 29 :925 (2003 ) ;Maggi , et al . Eur. J. Pharm . Biopharm . 55:99 binders such as starches, and / or lubricants such as talc or ( 2003) ; Khanvilkar , et al ., Drug Dev. Ind . Pharm . 228 :601 magnesium stearate and , optionally , stabilizers . In soft cap ( 2002) ; and Schmidt, et al. , Int . J. Pharm . 216 : 9 (2001 ) ) . 50 sules, the active compounds can be dissolved or suspended Sustained -release delivery systems can , depending on their in suitable liquids, such as fatty oils , liquid paraffin , or liquid design , release the compounds over the course of hours or polyethylene glycols . In addition , stabilizers can be added . days, for instance, over 4 , 6 , 8 , 10 , 12 , 16 , 20 , 24 hours or All formulations for oral administration should be in dos more . Usually , sustained release formulations can be pre ages suitable for such administration . pared using naturally occurring or synthetic polymers , for 55 Dragee cores are provided with suitable coatings . For this instance , polymeric vinyl pyrrolidones , such as polyvinyl purpose, concentrated sugar solutions can be used , which pyrrolidone (PVP ); carboxyvinyl hydrophilic polymers ; can optionally contain gum arabic , talc , polyvinyl pyrroli hydrophobic and /or hydrophilic hydrocolloids, such as done, carbopol gel , polyethylene glycol , and /or titanium methylcellulose, ethylcellulose, hydroxypropylcellulose , dioxide, lacquer solutions, and suitable organic solvents or and hydroxypropylmethylcellulose ; and carboxypolymeth- 60 solventmixtures . Dyestuffs or pigments can be added to the ylene. tablets or dragee coatings for identification or to characterize The sustained or extended -release formulations can also different combinations of active compound doses. be prepared using natural ingredients , such as minerals , The agents can be formulated for parenteral administra including titanium dioxide, silicon dioxide, zinc oxide, and tion by injection , e.g. , by bolus injection or continuous clay ( see , U .S . Pat. No. 6,638,521, herein incorporated by 65 infusion . For injection , the compound can be formulated into reference ) . Exemplified extended release formulations that preparations by dissolving, suspending or emulsifying them can be used in delivering a compound of the present inven in an aqueous or nonaqueous solvent, such as vegetable or US 10,493,080 B2 75 76 other similar oils , synthetic aliphatic acid glycerides , esters modulating compounds as described herein with an effective of higher aliphatic acids or propylene glycol; and if desired , amount of other therapeutic agents as combination partners , with conventional additives such as solubilizers, isotonic particularly those used for treating demyelinating diseases , agents , suspending agents , emulsifying agents , stabilizers such as immunomodulary agents . An effective amount of the and preservatives . In some embodiments , an agent of the 5 agent and/ or combination partner will, of course , be depen invention can be formulated in aqueous solutions, preferably dent on the subject being treated , the severity of the affliction in physiologically compatible buffers such as Hanks's solu and the manner of administration . Determination of an tion , Ringer's solution , or physiological saline buffer. For effective amount is well within the capability of those skilled mulations for injection can be presented in unit dosage form , in the art, especially in light of the detailed disclosure e.g., in ampules or in multi- dose containers , with an added 10 provided herein . Generally , an efficacious or effective preservative. The compositions can take such forms as amount of an agent is determined by first administering a suspensions, solutions or emulsions in oily or aqueous low dose or small amount, and then incrementally increasing vehicles , and can contain formulatory agents such as sus the administered dose or dosages until a desired therapeutic pending , stabilizing and /or dispersing agents . effect is observed in the treated subject , with minimal or no Pharmaceutical formulations for parenteral administra- 15 toxic side effects . Applicable methods for determining an tion include aqueous solutions of the active agents in water appropriate dose and dosing schedule for administration of soluble form . Additionally , suspensions of the active agents the present invention are described , for example , in Good can be prepared as appropriate oily injection suspensions . man and Gilman's The Pharmacological Basis of Thera Suitable lipophilic solvents or vehicles include fatty oils peutics, 11th Ed ., Brunton , Lazo and Parker, Eds. , McGraw such as sesame oil, or synthetic fatty acid esters , such as 20 Hill ( 2006 ) , and in Remington : The Science and Practice of ethyl oleate or triglycerides, or liposomes . Aqueous injection Pharmacy , 21st Ed ., Gennaro , Ed ., Lippencott Williams & suspensions can contain substances which increase the vis Wilkins ( 2003 ) , both of which are hereby incorporated cosity of the suspension , such as sodium carboxymethyl herein by reference . In some embodiments , one or both of a cellulose , sorbitol, or dextran . Optionally , the suspension neurotransmitter receptor modulating agent and an immu can also contain suitable stabilizers or agents which increase 25 nomodulary agent are formulated in a therapeutically effec the solubility of the compounds to allow for the preparation tive dose . In some embodiments , one or both of a neu of highly concentrated solutions. Alternatively, the active rotransmitter receptor modulating agent and an ingredient can be in powder form for constitution with a immunomodulary agent are formulated in a subtherapeutic suitable vehicle , e.g., sterile pyrogen - free water , before use . dose . In some embodiments , a neurotransmitter receptor Systemic administration can also be by transmucosal or 30 modulating agent is formulated in a therapeutically effective transdermal means . For transmucosal or transdermal admin dose and an immunomodulary agent is formulated in a istration , penetrants appropriate to the barrier to be perme subtherapeutic dose . In some embodiments, an immuno ated are used in the formulation . For topical administration , modulary agent is formulated in a therapeutically effective the agents are formulated into ointments , creams, salves , dose and a neurotransmitter receptor modulating agent is powders and gels . In one embodiment , the transdermal 35 formulated in a subtherapeutic dose . delivery agent can be DMSO . Transdermal delivery systems The pharmaceutical preparation is preferably in unit dos can include, e.g. , patches. For transmucosal administration , age form . In such form the preparation is subdivided into penetrants appropriate to the barrier to be permeated are unit doses containing appropriate quantities of the active used in the formulation . Such penetrants are generally component. The unit dosage form can be a packaged prepa known in the art. Exemplified transdermal delivery formu- 40 ration, the package containing discrete quantities of prepa lations that can find use in the present invention include ration , such as packeted tablets , capsules, and powders in those described in U.S. Pat. Nos. 6,589,549 ; 6,544,548 ; vials or ampoules. Also , the unit dosage form can be a 6,517,864 ; 6,512,010 ; 6,465,006 ; 6,379,696 ; 6,312,717 and capsule , tablet , cachet, or lozenge itself, or it can be the 6,310,177 , each of which are hereby incorporated herein by appropriate number of any of these in packaged form . reference . 45 Administration For buccal administration , the agents can take the form of Administration of a neurotransmitter receptor modulating tablets or lozenges formulated in conventional manner. agent and /or an immunomodulatory agent can be achieved In addition to the formulations described previously , an in various ways , including oral, buccal , parenteral, including agent of the present invention can also be formulated as a intravenous , intradermal, subcutaneous, intramuscular, depot preparation . Such long acting formulations can be 50 transdermal, transmucosal, intranasal , etc. , administration . administered by implantation ( for example subcutaneously When a neurotransmitter receptor modulating agent and an or intramuscularly ) or by intramuscular injection . Thus, for immunomodulatory agent are co -administered , the neu example , the agents can be formulated with suitable poly rotransmitter receptor modulating agent can be administered meric or hydrophobic materials (for example as an emulsion by the same or different route of administration as the in an acceptable oil) or ion exchange resins, or as sparingly 55 immunomodulatory agent. In some embodiments , a com soluble derivatives, for example , as a sparingly soluble salt . pound of the present invention ( e.g. , a neurotransmitter The pharmaceutical compositions also can comprise suit receptor modulating agent and /or an immunomodulatory able solid or gel phase carriers or excipients . Examples of agent) is administered systemically . such carriers or excipients include but are not limited to The dosages and frequency of administration will depend calcium carbonate , calcium phosphate , various sugars , 60 upon various factors generally appreciated by those of skill starches , cellulose derivatives , gelatin , and polymers such as in the art , including, e.g., the severity of a subject's disease . polyethylene glycols . Generally, daily doses can range from about 0.001-100 Pharmaceutical compositions suitable for use in the pres mg/ kg total bodyweight , from about 0.01-50 mg/ kg , or from ent invention include compositions wherein the active ingre about 0.01 mg/ kg - 10 mg/ kg . However , doses in the range of dients are contained in a therapeutically effective amount. 65 10-500 mg/ kg per day may be effective and well tolerated . The present invention also contemplates pharmaceutical The principal determining factor in defining the appropriate compositions comprising the neurotransmitter receptor dose is the amount of a particular compound necessary to be US 10,493,080 B2 77 78 therapeutically effective in a particular context. Repeated tifocal leukoencephalopathy, optic neuritis, leukodystrophy, administrations may be required in order to achieve longer Guillain -Barre syndrome, chronic inflammatory demyelinat lasting immune tolerance . Single or multiple administrations ing polyneuropathy, autoimmune peripheral neuropathy , of the compositions can be carried out with the dose levels Charcot- Marie - Tooth disease, acute disseminated encepha and pattern being selected by the treating physician . 5 lomyelitis , adrenoleukodystrophy, adrenomyeloneuropathy , In some embodiments , a compound of the present inven Leber's hereditary optic neuropathy, or human T - cell lym tion ( e.g., a neurotransmitter receptor modulating agent photropic virus (HTLV )-associated myelopathy ). In some and /or an immunomodulatory agent) is administered to a subject in need thereof over an extended period of time . The embodiments , the kit comprises a neurotransmitter receptor methods can be carried out for at least 20 days , in some 10 modulating agent selected from a muscarinic receptor embodiments for at least 40 , 60 , 80 or 100 days, and in some antagonist, a dopamine receptor antagonist, a histamine embodiments for at least 150 , 200 , 250 , 300 , 350 days, 1 receptor antagonist , a beta adrenergic receptor modulator, year or longer. In some embodiments , a compound of the and an opioid receptor modulator. In some embodiments , the present invention ( e.g. , a neurotransmitter receptor modu kit comprises a neurotransmitter receptor modulating agent lating agent and / or an immunomodulatory agent) is admin- 15 selected from a compound listed in Table 1. In some istered to a subject in need thereof at or after the onset of one embodiments , the kit comprises a neurotransmitter receptor or more symptoms of a demyelinating disease . In some modulating agent selected from benztropine, clemastine, embodiments , a compound of the present invention ( e.g. , a salmeterol , salbutamol, trifluoperazine , and salts thereof. In neurotransmitter receptor modulating agent and / or an immu some embodiments , the kit comprises benztropine or a salt nomodulatory agent ) is administered to a subject in need 20 thereof . thereof prior to the onset of symptoms of a demyelinating In some embodiments , a kit of the present invention disease ( i.e., prophylactically ). further comprises an immunomodulatory agent . In some Co -administration embodiments , the kit comprises a neurotransmitter receptor In some embodiments , the methods of the present inven modulating agent and an immunomodulatory agent for the tion comprise co - administering a neurotransmitter receptor 25 treatment of MS ( e.g., interferon - ß , e.g. , interferon beta - la modulating agent and an immunomodulatory agent. Co or interferon beta - 1b , glatiramer acetate , mitoxantrone, fin administered agents can be administered together or sepa golimod (FTY720 ), natalizumab , rituximab , daclizumab , or rately , simultaneously or at different times . When adminis alemtuzumab ) . In some embodiments , the kit comprises a teredthe immunomodulatory , the neurotransmitter agent receptor independently modulating can beagent admin- and 30 neurotransmitter receptor modulating agent selected from istered once , twice , three , four times daily or more or less benztropine , clemastine , salmeterol, salbutamol, trifluopera often , as needed . In some embodiments , the agents are zine, and salts thereof and an immunomodulatory agent administered once daily . In some embodiments , the agents selected from interferon beta - la , interferon beta - lb , glati are administered at the same time or times, for instance as ramer acetate , mitoxantrone , fingolimod (FTY720 ) , natali an admixture . One or more of the agents can be administered 35 zumab , rituximab , daclizumab , and alemtuzumab . In some in a sustained - release formulation . embodiments , the kit comprises benztropine or a salt thereof In some embodiments , co - administration includes admin and an immunomodulatory agent selected from interferon istering one active agent within 0.5 , 1, 2 , 4 , 6 , 8 , 10 , 12 , 16 , beta- la, interferon beta - 1b , and fingolimod (FTY720 ) . 20 , or 24 hours of a second active agent. Co -administration In some embodiments , a kit comprises a neurotransmitter includesapproximately administering simultaneously two active ( e.g. , agentswithin aboutsimultaneously 1 , 5 , 10 , 40 receptor modulating agent as described herein and an immu 15 , 20 , or 30 minutes of each other) , or sequentially in any nomodulatory agent in a single formulation . In some order . In some embodiments , co - administration can be embodiments , a kit comprises a neurotransmitter receptor accomplished by co - formulation , i.e., preparing a single modulating agent as described herein and an immunomodu pharmaceutical composition including both active agents 45 latory agent in separate formulations. Suitable formulations ( i.e., the neurotransmitter receptormodulating agent and the for the neurotransmitter receptor modulating agent and immunomodulatory agent are administered as a single for immunomodulatory agent are described herein . In some mulation ) . In other embodiments , the active agents can be embodiments , a kit provides a neurotransmitter receptor formulated separately ( i.e., the neurotransmitter receptor modulating agent agent as described herein and one or more modulating agent and the immunomodulatory agent are 50 additional therapeutic agents ( e.g., an immunomodulatory administered as separate formulations ). In another embodi agent) independently in uniform dosage formulations ment, the active and / or adjunctive agents may be linked or throughout the course of treatment. In some embodiments , a conjugated to one another. kit provides a neurotransmitter receptor modulating agent as In some embodiments , one or both of a neurotransmitter described herein and one or more additional therapeutic receptor modulating agent and an immunomodulatory agent 55 agents (e.g. , an immunomodulatory agent ) independently in can be administered prophylactically to prevent undesirable graduated dosages over the course of treatment , either recurrence of the symptoms of the demyelinating disease increasing or decreasing , but usually increasing to an effi ( e.g., to prevent or delay the recurrence of clinical attacks in cacious dosage level , according to the requirements of an MS ) , or therapeutically to achieve a desired reduction in individual . In some embodiments , a kit comprises a neu symptoms of the demyelinating disease and maintain such 60 rotransmitter receptor modulating agent as described herein reduction in symptoms of the demyelinating disease for a in a therapeutically effective dose and an immunomodula sustained period of time. tory agent in a therapeutically effective dose . In some V. Kits embodiments , a kit comprises a neurotransmitter receptor useIn inanother the treatment aspect, theof a present demyelinating invention disease provides ( e.g. kits, mul- for 65 modulating agent as described herein in a therapeutically tiple sclerosis , idiopathic inflammatory demyelinating dis effective dose and an immunomodulatory agent in a sub ease , transverse myelitis , Devic's disease , progressive mul therapeutic dose . US 10,493,080 B2 79 80 EXAMPLES For GBT12935, the percentage of resulting MBP -positive cells was over 15 % . These results show that carbetapentane , The following examples are offered to illustrate , but not clemastine , benztropine, trifluoperazine , salmeterol, and to limit the claimed invention . GBR12935 all promote OPC differentiation . 5 Example 1 Example 2 Small Molecule Screen to Identify Inducers of OPC Validation of Hits from Small Molecule Screen Differentiation 10 Several classes of neurotransmitter receptor modulating A large -scale small molecule screen was conducted to agents that were identified by the primary screen were identify small molecules that promote the differentiation of selected based on FDA approval status, known toxicity , and oligodendrocyte precursor cells (OPCs ) to a mature myeli brain pharmacokinetics for evaluation in subsequent in vitro nating fate . High content imaging was used to detect in -well and in vivo validation assays . The ability of compounds to differentiation of rat optic nerve derived OPCs. Rat optic 15 induce robust differentiation of OPCS to a fully mature nerve derived OPCs were expanded en masse in OPC media myelinating oligodendrocyte cell fate was evaluated in vitro containing 30 ng/ mL PDGFaa . To screen for small mol using quantitative RT- PCR , western blotting and immuno ecule inducers of OPC differentiation , OPCs (cultured for fluorescence analysis techniques . The ability of compounds fewer than 15 passages ) were plated in poly -D -lysine coated to induce remyelination in vivo ( in the presence of inflam 384 well culture plates in OPC Media containing 2 ng/ mL 20 matory insult) was evaluated using a proteolipid protein PDGFac and immediately treated with compounds at a final ( PLP) induced Experimental Autoimmune Encephalitis concentration of 6 UM ( 0.6 % DMSO ) . Compound treated (EAE ) mouse model of relapsing multiple sclerosis . cells were incubated at 37 ° C ., 5 % CO2 for 6 days. At the end Quantitative RT- PCR (“ qRT- PCR ” ) and Western blot of 6 days , cells were fixed in 4 % paraformaldehyde and analysis were performed on OPC samples that had been subjected to immunofluorescence analysis . Anti -Myelin 25 cultured with DMSO (negative control) , T3 (positive con Basic Protein monoclonal antibody (a.a. 129-138 , clone 1 trol ) , benztropine , carbetapentane , clemastine , trifluopera monoclonal antibody, Millipore ) ( 1: 1000 dilution ) in block zine , GBR12935 , or salmeterol to measure the levels of ing buffere solution (3 % BSA , 0.3 % Triton X - 100 in PBS ) expression of MBP and MOG , markers for myelinating was added to fixed and washed cells and incubated at 4 ° C. oligodendrocytes. As shown in FIG . 2A , OPC samples overnight. The primary antibody solution was removed and 30 cultured with benztropine, carbetapentane , clemastine , cells were washed with PBS prior to incubation with a GBR12935, or salmeterol all were positive for MBP and blocking buffer solution containing goat anti -mouse IgG MOG expression . qRT- PCR showed two - fold induction of Alexa fluor488 ( Invitrogen ) secondary antibody ( 1: 1000 expression , or greater , ofMBP and MOG for OPC samples dilution ) and DAPI ( 2 ug/ mL ) for 1 hr at 25 ° C. The cultured with benztropine , carbetapentane, clemastine, trif secondary antibody solution was washed with PBS and 35 luoperazine , GBR12935 , or salmeterol ( FIG . 2B - C ) . plates were imaged using an OPERA high content screening Immunofluorescence was also used to confirm that the system (Perkin Elmer) . A cell scoring based image analysis hits were able to induce oligodendrocyte maturation . As algorithm was used to identify and score MBP positive cells . shown in FIGS. 3 and 4 , immunofluorescence analysis Hits were identified as compounds that induced > 400 cells / confirmed the presence ofmultiple markers ofmature myeli field and > 5 % MBP positive cells/ field . The average values 40 nating oligodendrocytes (MBP , MOG , CNP, Galc , 01, and for DMSO negative controls were consistently < 0.5 % MBP 04 ) following treatment of in vitro cultures with benz positive. A total of 6058 compounds comprised of commer tropine , carbetapentane, clemastine , trifluoperazine , cially available small molecule screening collections GBR12935 , or salmeterol . (LOPAC , Tocris , Enzo ) were screened . A total of 104 Two of the compounds , trifluoperazine and benztropine , primary hits were identified . These consisted of a number of 45 were tested in vivo in proteolipid protein (PLP ) induced known OPC differentiating inducing agents ( e.g., retenoids , Experimental Autoimmune Encephalitis (EAE ) mice, a sterols , nucleoside analogues, Rho kinase inhibitors ) , as well model of multiple sclerosis . 10 week old SJL mice were as a number of previously unidentified OPC differentiation immunized with PLP emulsion and pertusis toxin to induce inducing agents ( e.g., neurotransmitter modulating agents ) . EAE . EAE symptoms developed in mice within 10 days Identified primary hits were subsequently evaluated by 50 after immunization . Animals that developed EAE showed determing EC50 values in multiple replicate experiments neurological deficits ranging from impaired tail movements using the primary assay format for determing OPC differ to paralysis in all 4 limbs with the most severe symptoms entiation . Cells were treated with compounds serially diluted lasting for about 4 days, followed by a period of remission . in DMSO ( 10 dilutions of 1 : 3 across a range of 71 uM to 4 Compounds were administered daily via intraperitoneal nM ) . Staining and imaging was performed as described for 55 injection of 100 ul in sterile saline at 10 mg/ kg alone or in the primary screen . EC50 values were determined using combination with mycophenolate motefil in sterile saline at appropriate curve fitting equations in Graphpad Prism 5.0 . pH 5 at 20 mg/ kg . Compound administration was initiated at FIG . 1A shows the EC50 for six identified hits ( carbetapen disease onset ( day 10 ) while mycophenolate motefil admin tane, clemastine , benztropine , trifluoperazine , salmeterol, istration was initiated on Day 14. The mice were scored and GBR12935 ) , all of which are FDA - approved blood- 60 daily on a standard EAE scale of 0-5 . MS- like symptoms brain barrier -penetrating drugs. FIG . 1 also shows the ability were induced in 65-100 % of the mice. Mycophenolate of these six identified hits to induce differentiation of OPCs motefil, used at a sub - optimal dose showed a decrease in (as measured by the percentage of myelin basic protein severity of the relapse (FIG . 5A ). Trifluoperazine showed a (MBP ) -positive cells ) at varying concentrations (FIG . 1B ) . modest decrease in recovery time following the acute phase For each of these agents , when the OPC sample was cultured 65 (FIG . 5D - E ) . Each of trifluoperazine and benztropine alone with an agent at maximum potency , the percentage of significantly reduced the intensity of relapse measured as resulting MBP -positive cells was at least 10 % ( FIG . 1C ) . significantly lower EAE scores for compound treated ani US 10,493,080 B2 81 82 mals compared to vehicle controls (FIG . 5B , D ). In the (FIG . 11 ) . Unfortunately , T3 has multiple physiological presence of mycophenolate motefil no relapse was observed effects that make it unattractive as an OPC differentiation in animals treated with compounds (FIG . 5C , E ). Thus, based therapeutic . trifluoperazine and benztropine prevented relapse and To identify small drug - like molecules that induce OPC improved motor and cognitive function as compared to the 5 differentiation , we developed a high content imaging assay controls . that is based on the induction MBP expression in primary rat In another set of experiments , four compounds (benz optic nerve derived OPCs cultured for 6 days under basal tropine , clemastine , trifluoperazine , and salbutamol) were differentiation conditions . The assay was adapted to 384 tested in vivo in a EAE model . As shown in FIG . 34A , mice well format and used to screen collections of known bio treated with benztropine ( 10 mg/ kg ) in a prophylactic mode 10 logicallystructurally active diverse compounds drug -like , as wellmolecules as a collection. This led of to -50K the showed a significantly decreased clinical severity in both identification of multiple previously identified inducers of acute and relapse phase of the disease as compared to OPC differentiation including retinoids , corticosteroids, vehicle treated mice . In a therapeutic mode , each of benz nucleoside analogs, Rho - kinase inhibitors and ErbB inhibi tropine (FIG . 34C ), trifluoperazine (FIG . 34D ) , clemastine 15 tors (M. J. Latasa et al. , Glia , 58 , 1451 (2010 ) ; C. E. Buckley ( FIG . 34E ), and salbutamol (FIG . 34E ) showed a signifi et al. , Neuropharmacology, 59 , 149 ( 2010 ) ; L. Joubert et al ., cantly decreased clinical severity in the relapse phase of the J Neurosci Res, 88 , 2546 ( 2010 ) ;A. S. Baer et al. , Brain 132 , disease as compared to vehicle treated mice . 465 ( 2009 ) ; A. S. Paintlia et al ., Mol Pharmacol, 73 , 1381 The pharmacological mechanisms of identified OPC dif (2008 ) ; C. Ibanez et al. , Prog Neurobiol, 71, 49 (2003 ) ; L. ferentiation - inducing agents was also investigated . It was 20 Giardino , C. Bettelli, L. Calza , Neurosci Lett, 295, 17 determined that multiple pharmacological mechanisms exist (2000 ) ) , which have limited therapeutic potential due to for inducing OPC differentiation with identified agents . For off -target activities , toxicity , or a demonstrated lack of in example , as shown in FIG . 6A ,muscarinic receptor agonism vivo efficacy . Surprisingly , among the most effective induc with carbachol inhibited the OPC differentiation induced by ers of OPC differentiation was benztropine (EC 50 ~ 350 nM ), benztropine , carbetapentane, and clemastine , suggesting the 25 for which OPC differentiation activity has not previously mechanism at least some OPC differentiation - inducing been reported (FIGS . 7A and 12 ). We chose to further agents is muscarinic receptor antagonism . Other muscarinic investigate the activity of this compound , as it is an orally receptor antagonists, atropine and ipratropium , also induce available , well- tolerated , FDA approved drug that readily OPC differentiation . However, carbachol did not inhibit crosses the blood brain barrier, and therefore has the poten OPC differentiation induced by salmeterol, GBR12935 , or 30 tial to move rapidly to proof of concept studies as a new trifluoperazine, suggesting these agents induce OPC differ treatment for MS . entiation by one or more pharmacological mechanisms. Benztropine - induced in vitro differentiation of rodent OPCS was confirmed by evaluating the transcription and Example 3 translation levels of the oligodendrocyte specific markers 35 MBP and myelin oligodendroglial glycoprotein (MOG ) by A Stem Cell -Based Strategy for the Treatment of RT- PCR and Western blot analysis , respectively ( FIGS. 7B Multiple Sclerosis and 13 ). Additionally , in vitro OPC differentiation activity was assessed by immunofluorescent analysis using multiple Studies aimed at evaluating the presence and relative markers specifically expressed in mature oligodendrocytes densities of OPCs at sites of chronically demyelinated MS 40 (including MBP, MOG , 2' , 3 '- cyclic - nucleotide 3 '- phos lesions indicate that it is not a failure of repopulation or phodiesterase (CNP ), galactocerebrosidase (GalC ), oligo migration of OPCs, but rather inhibition of OPC differen dendrocyte marker 01 (01 ) and oligodendrocyte marker 04 tiation at sites of injury that contributes to disease progres ( 04 )) following six days of compound treatment (FIG . 14 ) . sion (D. M. Chari, W. F. Blakemore, Glia , 37 , 307 ( 2002) ; Furthermore , global gene expression profiles of OPCS D. M. Chari et al. , J Neurosci Res, 73 , 787 ( 2003 ) ; G. 45 treated for 6 days with benztropine were found to cluster Wolswijk , J Neurosci, 18 , 601 ( 1998 ) ; A. Chang et al. , N with those obtained from T3 treated positive control cells Engl J Med , 346 , 165 ( 2002 ) ; T. Kuhlmann et al. , Brain , 131 , ( FIG . 15 ) . Downregulation of OPC specific genes required 1749 (2008 )) . As such , the identification of drug - like small for proliferation ( e.g. , Idr2 , Egr 1 , Sox 11 ; V. A. Swiss et al. , molecules that selectively induce differentiation of OPCs at PLoS One , 6 , el8088 (2011 ) ) , and upregulation of mature sites of demyelinated lesions would have a significant 50 myelinatingoligodendrocyte specific genes (e.g. , lipid impact on the development of new , effective treatments for metabolism , myelin related proteins (V. A. Swiss et al. , PLOS MS ( D. Kremer et al. , Ann Neurol, 69, 602 ( 2011 ) ) . One, 6 , e18088 (2011 ) ) was observed following treatment of Primary rodent and human OPCs proliferate in vitro when OPCS with benztropine (FIG . 16 ) . In order to determine the cultured in serum - free media containing PDGF (C. Ffrench stage of OPC differentiation at which benztropine is active Constant, M. C. Raff, Nature , 319 , 499 (1986 ) ; M. C. Raff 55 (Gard , Neuron , 3 , 615 ( 1990 ) ; A. L. Gard , S. E. Pfeiffer , Dev et al. , J Exp Biol , 132, 35 ( 1987 ) ) . Upon withdrawal of Biol, 159 , 618 ( 1993 ) ; D. Avossa , S. E. Pfeiffer , J Neurosci PDGF, immature OPCs cease to proliferate , but also fail to Res, 34 , 113 ( 1993 ) ; R. Aharoni et al. , Proc Natl Acad Sci efficiently differentiate to a mature myelin basic protein U.S.A., 102 , 19045 (2005 )) , we treated OPCS for differing (MBP ) positive fate (FIG . 11 ) . Addition of triiodothryonine durations starting atmultiple time points (FIG . 17 ) . Maximal ( T3 ), a known inducer of OPC differentiation (M. C. Nunes 60 induction ofMBP expression was observed when compound et al. , Nat Med , 9,439 (2003 ) ; N. Billon et al. , Dev Biol , 235 , was added within 48 hours of PDGF withdrawal and left to 110 ( 2001 ); N. Billon et al. , EMBOJ, 21 , 6452 ( 2002 ); Y. M. incubate for 5 days (FIG . 17 ) , indicating that this drug likely Tokumoto , D. G. Tang , M. C. Raff , EMBO J 20 , 5261 acts on immature A2B5 positive OPCS and not on an (2001 ) ; Fernandes , 2004 ; L. Calza , M.Fernandez , L. Giar intermediate “ pre -oligodendrocyte ” stage of differentiation . dino , J Mol Endocrinol , 44 , 13 ( 2010 ) , at the time of 65 Benztropine is used clinically for the management of mitogen withdrawal results in the differentiation ofOPCs to Parkinson's disease and its pharmacological activity is MBP positive oligodendrocytes following 6 days of culture thought to result from anticholinergic activity that decreases US 10,493,080 B2 83 84 the imbalance between dopamine and acetylcholine ( A. J. be used to determine the effectiveness of promyelinating Eshleman et al ., Mol Pharmacol , 45 , 312 ( 1994 ) ; R. Kat agents that function by enhancing OPC differentiation (M. senschlager et al. , Cochrane Database Syst Rev, CD003735 Fernandez et al. , Proc Natl Acad Sci U.S.A , 101, 16363 (2003 ) ; J. T. Coyle and S. H. Snyder , Science, 166 , 899 (2004 ) ; X. Lee et al. , J Neurosci, 27 , 220 (2007 ) ; reviewed ( 1969) ) . In addition to its anticholinergic activity , Benz- 5 in R. J. Franklin , C. Ffrench - Constant, Nat Rev Neurosci, 9 , tropine is a centrally acting anti -histamine and dopamine 839 (2008 )) . Benztropine ( 10 mg/ kg ) was dosed prophylac re - uptake inhibitor (G. E. Agoston et al ., J Med Chem , 40 , tically using a daily intraperitoneal (IP ) injection regimen 4329 ( 1997) ; J. L.Katz et al ., J Pharmacol Exp Therap , 309, initiated at the onset of immunization of 8 week old SJL 605 (2004 ) ; D. Simoni et al. , J Med Chem , 48 , 3337 (2005 ) ). mice with PLP. Benztropine dramatically decreased the In order to determine the mechanism of action of benz- 10 severity of the acute phase of disease and virtually elimi tropine, we evaluated the ability of selective agonists of nated the observation of a relapse phase as compared to muscarinic acetylcholine receptors (mAChRs ) ( e.g., carba vehicle treated controls ( FIGS. 8A and 25 ) . We next evalu chol) or histaminergic receptors ( e.g. , histamine and hista ated efficacy when the drug is dosed therapeutically , by mine trifloromethyl- toluidine (HTMT ) ) to block benztropine starting daily injections at the first sign of disease onset . activity . Potent inhibition of benztropine induced OPC dif- 15 Treatment with benztropine in this mode again led to func ferentiation was observed in the presence of carbachol tional recovery, with significant decreases in clinical sever (FIGS . 7C and 18 ), whereas histamine or HTMT showed no ity in remission phases observed and the occurrence of observable effect (FIG . 19 ). In addition , neither the dop relapse again virtually eliminated (FIG . 8A ) . In fact , treat amine receptor antagonist haloperidol , nor the dopamine ment with benztopine in this mode resulted in decreases in receptor agonist quinpirole affected the induction of OPC 20 observed clinical severity that exceeded those observed for differentiation by benztropine ( FIG . 20) . Quinpirole did not the immunomodulating MS drugs FTY720 or interferon -B induce significant differentiation when used alone either (dosed at near maximally tolerated doses ) ( FIG . 8A ) . ( data not shown ). We therefore next evaluated a panel of In parallel experiments , during relapse phases (day mACHR antagonists ( atropine, oxybutynin , scopolamine , 23-25 ), we isolated spinal cords from benztropine or vehicle ipratropium , and propiverine ) and found that all induced 25 treated mice and stained sections from multiple regions of OPC differentiation in a dose dependent manner (FIG . 21) . each spinal cord with luxol fast blue ( LFB ) ( to visualize OPCs express mACHRs, predominantly subtypes M1, M2, myelin ) or H & E ( to visualize infiltrating immune cells ). and Ms( F. Ragheb et al. , J Neurochem , 77, 1396 (2001 ) ). We Sections from both vehicle and benztropine treated mice confirmed expression of these receptors , as well as the showed significant infiltration by H & E reactive immune acetylcholine synthesizing enzyme, choline acetyl trans- 30 cells (FIG . 26 ) . In vehicle treated mice, areas of infiltration ferase , in OPCs by RT- PCR ( FIG . 22 ) . Carbachol induced corresponded to areas with significant demyelination (FIG . activation of mAChRs triggers protein kinase -C dependent 26 ). In contrast , in benztropine treated mice, a large number activation of the MAPK / ERK pathway leading modula of immune cell infiltrated areas stained positive for LFB , tion of c- Fos expression (F. Ragheb et al. , J Neurochem , 77 , consistent with a stem cell versus immunomodulatory 1396 ( 2001) ) . Western blot analysis of benztropine treated 35 mechanism (FIG . 26 ). We further evaluated drug -enhanced OPCswas consistent with general inhibition of this pathway remyelination using confocal microscopy, by examining ( i.e., decreased phospho - p42 / 44 MAPK and phospho - Akt regions of T -cell infiltration in spinal cord sections stained and stimulated phosphorylation of p38 MAPK and CREB ) with markers of mature oligodendrocytes (GST -ht ) and (FIG . 23A ). Additionally , transcript levels of cyclin Di , immature OPCs (NG2 ) (FIG . 8B ). Quantitative image analy cyclin D2 , C - Fos, and c - Jun were significantly decreased in 40 sis of multiple random fields per group indicates that ben benztropine treated OPCs , consistent with a mechanism ztropine treatment causes a significant increase in the num involving general inhibition of MAPK /ERK dependent cell ber ofGST- , positivemature oligodendrocytes from ~ 500 to cycle progression (FIG . 23B ) . Activation of My and M3 ~ 1100 per field ( FIG . 8C ) , whereas the number of NG2 MAChRs is coupled to downstream signal transduction positive cells did not differ significantly with treatment (FIG . events through phospholipase C , which results in increased 45 8C ) . The observed increase in mature oligodendrocyte num intracellular calcium concentrations (F. Ragheb et al. , J bers at sites of T - cell infiltration is consistent with a mecha Neurochem , 77 , 1396 ( 2001 )) (FIG . 23C ) . M , and M4 nism of benztropine - induced clinical recovery that involves mACHR activation inhibits adenylatecyclase , leading to the stimulation of OPC differentiation , leading to enhanced decreased intracellular cAMP levels (C. C. Felder , FASEB J , remyelination , in the context of an inflammatory environ 9 , 619 (1995 ) ; M. Lopez - llasaca et al ., Science , 275, 394 50 ment. Notably , general toxicity was not observed in drug (1997 ) ) . In OPCs, benztropine inhibits carbachol induced treated mice, nor was it observed in this histological analysis calcium influx , but has no effect on CAMP levels (FIG . 24 ) . ( i.e., drug induced demyelination was not observed ) follow Together , these results suggest that benztropine induces ing 4 weeks of daily injections at 10 mg/ kg . OPC differentiation by a mechanism involving direct The primary immunological processes involved in MS antagonism ofMy /Mz muscarinic receptors. Acetylcholine is 55 and EAE are T- cell mediated and the primary treatment a known regulator of OPC proliferation and , as such , mus paradigms are based on immunomodulatory drugs ( T. carinic receptor subtypes represent a reasonable class of Kopadze et al ., Arch Neurol, 63, 1572 ( 2006 ) ; J. M.Greer et therapeutic targets for the modulation of OPC proliferation al. , J Immunol, 180 , 6402 (2007 ) , M. P. Pender and J. M. and differentiation ( F. De Angelis et al. , Dev Neurobiol, Greer, curr Allergy Asthma Rep , 7 , 285 (2007 )) . In order to ( 2011 ) ) . 60 determine whether the efficacy of benztropine in the EAE We next examined the activity of benztropine in the model results at least in part from a T- cell inhibitory activity, myelin proteolipid protein (PLP )-induced experimental we evaluated the effects of benztropine on T -cell activation autoimmune encephalomyelitis (EAE ) rodent model of and proliferation . Benztropine had no effect on T- cell acti relapsing- remitting MS (D. A. Sipkins , J Neuroimmunol, vation or proliferation in vitro , as determined by evaluating 104, 1 (2000 ) ; T. Owens, S. Sriram , Neurol Clin , 13 , 51 65 CD4 + /CD69 + and CD4 + /CD25 + populations and using a ( 1995 ) ) . This model is most commonly used to evaluate the CFSE assay , respectively (FIG . 27 ) . We evaluated the effect potential efficacy of immunomodulatory agents , but can also of benztropine on the immune system in vivo in SJL mice in US 10,493,080 B2 85 86 which EAE had or had not been induced with PLP. In either also enhance remyelination by exerting a protective effect on diseased or healthy animals , benztropine had no effecton the a " pre -oligodendrocyte ” intermediate cell type or by a number of circulating splenocytes, CD4 + cells , CD8 + cells , process involving a combination of both effects . Caspase - 3 CD4 + /CD44Hi cells and CD8 + /CD44Hi cells ( FIGS. 28 and activation results from both intrinsic and extrinsic apoptotic 29 ). A minor but significant decrease in B -cell numbers was 5 pathways and serves as a general indicator of stress induced observed following treatment with benztropine (FIGS . 28 cell death . Benztropine treatment was not found inhibit and 29 ) . Treatment with benztropine had no effect on caspase - 3 activation on days 2 , 4 , and 6 of differentiation , as cytokine production , as determined by evaluating CD4 + / determined by western blot and immunofluorescence analy IL2 + , CD4 + / IFN - y + , CD4+ / IL - 10 + and CD4 + /TNF - a + T -cell sis (FIG . 32 ) . However , cleaved caspase - 3 was barely populations isolated from drug or vehicle treated normal or 10 detectable in OPCs at any time point in DMSO treated diseased mice (FIGS . 28 and 29 ) . We also tested the effects controls , indicating that failed OPC differentiation does not of benztropine on T -cell dependent responses induced by likely result from stress induced cell death . TNP - LPS and TNP -Ficoll and on T -cell independent For the treatment ofMS , an OPC differentiation inducing responses produced by TNP- KLH . Benztropine had no drug would most likely be introduced clinically as part of a effect on IgG and IgM production in response to any of these 15 combination therapy with an immunosuppressive drug . antigens (FIG . 30 ) . Using the PLP induced EAE model, we therefore evaluated We further evaluated the ability of benztropine to induce the clinical efficacy of benztropine when combined with OPC differentiation and enhance remyelination in vivo using either of two immunomodulating drugs approved for the the T -cell independent cuprizone - induced model of demy treatment of MS, interferon - ß and FTY720 (L. Kappos et al. , elination . In this model , mice are fed the copper chelatorbis 20 N Engl J Med , 355 , 1124 ( 2006 ) ; M. Fujino et al ., J ( cyclohexylidenehydrazide ) (cuprizone ), which results in Pharmacol Exp Ther, 305 , 70 (2003 ); L. Jacobs et al. , Arch oxidative /nitrative stress that causes mitochondrial dysfunc Neurol, 39 , 609 (1982 ); M. Huber et al. , J Neurol, 235 , 171 tion and leads to CNS demyelination ( P. Mana et al. , Am J ( 1988 ) ) . The former reduces T -cell proliferation and alters Pathol, 168 , 1464 ( 2006 ) ; M. Lindner et al ., Glia , 56 , 1104 cytokine expression ( A. Noronha, A. Toscas, M. A. Jensen , ( 2008 ) ; P.Mana et al. , J Neuroimmunol, 210 , 13 ( 2009 ) ; L. 25 J Neuroimmunol, 46 , 145 ( 1993) ; A. Noronha , A. Toscas, M. Liu et al. , Nat Neurosci, 13 , 319 ( 2010 ) ; A. J. Steelman , J. A. Jensen , Ann Neurol, 27 , 207 ( 1990 )) , while the latter is an P. Thompson , J. Li, Neurosci Res, 72 , 32 (2012 ) ) . The S1P agonist that regulates T -cell trafficking (V. Brinkmann et demyelinated lesions observed in this model are reminiscent al. , J Biol Chem , 277 , 21453 (2002 ) ; V. Brinkmann et al . , of pattern III MS lesions and involve minimal contribution Nat Rev Drug Discov, 9 , 883 (2010 )) . We determined from hematogenous immune cells (L. Liu et al. , Nat Neu- 30 whether the combination of an OPC inducing drug with rosci, 13 , 319 (2010 ); C. Lucchinetti et al. , Ann Neurol 47 , either interferon - B or FTY720 would improve efficacy and / 707 ( 2000 ); L. T. Remington et al. , Am J Pathol, 170 , 1713 or decrease the dose of the immunosuppressive agent (2007 ) ) . Inclusion of 0.2 % ( w / v ) cuprizone in the diet of required to achieve maximal benefit . Initially , all three drugs C57BL /6 mice induces a demyelination program that pro were dosed individually over a range of concentrations, to ceeds with a defined series of events over a characteristic 35 determine suboptimal and maximal effective / tolerated doses time course , wherein the corpus callosum shows peak demy of each in the models (FIGS . 25 and 33 A , B ) . Benztropine elination following 6-7 weeks of feeding ( T. Skripuletz et was then administered with either of two existing immune al. , Am J Pathol, 172 , 1053 ( 2008 )) . Spontaneous remyeli modulating treatments for MS, and as described below , these nation is observed 2-4 weeks following cuprizone with combinations resulted in significant benefits in the EAE drawal ( T. Skripuletz et al. , Am J Pathol, 172 , 1053 (2008 ) ) . 40 model. Addition of 2.5 mg/ kg benztropine to 1 mg/ kg By administering drugs at the time when a cuprizone - free FTY720 ( FIG . 10A ) or 10,000 U /mouse interferon - B ( FIG . diet is reintroduced , the efficacy of promyelinating agents 10B ) resulted in a significant decrease in observed clinical can be examined by evaluating the relative kinetics of OPC severity, as compared to that observed when either FTY720 dependent remyelination (T. Skripuletz et al ., Am J Pathol, or interferon - ß was dosed alone. Further , the combination of 172, 1053 (2008 )) . Following 7 weeks of exposure , upon 45 2.5 mg/ kg benztropine with a suboptimal dose of FTY720 withdrawal of cuprizone , we administered benztropine ( 10 (0.1 mg/ kg ) resulted in a significant decrease in clinical mg/ kg ) intraperitoneally to 15 week old C57BL /6 mice. severity that was greater than that observed when either drug Drug or vehicle treated mice were sacrificed in groups of 5 was dosed alone and was comparable to the clinical efficacy weekly for 5 weeks , following drug treatment , and remy observed when FTY720 was dosed alone at near the maxi elination was quantitatively evaluated by staining the corpus 50 mal observed tolerated dose of 1 mg/ kg (FIG . 10C ). This callosum regions ofharvested brains with LFB (FIG .9A , B ). observation may prove clinically relevant as FTY720 treat Significant demyelination was clearly observed following ment is associated with bradycardia which is dose depen seven weeks of treatment with cuprizone , as compared to dent . control animals . Consistent with an enhancement of OPC We have identified a centrally acting FDA approved drug differentiation and accelerated remyelination , a significant 55 that, when dosed alone in themost clinically relevantmodel increase in myelin staining in the corpus callosum was of MS , significantly decreases disease severity . Benztropine observed at week 2 following treatment with benztropine, as enhances remyelination , leading to functional recovery, by compared to the spontaneous remyelination observed in directly stimulating the differentiation of OPCs by a mecha vehicle controls (FIG . 9A , B ) . As expected , at later time nism involving antagonism ofM2 / M3 muscarinic receptors points spontaneous remyelination was relatively complete 60 expressed on immature OPCs. Evidence that this drug and significant differences were not observed between drug functions by directly stimulating remyelination , and not by and vehicle treated animals . A lack of difference at these inhibiting demyelination , is provided by the observed lack later time points indicates that , even following 5 weeks of of effect of benztropine on in vitro and in vivo T - cell biology treatment at efficacious doses, benztropine is not toxic to and by the observed promyelinating activity of benzotropine mature oligodendrocytes . These data again suggest that 65 in the T- cell independent cuprizone -induced model of demy benztropine enhances the process of in vivo remyelination , elination . Inclusion of benztropine in treatment regimens by directly inducing OPC differentiation . Benztropine could involving existing approved immunomodulatory drugs US 10,493,080 B2 87 88 resulted in enhanced functional recovery and significantly elinating disease , transverse myelitis , Devic's disease , pro decreases the dosages of the latter that are required to gressive multifocal leukoencephalopathy, optic neuritis , leu achieve an equivalent level of efficacy. To our knowledge , kodystrophy , Guillain - Barre syndrome, chronic these results provide the first in vivo evidence supporting the inflammatory demyelinating polyneuropathy, autoimmune notion that a benefit can be achieved by treating MS- like 5 peripheral neuropathy, Charcot- Marie - Tooth disease , acute symptoms using the combination of an immunomodulator disseminated encephalomyelitis , adrenoleukodystrophy, with a remyelination enhancer . adrenomyeloneuropathy , Leber's hereditary optic neuropa It is understood that the examples and embodiments described herein are for illustrative purposes only and that thy , or HTLV - associated myelopathy . various modifications or changes in light thereof will be 10 5. The method of claim 4 , wherein the demyelinating suggested to persons skilled in the art and are to be included disease is multiple sclerosis . within the spirit and purview of this application and scope of 6. The method of claim 1 , wherein the subject has been the appended claims. All publications , patents , and patent administered an immunomodulatory agent. applications cited herein are hereby incorporated by refer 7. The method of claim 6 , wherein the immunomodula ence in their entirety for all purposes . 15 tory agent is fingolimod (FTY720 ) , interferon beta - la , inter What is claimed is : feron beta - 1b , glatiramer acetate , mitoxantrone, or natali 1. A method of stimulating increased myelination of zumab . nerves in a human subject having a demyelinating disease , 8. The method of claim 2 , wherein the demyelinating the method comprising: disease is multiple sclerosis , idiopathic inflammatory demy administering to the subject a muscarinic receptor antago- 20 elinating disease , transverse myelitis , Devic's disease, pro nist as a neurotransmitter receptor modulating agent in gressive multifocal leukoencephalopathy, optic neuritis , leu an amount sufficient to stimulate oligodendrocyte pre kodystrophy, Guillain - Barre syndrome, chronic cursor cell differentiation in the subject, thereby stimu inflammatory demyelinating polyneuropathy , autoimmune lating increased myelination of nerves in the subject, peripheral neuropathy, Charcot- Marie - Tooth disease, acute wherein the muscarinic receptor antagonist is selected 25 disseminated encephalomyelitis , adrenoleukodystrophy, from benztropine, carbetapentane , clemastine , and salts adrenomyeloneuropathy, Leber's hereditary optic neuropa thereof. thy, or HTLV -associated myelopathy. 2. A method of stimulating increased myelination of 9. The method of claim 8 , wherein the demyelinating nerves in a human subject having a demyelinating disease , disease is multiple sclerosis . the method comprising : 30 administering to the subject a dopamine receptor antago 10. The method of claim 2 , wherein the subject has been nist as a neurotransmitter receptor modulating agent in administered an immunomodulatory agent. an amount sufficient to stimulate oligodendrocyte pre 11. The method of claim 10 , wherein the immunomodu cursor cell differentiation in the subject, thereby stimu latory agent is fingolimod (FTY720 ), interferon beta -la , lating increased myelination of nerves in the subject , 35 interferon beta - 1b , glatiramer acetate , mitoxantrone, or wherein the dopamine receptor antagonist is selected from natalizumab . benztropine , GBR12935 , trifluoperazine , and salts 12. The method of claim 3 , wherein the demyelinating thereof. disease is multiple sclerosis, idiopathic inflammatory demy 3. A method of stimulating increased myelination of elinating disease, transverse myelitis , Devic's disease, pro nerves in a human subject having a demyelinating disease , 40 gressive multifocal leukoencephalopathy, optic neuritis , leu the method comprising: kodystrophy, Guillain -Barre syndrome, chronic administering to the subject a neurotransmitter receptor inflammatory demyelinating polyneuropathy , autoimmune modulating agent selected from the group consisting of peripheral neuropathy, Charcot- Marie - Tooth disease , acute a histamine receptor antagonist, a beta adrenergic disseminated encephalomyelitis , adrenoleukodystrophy , receptor antagonist, and an opioid receptor modulator 45 adrenomyeloneuropathy , Leber's hereditary optic neuropa in an amount sufficient to stimulate oligodendrocyte thy , or HTLV -associated myelopathy . precursor cell differentiation in the subject , thereby 13. The method of claim 12 , wherein the demyelinating stimulating increased myelination of nerves in the disease is multiple sclerosis . subject , 14. The method of claim 3 , wherein the subject has been wherein the histamine receptor antagonist is clemastine or 50 administered an immunomodulatory agent. a salt thereof, or 15. The method of claim 14 , wherein the immunomodu wherein the beta adrenergic receptor antagonist is salbu latory agent is fingolimod (FTY720 ), interferon beta -la , tamol or a salt thereof, or interferon beta - 1b , glatiramer acetate , mitoxantrone , or wherein the opioid receptor modulator is carbetapentane natalizumab . or a salt thereof. 55 16. The method of claim 1 , wherein the muscarinic 4. The method of claim 1 , wherein the demyelinating receptor antagonist is selected clemastine or a salt thereof. disease is multiple sclerosis , idiopathic inflammatory demy