sign in sign up
<<
Home , Amibegron, Amidephrine, Anisodamine, Anisodine, Betazole, Burimamide

USO09592288B2

(12) United States Patent (10) Patent No.: US 9,592.288 B2 Schultz et al. (45) Date of Patent: Mar. 14, 2017

(54) DIRECTED DIFFERENTATION OF (56) References Cited OLGODENIDROCYTE PRECURSOR CELLS TO A MYELINATING CELL FATE U.S. PATENT DOCUMENTS 7.301,071 B2 * 1 1/2007 Zheng ...... CO7K 14,415 (75) Inventors: Peter Schultz, La Jolla, CA (US); Luke 435/320.1 Lairson, San Diego, CA (US); Vishal 2003,0225.072 A1 12/2003 Welsh et al. 2006/0258735 A1 11/2006 Meng et al. Deshmukh, La Jolla, CA (US); Costas 2009. O1552O7 A1 6/2009 Hariri et al. Lyssiotis, Boston, MA (US) 2010, O189698 A1 7, 2010 Willis (73) Assignees: The Scripps Research Institute, La FOREIGN PATENT DOCUMENTS Jolla, CA (US); IRM LLC, Hamilton WO 2009/068668 A1 6, 2009 (BM) WO 2009,153291 A1 12/2009

(*) Notice: Subject to any disclaimer, the term of this OTHER PUBLICATIONS patent is extended or adjusted under 35 U.S.C. 154(b) by 445 days. Mendes et al. “Classical immunomodulatory therapy in multiple sclerosis' Arq Neuropsiquiatr, 2011, vol. 69, No. 3, pp. 536-543.* The TMA. “Animal Models to Study NMO and TM, Nov. 23, (21) Appl. No.: 13/985,342 2015. https://myelitis.org/animal-models-to-study-nmo-and-tim?, printed Jul. 14, 2016, pp. 1-7.* Tian et al. “Neuropathic Pain in Animal Models of Nervous System (22) PCT Fed: Feb. 17, 2012 Autoimmune Diseases' Mediators of Inflammation, 2013, pp. 1-13. Kraker et al. “Autoimmune Neuromuscular Disorder'. Current (86) PCT No.: PCT/US2012/O25712 Neuropharmacology, 2011, vol. 9, pp. 400-408.* Lehmann et al. "Pathogenesisi and treatment of immune-mediated S 371 (c)(1), neuropathies' Therapeutic Advances in Neurological Disorders, (2), (4) Date: Oct. 18, 2013 2009, vol. 2, issue 4, pp. 261-281.* Walgaard et al. "Emerging for Guillain-Barre syndrome' PCT Pub. No.: WO2O12A112933 Expert Opinion of Emerging Drugs, Feb. 25, 2011, vol. 16, issue 1. (87) pp. 105-120.* PCT Pub. Date: Aug. 23, 2012 Collongues et al. “Current and future treatment approaches for neuromyelitis optica. Therapeutic Advances in Neurological Dis orders, 2011, vol. 4, issue 2, pp. 111-121.* (65) Prior Publication Data Oh et al. Treatment of HTLV-1-Associated Myelopathy/Tropical US 2014/OO38949 A1 Feb. 6, 2014 Spastic Paraparesis: Towards Rational Targeted Therapy, Neurol Clin., 2008, vol. 26, issue 3, pp. 781-796 (printed as 1-15).* Alexander et al. "Acute disseminating encephalomyelitis: Treatment guidelines' Ann Indian Acad Neurol, 2011, vol. 14, suppl1, S60 Related U.S. Application Data S64 (printed as pp. 1-12).* Chang et al., “Premyelinating Oligodendrocytes in Chronic Lesions (60) Provisional application No. 61/444,666, filed on Feb. of Multple Sclerosis.” N Engl J Med, 2002, vol. 346, 165. 18, 2011. Chari et al., “Efficient Recolonisation of Progenitor-Depleted Areas of the CNS by Adult Oligodendrocyte Progenitor Cells.” Glia, 2002, 37: 307-313. (51) Int. C. Chariet al., “Dysfunctional Oligodendrocyte Progenitor Cell (OPC) A6 IK3I/545 (2006.01) Populations May Inhibit Repopulation of OPC Depleted Tissue.” J. A6 IK3I/35 (2006.01) Neurosci Res, 2003, 73: 787-793. A6 IK3I/37 (2006.01) A6 IK3I/38 (2006.01) (Continued) A6 IK 3/40 (2006.01) Primary Examiner — Kendra D Carter A6 IK 3/439 (2006.01) (74) Attorney, Agent, or Firm — Kilpatrick Townsend & A 6LX 39/395 (2006.01) Stockton LLP A6 IK 38/2 (2006.01) (52) U.S. C. (57) ABSTRACT CPC ...... A61K 39/3955 (2013.01); A61 K3I/I35 The present invention provides methods of inducing differ (2013.01); A61 K3I/137 (2013.01); A61 K entiation of oligodendrocyte progenitor cells to a mature 3 1/138 (2013.01); A61K 31/40 (2013.01); myelinating cell fate with a modu A61K 31/439 (2013.01); A61K 3.1/5415 lating agent. The present invention also provides methods of (2013.01); A61K 38/215 (2013.01) stimulating increased myelination in a subject in need (58) Field of Classification Search thereof by administering said neurotransmitter receptor CPC ...... A61K 3.1/5415: A61K 31/135; A61 K modulating agent. Methods of treating a Subject having a 3 1/137; A61K 31/138: A61K 31/40: demyelinating disease using a neurotransmitter receptor A61K 31/439; A61K 38/215: A61 K modulating agent are also provided. 39/3955 See application file for complete search history. 14 Claims, 52 Drawing Sheets US 9,592.288 B2 Page 2

(56) References Cited tion'. International Review of Neurobiology, 2009, vol. 07, pp. 295-312, DOI: 10.1016. Butt et al., “Neurotransmitter-Mediated Calcium Signalling in OTHER PUBLICATIONS Oligodendrocyte Physiology and Pathology.” Sep. 2006, Wiley InterScience, pp. 665-675. Chong et al., “Tapping into the glial reservoir: cells committed to Bangham et al., HTLV-1-associated myelopathy?tropical spastic remaining uncommitted,” J Cell Biol 2010, 188(3), pp. 305-312. paraparesis. Nature Reviews, Article No. 15012, Published online: De Angelis et al., “Muscarinic Receptor Subtypes as Potential doi:10 1038/nrpd.2015.12, 16 pages, Jun. 18, 2015. Targets to Modulate Oligodendrocyte Progenitor Survival, Prolif Guerreiro et al., Levels of serum chemokines discriminate clinical eration, and Differentiation, Dev Neurobiol, 2012, 72: 713-728. myelopathy associated with human T lymphotropic virus type 1 Gobert et al., "Convergent functional genomics of oligodendrocyte (HTLV-1)/tropical spastic paraparesis (HAM/TSP) disease from differentiation identifies multiple autoinhibitory signaling circuits.” HTLV-1 carrier state. Clinical and Experimental Immunology, Molecular and Cellular Biology, 2009, 29:1538. 145:296-301, 2006. Joubert et al., "Chemical inducers and transcriptional markers of Linker and Lee, Models of autoimmune demyelination in the central oligodendrocyte differentiation.” Journal of Neuroscience Research, nervous system: on the way to translational medicine. Experimental 2010, 88: 2546-2557. & Translational Stroke Medicine, 1(5):10 pages, 2009. Kremer et al., “The Complex World of Oligodendroglial Differen Mayo et al., The Innate immune system in demyelinating disease. tiation Inhibitors.” Ann Neurol, 2011, 69: 602-618. Immunol Review, 248(1): 170-187, 2012. Kuhlmann et al., “Differentiation block of oligodendroglia progeni Merrill. In Vitro and In Vivo pharmacological models to access tor cells as a cause for remyelination failure in chronic multiple demyelination and remyelination. Neuropsychopharmacology sclerosis, , 2008, 131, 1749-1758. Reviews, 34:55-73, 2009. Patel et al., “Mediators of oligodendrocyte differentiation during Rangachari and Kuchroo. Using EAE to better understand prin remyelination.” FEBS Lett, 2011, 585, 3730-3737. ciples of immune function and autoimmune pathology. J. Autoim Stevens et al., “Adenosine: A Neuron-Glial Transmitter Promoting mun., 45:31-39, 2013. Myelination in the CNS in Response to Action Potentials.” Neuron, Reeves and Swenson, Disorders of the Nervous System. Chapter 23, Dec. 2002, vol. 36(5), pp. 855-868. “Demyelinating diseases of the nervous system.” reprinted from the Wolswijk, "Chronic Stage Multiple Sclerosis Lesions Contain a internet: http//www.dartmouth.edu/~dons/part3/chapter 23.htm Relatively Quiescent Population of Oligodendrocyte Precursor Apr. 27, 2016, 7 pages. Cells,” J. Neurosci, 1998, 18(2), pp. 601-609. Spalice et al., Clinical and pharmacological aspects of inflammatory PCT application No. PCT/US 12/25712, International Search Report demyelinating diseases in childhood: An update. Current and Written Opinion, 6 pages. Neuropharmacology, 8:135-148, 2010. Deshmukh et al., “A regenerative approach to the treatment of Steinman, Assessment of animal models for MS and demyelinating multiple sclerosis.” Nature, Oct. 2013, vol. 502(7471), pp. 327-332, disease in the design of rational therapy. Neuron, 24:511-5.14, 1999. DOI: 10.1038/nature 12647. Swanborg, Short Analytical Review. Animal models of human Stangel et al., “Remyelination Strategies: New Advancements disease. Experimental Autoimmune encephalomyelitis in rodents as Toward a Regenerative Treatment in Multiple Sclerosis.” Current a model for human demyelinating disease. Clinical Immunology Neurology and Neuroscience Reports, May 2006, vol. 6(3), pp. and Immunopathology, 77(1):4-13, 1995. 229-235. Khoury et al., “A Randomized Controlled Double-Masked Trial of EP Patent Application No. 12 74.7134, Supplementary Search Albuterol Add-on Therapy in Patients With Multiple Sclerosis.” Report, Jun. 18, 2014, 3 pages. Arch Neurol. 2010:67(9): 1055-1061. Magnaghi et al., “Novel Pharmacological Approaches to Schwann cells as Neuroprotective Agents for Peripheral Nerve Regenera * cited by examiner U.S. Patent Mar. 14, 2017. Sheet 1 of 52 US 9,592.288 B2

Fig. IA

1.s K f -o- -N s a r

Ceaste; Esq: 8 NYf f s-N- 1SF

Berztropine: ECsas 250 V rifluoperazine: Eso's 30 M

1s 2-s! (----->-YN-Y-di loh -N Safeter: Coxe is - OutSa - S-N-ro a s SSR 33: Cses 8 BW U.S. Patent Mar. 14, 2017 Sheet 2 of 52 US 9,592.288 B2

Fig. IB

w ax (Easte (xx (SER 335 rir rifluropera zine - Carbetapentarie A -- Benztropine ? xx Safetail

o re- compound tail

U.S. Patent Mar. 14, 2017. Sheet 3 of 52 US 9,592.288 B2

Fig. 2A

$8 S Š S Š 8 y 4: Carbapentane Sr. 5: Cerasire 6: GBR12935

& U.S. Patent Mar. 14, 2017 Sheet 4 of 52 US 9,592.288 B2

Fig. 2B-C

MBP qRT PCR

U.S. Patent Mar. 14, 2017 Sheet S of 52 US 9,592.288 B2

?uequadeque0

55WIRT U.S. Patent Mar. 14, 2017 Sheet 6 of 52 US 9,592.288 B2

OSWICI

**** U.S. Patent Mar. 14, 2017. Sheet 7 of 52 US 9,592.288 B2

Fig. 5A Mycophenolate 3.

-- - Weisis: H Mycopieiolate

E.

.

, 3 S is 8 it 3 SS F 18 is 324 SS 8. Days U.S. Patent Mar. 14, 2017. Sheet 8 of 52 US 9,592.288 B2

Fig. 5B-C B. 3. Weisie Hierzatropine 2.5

2.

S

S

8. 1 2 3 S S 8 234 SS 118 13:222324 SS 28S 3G 3 Days

Benzatropine + Mycophenolate 3.

--Wsics 2S H. Senzairpire.--&iyeopsis:

. 8

1.

S

8 3 S S 23 as S. S. EEESSE228233 Days U.S. Patent Mar. 14, 2017. Sheet 9 of 52 US 9,592.288 B2

Fig. 5D-E D. 3.

--Wsici; S. -Hirtitioperazirie

S. 8 -

s - a 1.5 - - g H HE 1.0 s -- a

. r 3 & S S : R S S S F 89.325iS 83.333 Days

E. Trifluoperazine + Mycophenolate 3.

-- Weice S -- rifisoperazie--Mycophenoiate

.

1

S

E. 3 S S 6 F S 9 - 23.4 S S ASSESS383 Days U.S. Patent Mar. 14, 2017 Sheet 10 of 52 US 9,592.288 B2

Fig. 6A-B

2 5. Celastine 2 GBR 2935 risticefazine s Cabaretafe . Beizatiojine & Saifeter

o Conc, Carbachoi tail

Saetro, GBR.2935, a rifluoperazine, 3

Benztropine, Carbapentane, Cernastine

U.S. Patent Mar. 14, 2017. Sheet 11 of 52 US 9,592.288 B2

Fig. 7A-C

Benztropi ne 5 uM) Benztropine

B. C. 20

8 Carbachod (Ouri) s OG Carbacho (0.6LA) s 8 Carbachal (4.7ui) g 120 to igg 20 d

15 S. 5

E8S & S. S. PC SO BA H Benztropine (2.3uM) U.S. Patent Mar. 14, 2017. Sheet 12 of 52 US 9,592.288 B2 Fig. 8A-B

A. 5 -e-Weice as Benztropine Therapeutic -- Benztropine Prophylactic 4. -- FTY720 Therapeutic ; as interferon-3 Therapeutic ; 3

2

s

21

St. U.S. Patent Mar. 14, 2017 Sheet 13 Of 52 US 9,592.288 B2

Fig.

U.S. Patent Mar. 14, 2017 Sheet 14 of 52 US 9,592.288 B2

U.S. Patent Mar. 14, 2017. Sheet 15 of 52 US 9,592.288 B2

Fig. 10A-B

A. nee Weice -S FTY720 mg/kg - BA 2.5mg/kg * FTY720 mg/kg + BA 2.5mg/kg

-- Weice ..s. FN-3 OOOOU I - BA 2.5mg/kg i.S. - FN-OOOOU BA 2.5mg/kg

O S-S-S-S-S-S-S - - I - s 5 3. U.S. Patent Mar. 14, 2017 Sheet 16 of 52 US 9,592.288 B2

Fig. 10C-D

5 again Vehicle .- -- FTY720 0.1mg/kg 3.5- w FTY720 mg/kg rBA 2.5mg/kg 3- - 2.5-

S - 5- I 0.0 isssssss- T - 5 S 2 5 3 Days

A5 4.0 ------Weice

i w FN-83000 SS mas FN-B 0000U S. ---w BA 2.5ig/kg

2.

S

O.O. - s S 2 is 3. days U.S. Patent Mar. 14, 2017. Sheet 17 of 52 US 9,592.288 B2

Fig. 10E-F

E. men feice (NFTY720 ingkg me FY720 (.img/kg BA 2.5mg/kg

s S. s s Days

F. i. A. main Weice * it is FN-3 0000 3. eIFN-s30000U t 2.5 E 2.0- 5 .5 Y . s 0.0 essessed ---T----T---- 5 s 25 S. days U.S. Patent Mar. 14, 2017 Sheet 18 of 52 US 9,592.288 B2

Fig. I IA-B

SS Thyro cre PGF

SS s SS S s Oligodendrocyte U.S. Patent Mar. 14, 2017. Sheet 19 of 52 US 9,592.288 B2

Fig. 12A-B

-- Benztropine g 20 EC 350 nt

, . 0.8 . Conc, (ii)

B. Benzopine is

S ES

::::::::::::::::::::::::::::: S: s" Thyroid Hormoner." U.S. Patent Mar. 14, 2017. Sheet 20 of 52 US 9,592.288 B2

Fig. 13

U.S. Patent Mar. 14, 2017 Sheet 21 of 52 US 9,592.288 B2

Fig. 14 Thyroics XV Benztropine Crose

SS. XXXX

R '',

U.S. Patent Mar. 14, 2017. Sheet 22 of 52 US 9,592.288 B2

l. t td

U.S. Patent Mar. 14, 2017. Sheet 23 of 52 US 9,592.288 B2

Fig. 16A-B

A. Benztropinel Thyroid Hormonel OPC/DMSO DMSO DMSO

Apod O.58 10.79 11.21 Enpp6 Mac Mbp Mobp Mog OliC1 Plp Pnlip Tpd52

B. OPC/DMSO Benztropinel Thyroid Hormonel DMSO DMSO CCnd2 1.93 Cited2 0.99 1.10 0.98 E2f1 0.96 0.75 0.80 ECr1 1.19

NotXr SCC1 SOX11 TOO2a 1001.OO U.S. Patent Mar. 14, 2017. Sheet 24 of 52 US 9,592.288 B2

Fig. 17A-B

A. Day 6 Day ( d & '8 & 8 is Day 2 ) $$$$$8 {

Day 3 artia y Ratsie O. Day 4 d $$$$$$$$$8 & Day 5 $$$$88

B. lay d Bay, $$$8

&S$8 : U.S. Patent Mar. 14, 2017 Sheet 25 Of 52 US 9,592.288 B2

Fig. 17C-D

U.S. Patent Mar. 14, 2017. Sheet 26 of 52 US 9,592.288 B2

Fig. 18

S. Caract as

S

Benztropine (2.3M) as ...... Berztropine (2.3.IV)-- aracci s. U.S. Patent Mar. 14, 2017. Sheet 27 of 52 US 9,592.288 B2

Fig. 19A-B

A. * }

8

3wn s is a S

Benztropine 0.8 u?i (i.7 u?i) State 3.5 Histamine {0 uši)

B.

8

36 s E 4 SS 2

Benztropine E0.8 t) HTMF (i.7 usi Hi , HT&T (out) U.S. Patent Mar. 14, 2017. Sheet 28 of 52 US 9,592.288 B2

Fig. 20A-B i 5

Benztropine I0.8 till (4.7 uRI) (Ruinspiroie (0.8 us) Quinpirole (0 tuit)

i 5

Benztropine 0.8 .il/ Haiaperidol (4.7 a M3 Haiaperidod (.6 u} Haiaperidol (Quia) U.S. Patent Mar. 14, 2017. Sheet 29 of 52 US 9,592.288 B2

Fig. 21

-- (-EC: 0.8 unt on (-ECs: 5.7 avi w lpratropium i-ECs: 5.8 u?) to Propiwerine (-EC: 0.42 u} w Scopodamine (-ECs; 5. At

S^SX& * SS Coac. Italy

R2 Receptor

Rii Receptor

A3 Receptor

C

GA U.S. Patent Mar. 14, 2017. Sheet 30 of 52 US 9,592.288 B2

Fig. 23A

A. in titles S 3 st Phospho p38 MAPix ata p38 APK hospha Akt :

sta Akt Phospho p42.f44. MAPK s& sta p42.f44 & s::::::::::::::::::::::::::s yapks Yxx. ext S

Total cREB U.S. Patent Mar. 14, 2017. Sheet 31 of 52 US 9,592.288 B2

Fig. 23B

.3 Cyclin D1 t S. Š 3. ,

S. &

i

.3

s

3. 8.

s

S., s: U.S. Patent Mar. 14, 2017. Sheet 32 of 52 US 9,592.288 B2

Fig. 23C

G-Rotein itar&sitio's

U.S. Patent Mar. 14, 2017. Sheet 33 of 52 US 9,592.288 B2

Fig. 24A-B

a SSS is wr SS-Sit F is

on Cartacial 2 us

S

s : s s Se

a site is m Seitztrupine 20tsi} + Catache ESEER & wer Serztropie Fifi + Cartacha Stuss Yes, - Berztropine 2u5A) + Carbaeho (souri

S

s s : U.S. Patent Mar. 14, 2017. Sheet 34 of 52 US 9,592.288 B2

Fig. 24C-D

N if RSS ^ Atropine 2Citi - Catachat ESGN re. At rapine sii - Cattaches 5(it

|S

s S|

s is Sist

ODMso Forskolin Berztropine

4Gufi 3.3 ul. U.S. Patent Mar. 14, 2017. Sheet 35 of 52 US 9,592.288 B2

Fig. 25

-H Wehicle Saie -E-Benztropine (8.1 mg/kg -- Benztropine (8.5 mg/kg) -e-Benztrepine (2.5 mg/kg) -- Benztropine (2.5 g/kg)

i U.S. Patent Mar. 14, 2017. Sheet 36 of 52 US 9,592.288 B2

Fig. 26

XC Fast Site - U.S. Patent Mar. 14, 2017 Sheet 37 of 52 US 9,592.288 B2

Fig. 27A-D

~~~~); ~*~~~~ ....….…--~~~~* ...... --~~~^^***^ ~~~~);

------s&SSSSSSSSSS3

ºzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzºzzz!

xxx xxxxx xxxxx xxxxx xxxxxx xxxxx xxxxx xxxxx xxxxx xxxxx xxxxx xxxx xxxx xxxxxx xxxxxx xxx xxxxx xxxxx xxxxxxxx xxxxx xxxxx xxxx xxxxx xxxxx xxxxxx xxxxxxxx xxxxx xxxxx xxxx S. 8S.

W ~~~~!) & S ~~~~~~~~~~~~~~~~~~~e,~~~~~~~~~~~~); 8 s

U.S. Patent Mar. 14, 2017. Sheet 38 of 52 US 9,592.288 B2

Fig. 27E-F

S Benztropine U.S. Patent Mar. 14, 2017 Sheet 39 of 52 US 9,592.288 B2

Fig. 28A-F

Total Spienocytes

i.2xio's is 88. Six 8. six 8F g5s if Vehicle Benztropine

r Cat CES

is 8

F.

x8

self

Vehicle Benztropine Vehicie Benztropine

F. CBC is H Ces City as Ces 2S: 18.x if it.

s 23 °F St. .s S. Fs its S. Six it St. 3.5:18 Vehicle Benztropine Weiicle Benztropine U.S. Patent Mar. 14, 2017 Sheet 40 of 52 US 9,592.288 B2

Fig. 28G-K G. Oxf is H. at is 2.8.x if 3.x:

$.5x R s.ax of i x 87 . s.s 3 six is

Vehiclew Benztropine Vehicie Benztropine . .x: Cisit Ces CD4AFNyt Cells 2. Exit

1.5x to SS s s e 1.0x to 7 text of i Six 8.a. Six 38

: Vehicle Berztrepine Vehicle Beaztropine

U.S. Patent Mar. 14, 2017 Sheet 41 of 52 US 9,592.288 B2

Fig. 29A-F

A. Total Splenocytes

7w 5.3. 8): f . sex of ...sixf

Vehicle Benztropine Weihiele Berztropine

C-8 Ces

C4 Celis

s X

S. X 8. s

3. 8 8. ru S S.

Veiicie Berztropine Wehicle Benztropine

Catfás titles Caifa Hi Ces E. 3333333

8:38. 3.

333

Weaicle Berztropire Wehicle Berztropine U.S. Patent Mar. 14, 2017 Sheet 42 of 52 US 9,592.288 B2

Fig. 29G-J

G. as to (Caf2 Celis H. CD4*IL10 Celis

08

o f

Vehicle Benztropine Vehicle Benztropine

CAiif NFt Ces J CD4+/FNyt Cells 2.5.x if s.3.x if 3.x if 20x1097 8x if 21S. ran 7 a 1.5x10° s1.5x10 Saxtotif x & x is st.ox of is 10x10 K. f.5x8 5.8 x 388 5.8x 36 5x8 . i. Vehicle Benztropine Vehicle Berztropine U.S. Patent Mar. 14, 2017. Sheet 43 of 52 US 9,592.288 B2

Fig. 30A-C A. ign. TNP-LPS - - Weirii - Berzt. Opine

Days . 8 B IgG TNP-LPS SS -- Weice s - Besztropire 258

s. s

S.

...... 8 Days C gM TNP-Ficoi -- Weihsie - Benztrupiae 3000 200

.5 Days U.S. Patent Mar. 14, 2017. Sheet 44 of 52 US 9,592.288 B2 Fig. 30D-F igG TNP-Ficoi D. 3. -- Whe - Berzi'opine S

G

Day. E IgM TNP-KLH

-- Weice - Benztropine FS E 58 r

25

t s 25. Days F gG TNP-KLH Fs -- Weisie - Bezi opine 5000

as

25 U.S. Patent Mar. 14, 2017. Sheet 45 of 52 US 9,592.288 B2

Fig. 3 IA-D

s

to S ist 200

S.

S U.S. Patent Mar. 14, 2017 Sheet 46 of 52 US 9,592.288 B2

Fig. 32

Ceawed Caspase 3

Acti U.S. Patent Mar. 14, 2017. Sheet 47 of 52 US 9,592.288 B2

Fig. 33A-B

A. s s s -- ehicise Salise s St BA 1 Girg?kg - BA 2.5rigg 3. s 3.

N. s

s s s 3. lays

-E- Wehicle Sisie

- N IFN-S iOOOGU - FN-B 3000

S U.S. Patent Mar. 14, 2017. Sheet 48 of 52 US 9,592.288 B2

Fig. 33C-D

-E- elicise Scies “St. BA 2.5iigikg + FTY ingkg & BA 2.5mg/kg FY S. gig : “r BA 2.5mgkg FYS.iiigikg

D. s fe exie Site

-N BA 2.5 g/kg - FN-S i(SOU s -- SA 2.5irgig - FN- 3000 se S U.S. Patent Mar. 14, 2017. Sheet 49 of 52 US 9,592.288 B2

Fig. 33E-F -- faii SS rS. FTY.0 mg/kg s' BA 2.5irgkg BA 2.5ingkg + FTY G.O img/kg

S.

. s

s S. s Sys

-d- is SS

Ne FN-B iOOOU r^ SA 2.5iigikg BA 2.5 gfxg

$& i-ra-i-, -, -- s s s says U.S. Patent Mar. 14, 2017. Sheet 50 of 52 US 9,592.288 B2

Fig. 34A-B

-- Vehicle (Saline) -O- Benztropine Therepeutic 3 N

2

OH O 5 10 15 20 25 30 Days

B. 4. -- Vehicle f -O- Benztropine Prophylactic 9. 8 3 V C) N

s y N al O 2 N a Nurs M w 5

s 1

0 T51o 15 20 25 30 Days U.S. Patent Mar. 14, 2017. Sheet 51 of 52 US 9,592.288 B2

Fig. 34C-D C. 5 -- Vehicle -O- Benztropine 0.1 mg/kg 24 -H Benztropine 0.5 mg/kg 8 - A - Benztropine 2.5 mg/kg D \ -- Benztropine 12.5 mg/kg I 3 N 9. 2

t O) E

O O 5 30 Days

D 3.5 -- Vehicle 3.0 -O- Trifluoperazine 32.5 C 2.0

9 E1.5 al O H H7 1.0 VHH 0.5 St. N7 O.OH O 5 20 25 30 U.S. Patent Mar. 14, 2017 Sheet 52 of 52 US 9,592.288 B2

Fig. 34E-F

-- Vehicle -O-

O.1.1.2.2.3.3.0. 5O5O5O5O 510 15 20 25 30 Days

- Vehicle -O-

0. O 0.1.1.2.2.3.3. 5O5O5O5 5 10 15 20 25 3O Days US 9,592,288 B2 1. 2 DIRECTED DIFFERENTATION OF Blakemore, Glia, 37, 307 (2002); D. M. Chari et al., J OLGODENIDROCYTE PRECURSORCELLS Neurosci Res, 73,787 (2003); G. Wolswijk, J Neurosci, 18, TO A MYELINATING CELL FATE 601 (1998); A. Chang et al., N Engl J Med, 346, 165 (2002); T. Kuhlmann et al., Brain, 131, 1749 (2008)). CROSS-REFERENCES TO RELATED There is no known cure for MS. For treating acute APPLICATIONS inflammatory attacks, intravenous corticosteroids are typi cally administered. Other treatments for MS involve the The present application is a national stage application administration of an immunomodulator. Although immuno under 35 U.S.C. 371 of International Application No. PCT/ modulators are able to reduce the frequency and severity of US 12/25712, filed Feb. 17, 2012, which claims benefit of 10 attacks or accumulation of lesions, they do not promote priority to U.S. Provisional Application No. 61/444,666, remyelination of damaged axons. filed Feb. 18, 2011, the entire content of each of which is incorporated by reference in its entirety. BRIEF SUMMARY OF THE INVENTION

BACKGROUND OF THE INVENTION 15 In one aspect, the present invention provides for methods of stimulating increased myelination of nerves in a subject Multiple sclerosis (MS) afflicts approximately 400,000 in need thereof. In some embodiments, the method com people in the United States and 2.5 million worldwide. MS prises administering to the Subject a therapeutically effective is an inflammatory disease in which myelin sheaths around dose of a neurotransmitter receptor modulating agent the axons of the brain and spinal cord are damaged. In MS selected from a muscarinic , a as well as other demyelinating diseases, autoimmune receptor antagonist, a antagonist, a beta inflammatory attack against myelin and oligodendrocytes , and an receptor causes demyelination. The thinning or loss of myelin Sur modulator; thereby stimulating increased myelination of rounding axons impairs the ability of the axons to effectively nerves in the subject. conduct signals and results in progressive neuronal damage. 25 In another aspect, the present invention provides for Remyelination is the process by which new myelin methods of treating a subject having a demyelinating dis sheaths are generated around axons. Remyelination persists ease. In some embodiments, the method comprises admin throughout adulthood in the CNS and involves the genera istering to the subject a therapeutically effective dose of a tion of new myelinating oligodendrocytes (C. Ffrench neurotransmitter receptor modulating agent selected from a Constant, M. C. Raff, Nature, 319, 499 (1986)). Despite 30 muscarinic receptor antagonist, a antago controversy regarding their intrinsic in vitro and in vivo nist, a histamine receptor antagonist, a beta adrenergic lineage potential (M. C. Nunes et al., Nat Med, 9, 439 receptor modulator, and an modulator; (2003); S. Belachew et al., J Cell Biol, 161, 169 (2003); T. thereby treating the demyelinating disease. Kondo, M. Raff, Science, 289, 1754 (2000); Jackson, 2006: In yet another aspect, the present invention provides for Zhu, 2011; Richardson, 2011; R. J. Franklin, C. Ffrench 35 methods of enhancing the therapeutic effect of an immuno Constant, Nat Rev Neurosci, 9, 839 (2008)), compelling modulatory agent in a Subject in need thereof. In some evidence indicates that a widespread proliferating popula embodiments, the method comprises administering to the tion of nerve/glial antigen-2 (NG2), platelet-derived growth Subject the immunomodulatory agent and a neurotransmitter factor receptor (alpha subunit, PDGFRa) positive cells, receptor modulating agent; thereby enhancing the therapeu termed NG2-glia or oligodendrocyte precursor cells (OPCs), 40 effect of the immunomodulatory agent. are the major source of newly formed mature oligodendro In some embodiments, the neurotransmitter receptor cytes required for remyelination (P. J. Homer et al., J modulating agent is a muscarinic receptor antagonist. In Neurosci, 20, 2218 (2000); M. C. Nunes et al., Nat Med, 9, Some embodiments, the muscarinic receptor antagonist is a 439 (2003); J. M. Gensert, J. E. Goldman, Neuron, 19, 197 muscarinic receptor modulator compound listed in Table 1. (1997); M. S. Windrem et al., Nat Med, 10, 93 (2004); R. J. 45 In some embodiments, the muscarinic receptor is selected Franklin, C. Ffrench-Constant, Nat Rev Neurosci, 9, 839 from benztropine, carbetapentane, clemastine, ipratropium, (2008); Richarson, 2011). atropine, and salts thereof. Remyelination can occur following the loss of myelin in In some embodiments, the neurotransmitter receptor diseases such as MS, thus restoring neurological function to modulating agent is a dopamine receptor antagonist. In some axons. However, although remyelination can occur in the 50 embodiments, the dopamine receptor antagonist is a dop early stages of MS, oligodendrocytes are unable to com amine receptor modulator compound listed in Table 1. In pletely rebuild the myelin sheath, and repeated inflammatory Some embodiments, the dopamine receptor antagonist is attacks ultimately lead to fewer effective remyelinations selected from benztropine, GBR12935, trifluoperazine, and until plaques build up around the damaged axons. A primary salts thereof. cause of remyelination failure is the progressive inability of 55 In some embodiments, the neurotransmitter receptor Somatic oligodendrocyte precursor cells to differentiate at modulating agent is a histamine receptor antagonist. In some the sites of injury. Thus, remission in MS is largely depen embodiments, the histamine receptor antagonist is a hista dent upon OPCs migrating to sites of injury, and Subse mine receptor modulator compound listed in Table 1. In quently differentiating to a mature cell fate capable of repair Some embodiments, the histamine receptor antagonist is (J. R. Patel, R. S. Klein, FEBS Lett, 585, 3730 (2011); D. 60 clemastine or a salt thereof. Kremer et al., Ann Neurol, 69, 602 (2011); A. Chang et al., In some embodiments, the neurotransmitter receptor NEngll Med, 346, 165 (2002)). Studies aimed at evaluating modulating agent is a beta modulator. In the presence and relative densities of OPCs at sites of Some embodiments, the beta adrenergic receptor modulator chronically demyelinated MS lesions indicate that it is not a is a beta adrenergic receptor modulator compound listed in failure of repopulation or migration of OPCs, but rather 65 Table 1. In some embodiments, the beta adrenergic receptor inhibition of OPC differentiation at sites of injury that modulator is selected from , , Salbutamol. contributes to disease progression (D. M. Chari, W. F. albuterol, and salts thereof. US 9,592,288 B2 3 4 In some embodiments, the neurotransmitter receptor method comprises administering the neurotransmitter recep modulating agent is an opioid receptor modulator. In some 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 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 10 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, 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 15 a neurotransmitter receptor modulating agent as described mesylate). herein and an immunomodulatory agent as described herein. In some embodiments, the Subject has a demyelinating 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 modulator, and an multifocal leukoencephalopathy, optic neuritis, leukoystro opioid receptor modulator. In some embodiments, the neu phy, Guillain-Barre Syndrome, chronic inflammatory demy 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 25 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 (FTY720), interferon beta-1a, interferon beta-1b, 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 30 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 fingolimod (FTY720), interferon beta-1a, 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 relapsing multiple scle 35 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 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 40 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-1a, interferon beta-1b, glati 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-1a, or interferon beta-1b. 45 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 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 50 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 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 55 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 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 60 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 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 Some embodiments, the method comprises administering the 65 tified as a neurotransmitter receptor modulating agent (e.g., neurotransmitter receptor modulating agent and the immu a compound described in Table 1 herein), it is also contem nomodulatory agent sequentially. In some embodiments, the plated that any pharmaceutically acceptable salts, , US 9,592,288 B2 5 6 racemic mixtures, conformational and/or optical isomers, nated encephalomyelitis, adrenoleukodystrophy, adrenomy crystalline polymorphs and isotopic variants of the com 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 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 10 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 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 15 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 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-C. 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), 23'-cyclic stabilizing compounds, addressable compounds, and other nucleotide 3' phosphodiesterase (CNP), galactocebroside functional moieties. Conventionally, compounds are (GalC). O1 antigen (O1), or O4 antigen (O4). screened by identifying a test compound (called a "screening As used herein, the terms “stimulating increased myeli 25 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, 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 “” refers to an agent that stimulates, increases, nating cell fate, as compared to the amount of myelin 30 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 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 35 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 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 40 amount or dose' or “therapeutically sufficient amount or the agent. The amount of 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 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 or more will depend on the purpose of the treatment, and will be characteristics of a demyelinating disease (e.g., multiple 45 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 of OPCs to a mature myelinating (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 50 of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, in a Subject having multiple Sclerosis when the frequency Williams & Wilkins). and/or severity of inflammatory attacks decreases Subse 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. 55 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 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 60 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 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 leukoystrophy, Guillain-Barre Syndrome, chronic inflamma 65 making the symptom, injury, pathology or condition more tory demyelinating polyneuropathy, autoimmune peripheral tolerable to the patient; decreasing the frequency or duration neuropathy, Charcot-Marie-Tooth disease, acute dissemi of the symptom or condition; or, in some situations, pre US 9,592,288 B2 7 8 venting the onset of the symptom or condition. The treat pound treatment of OPCs at EC concentrations for 6 days ment or amelioration of symptoms can be based on any 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 for galactocereberoside (GalC), oligodendrocyte marker O1 actual level of pharmacologically active agent in a subject, (O1) and oligodendrocyte marker O4 (O4). 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 10 ECoo 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 FIG. 5. Representative neurotransmitter receptor modu by a person of skill, for example, doses for a pharmacologi lating agents benzatropine and trifluoperazine ameliorate cal agent published in the Physicians’ Desk Reference, 66th symptoms in a PLP induced relapsing EAE mouse model. Ed., 2012, PDR Network, LLC; or Brunton, et al., Goodman 15 SJL mice were immunized with proteolipid peptide (PLP) & Gilman's The Pharmacological Basis of Therapeutics, and pertussis toxin and monitored daily with scoring on the 12th edition, 2011, McGraw-Hill Professional) when admin standard clinical EAE scale (0-5). Compounds were admin istered on its own. A “subtherapeutic dose” can be defined istered daily for the duration of the study in saline at 10 in relative terms (i.e., as a percentage amount (less than mg/kg via intra-peritoneal injection (0.1 ml), starting on the 100%) of the amount of pharmacologically active agent day of the appearance of clinical EAE Symptoms (day 10). conventionally administered). For example, a subtherapeutic Sub-optimal dosing of mycophenolate mofetil at 20 mg/kg dose amount can be about 1% to about 75% of the amount in sterile saline (pH 5) was administered in combination of pharmacologically active agent conventionally adminis with benzatropine and trifluoperazine respectively, starting tered. In some embodiments, a Subtherapeutic dose can be on the day of peak clinical EAE Symptoms (day 14) post about 75%, 50%, 30%, 25%, 20%, 10% or less, of the 25 PLP injection. Mean clinical EAE score and standard error amount of pharmacologically active agent conventionally of mean (SEM) are shown for each study group. Mice in the administered. vehicle control group, n=6 (FIG. 5A-E, open boxes) show appearance of acute phase (mean maximal clinical EAE BRIEF DESCRIPTION OF THE DRAWINGS score of 2+0.8 on day 11) followed by remission (mean 30 maximal clinical EAE score of 0.3-0.3 on day 19) and FIG.1. Neurotransmitter receptor modulating agents dose relapse of EAE symptoms (mean maximal clinical EAE dependently induce OPC differentiation. (A) Chemical 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 ECso values were determined severity of relapse (mean maximal EAE score 0.7+0.3 on using appropriate Graphpad Prism 5.0 curve fitting software. 35 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 show a significant reduction in the severity of relapse (mean in OPC media containing basal PDGF-C.C. (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 40 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 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 values imaging system. (C) Maximal OPC differentiation inducing <0.001). activity of identified compounds compared to DMSO con 45 FIG. 6. Muscarinic receptor antagonism induces OPC trol. differentiation but is not the Sole pharmacological mecha FIG. 2. Neurotransmitter receptor modulating agents nism of all identified differentiation inducing agents. (A) induce the differentiation of OPCs to a myelinating oligo The muscarinic receptoragonist carbacholantagonizes com dendrocyte cell fate. (A) Western blot analysis of OPCs pound induced OPC differentiation in some cases. OPCs treated with compounds at ECoo concentrations for 6 days. 50 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 ECoo concentrations and myelin basic protein (MBP) and myelin oligodendrocyte treated with 1:3 serial dilutions of . 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 ECoo concentrations for 6 days. Total RNA 55 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 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 B-actin and GAPDH. Fold change in tiation activity of salmeterol, GBR12935 and trifluopera gene expression over DMSO treated control cells is plotted 60 zine. (B) Carbachol-induced calcium (Ca") 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. 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-C. Cells treated OPCs using mature oligodendrocyte specific mark were equilibrated with Ca" sensitive Fluo-3AMR dye in ers. Immunofluorescence staining using specific antibodies 65 HBSS for 30 min. Multiple concentrations of OPC differ for MBP, MOG, and 2',3'-cyclic nucleotide 3' phosphodies entiation-inducing compounds (3 times the ECo ECoo and terase (CNP). Nuclei were identified using DAPI. Com ECs) were added followed by addition of 1:3 serial dilu US 9,592,288 B2 10 tions of the muscarinic receptor agonist Carbachol. Ca" interferon-?3(IFN: 10,000 U/mouse) or benztropine (BA; 2.5 influx was immediately measured using the FLIPR mg/kg). (E) Clinical EAE scores for mice treated with TETRAR) system. Shown are representative data for carba FTY720 (0.1 mg/kg) in combination with benztropine (BA: chol treatment at 100 uM and compound treatment at ECo. 2.5 mg/kg) show a comparable decrease in clinical severity Relative light units (RLU) indicating Ca" influx are plotted scores as compared to FTY720 (1 mg/kg), facilitating the on the y-axis and time (in sec) after addition of carbachol reduction in the dose of FTY720. (F) Clinical EAE scores plotted on the x-axis. GBR12935, trifluoperazine, and sal for mice treated with interferon-?3 (IFN: 3000 U/mouse) in meterol (top) show no effect on carbachol induced Ca" combination with benztropine (BA; 2.5 mg/kg) do not show influx while clemastine, benzatropine, and carbapentane a comparable decrease in clinical severity scores as inter (bottom) inhibit Ca" influx induced by carbachol. 10 feron-B (IFN: 10,000 U/mouse). Error bars indicate standard FIG. 7. A high throughput screen identified muscarinic deviation of the mean within each group of 8 mice. receptor antagonist benztropine as an inducer of OPC dif FIG. 11. Screen to identify inducers of OPC differentia ferentiation. (A) Benztropine 1.5 uM and positive control tion. (A) OPCs were maintained as proliferating A2B5 thyroid hormone 1 uM treated rat OPCs were cultured positive cells under basal growth conditions (Neurobasal under basal differentiation conditions (2 ng/ml PDGF) for 6 15 medium, B27 supplement without Vitamin A, non-essential days and stained for MBP (green). The structure of benz amino acids, L-Glutamine, 30 ng/ml PDGF). OPCs were tropine is also shown. (B) Benztropine 1.5 uM treated plated in basal differentiation media (Neurobasal medium, OPCs were analyzed for MBP and MOG expression after 6 B27 supplement without Vitamin A, non-essential amino days in culture using qRT-PCR. (C) OPCs were co-treated acids, L-Glutamine, 2 ng/ml PDGF), treated with DMSO with benztropine 2.3 uM and carbachol OuM, 0.6 uM and (<0.1%) or thyroid hormone (1 uM), fixed after 6 days in 4.7 uM for 6 days under basal differentiation conditions and culture, and stained using antibodies for CNP. O4, or MBP. Stained for MBP. A2B5-positive OPCs differentiate into immature oligoden FIG. 8. Benztropine decreases the clinical severity of drocytes that express CNP and O4, but not MBP, upon disease in the PLP induced EAE model for MS. (A) Benz withdrawal of PDGF. Addition of thyroid hormone induces tropine decreased the clinical severity of disease in the PLP 25 the differentiation of OPCs into mature oligodendrocytes induced EAE model when dosed prophylactically (starting that express MBP (B) Screen strategy using A2B5 positive on the day of PLP injection) as well as therapeutically (at the OPCs cultured under basal differentiation conditions and start of EAE symptoms) and showed efficacy comparable to treated with compounds for 6 days to identify small mol FTY720 (1 mg/kg) and interferon-B (10,000 U/mouse). (B) ecules that induce the differentiation of OPCs to mature, Quantification of confocal images of spinal cord sections 30 MBP expressing oligodendrocytes. from EAE mice treated with benztropine and stained with FIG. 12. Primary hit confirmation. (A) Dose response specific antibodies for GST-It (a marker of mature oligo assay used to confirm primary screening hits and determine dendrocytes) and NG2 (a marker of OPCs) showed potency (ECs). (ECs) is defined as the concentration that increased GST-IL positive cells as compared to vehicle results in a half maximal increase in the percentage of total treated mice, with no change in the number of NG2 positive 35 cells that express MBP as detected by immunostaining OPCs cells. (C) Representative confocal images of spinal cord were cultured in differentiation medium and treated with sections from EAE mice treated with benztropine and benztropine or control (DMSO <0.01%) for 6 days. Cells stained with specific antibodies for GST-It (mature oligo were fixed and immunostained using antibodies for MBP. dendrocytes) and NG2 (OPCs). Error bars represent standard deviations from 3 replicate FIG. 9. Benztropine treatment induces remyelination in 40 experiments. (B) Representative images show dose depen Vivo in the cuprizone model. (A) Representative images dent activity of benztropine. from the corpus callosum region of the brain at various time FIG. 13. Benztropine induces differentiation of OPCs to points show increased remyelination observed in benzatro mature oligodendrocytes. OPCs were plated in differentia pine treated mice as compared to vehicle treated mice 2 tion medium (Neurobasal medium, B27 supplement without weeks after cuprizone withdrawal and commencement of 45 Vitamin A, non-essential amino acids, L-Glutamine, 2 ng/ml administration. (B) Quantification of the myelinated PDGF) and treated with DMSO (<0.01%), benztropine 1.5 areas in the corpus callosum region shows a significant uM or thyroid hormone 1 uM. After 6 days in culture, (~2-fold) increase in myelin staining in benztropine treated cells were analyzed for MBP and MOG expression by mice as compared vehicle controls 2 weeks after drug Western blot. treatment. Data is represented in terms of threshold bins on 50 FIG. 14. Compound treatment induces the differentiation the grey scale (0-50) as described. Error bars represent of OPCs to mature oligodendrocytes. OPCs were plated in standard deviations of at least 6 corpus callosum regions. differentiation medium (Neurobasal medium, B27 supple FIG. 10. Combination with benztropine improves efficacy ment without Vitamin A, non-essential amino acids, L-Glu and allows for a reduction in the dose of FTY720 and tamine, 2 ng/ml PDGF) and treated with DMSO (<0.01%), interferon-?3. (A) Clinical EAE scores for mice treated with 55 benztropine 1.5 uM or thyroid hormone 1 uM for 6 days. FTY720 (1 mg/kg) in combination with benztropine (BA: Cells were fixed and immunostained for MBP, MOG, CNP, 2.5 mg/kg) show a significantly decreased clinical severity GalC, O1, or O4. Representative images of DMSO, benz as compared to mice treated with FTY720 (1 mg/kg) or tropine and thyroid hormone treated cells show expression benztropine (2.5 mg/kg) alone. (B) Clinical EAE scores for of mature oligodendrocyte markers in benztropine and thy mice treated with Interferon-?3 (IFN: 10,000 U/mouse) in 60 roid hormone treated cells, but not in DMSO treated cells. combination with benztropine (BA, 2.5 mg/kg) show a FIG. 15. Gene expression profile of OPCs differentiated to significantly decreased clinical severity as compared to mice oligodendrocytes by benztropine treatment. OPCs were treated with Interferon-Y (IFN: 10,000 U/mouse) or Benz plated in differentiation medium and treated with DMSO tropine (2.5 mg/kg) alone. (C) Clinical EAE scores for mice (<0.1%), benztropine 2.3 uM or thyroid hormone 1 uM treated with FTY720 (0.1 mg/kg) or FTY720 (0.1 mg/kg) or 65 for 6 days. OPCs from the same passage were also pelleted benztropine (BA; 2.5 mg/kg). (D) Clinical EAE scores for and frozen. Total RNA was isolated from the cells and gene mice treated with interferon-B (IFN: 3000 U/mouse) or expression analysis was performed using rat genome arrays US 9,592,288 B2 11 12 from Affymetrix. Global clustering analysis of mRNA scribed to cDNA and gene expression of muscarinic recep expression probe sets that displayed a >2-fold change in tors M. M., or M was detected by PCR using gene specific expression across samples shows clustering of benztropine primers. treated samples with thyroid hormone treated samples while FIG. 23. Antagonism of M/M 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 (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 M/M muscarinic receptors by down-regulating 10 phosphorylation of Akt, p42MAP Kinase, and increasing FIG. 16. Gene expression profiles of benztropine treated phosphorylation of p38 MAP Kinase and CREB. (B) OPCs cells show a downregulation of OPC genes and an upregu were plated in basal differentiation conditions and treated lation of mature oligodendrocyte genes. (A) Increased with benztropine 1.5 uM, thyroid hormone 1 uM or expression (fold change) of mature oligodendrocyte genes in DMSO (<0.1%). OPCs from the same passage were also compound treated cells as compared to DMSO treated cells. 15 pelleted and frozen. Total RNA was isolated from the cells, (B) Decreased expression (fold change) of OPC genes in reverse transcribed to first strand cDNA and used as a compound treated cells as compared to DMSO treated cells. template for qRT-PCR. Gene specific FAM labeled probes FIG. 17. Timing of compound treatment determines the were used to detect expression levels of various genes, with efficiency of differentiation. (A) OPCs were plated in dif probes for beta-actin and GAPDH as internal controls. Gene ferentiation medium on day 0 and treated with compounds expression shows a downregulation in cell cycle genes Such on various days. Cells were fixed and immunostained for as Cyclin D1, Cyclin D2, c-Fos, c-Jun indicating an exit MBP on day 6 after plating. (B) OPCs were plated in from cell cycle. (C) Signaling pathway downstream of the differentiation medium on day 0 and treated with com M/M muscarinic receptors. pounds 12 hours later. Cells were fixed on various days FIG. 24. Benztropine antagonizes the M/M muscarinic following compound treatment and immunostained for 25 receptors but not the M/M muscarinic receptors. OPCs MBP. (C) Treatment with benztropine within 48 hours of were plated in differentiation media for 12 hours. Media was plating in differentiation medium induced efficient differen changed to Hank's Balanced Salt Solution (HBSS) with tiation of OPCs to mature oligodendrocytes. Compound 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid treatment 72 hours or more after plating in differentiation (HEPES) and cells were treated with benztropine at various 30 concentrations for 1 hour. Carbachol was added and calcium medium reduced the efficiency of differentiation of OPCs. flux was measured for 186 seconds using the FLIPR (D) Benztropine treatment for a minimum of 5 days was TETRAR) system. (A) Carbachol induced a dose dependent necessary to induce efficient differentiation of OPCs to increase in intracellular Ca" levels. (B) Benztropine dose mature oligodendrocytes. dependently blocked carbachol induced calcium influx FIG. 18. Carbachol antagonizes benztropine induced OPC 35 through antagonism of M/M muscarinic receptors. (C) differentiation. OPCs were plated in basal differentiation Atropine, a , serves as a positive media and co-treated with benztropine 2.3 uM and carba control. (D) OPCs were plated in differentiation medium for chol OuM, 0.6 uM or 4.7 uM for 6 days and stained for 12 hours. A cAMP-HTRF assay was performed using the MBP (green). cAMP dynamic 2 kit. Benztropine had no effect on the levels FIG. 19. Benztropine has no effect on histamine receptor 40 of cAMP, IBMX was added as a cAMP stabilizer, and signaling. OPCs were plated in basal differentiation medium forskolin was a positive control. (Neurobasal medium, B27 supplement without Vitamin A, FIG. 25. Dose dependent activity of prophylactic benz non-essential amino acids, L-Glutamine, 2 ng/ml PDGF) tropine in the EAE model. EAE was induced in mice using and co-treated with various concentrations of benztropine PLP and pertussis toxin. Benztropine dissolved in saline at and (A) histamine or (B) the histamine receptor agonist 45 various doses was injected via I.P. injection daily using the histamine trifluoromethyltoluidide (HTMT). prophylactic mode followed by scoring clinical symptoms. FIG. 20. Benztropine has no effect on dopamine D2 and Prophylactic dosing is defined as administration of com D3 receptor signaling. OPCs plated in basal differentiation pound commenced on the day of PLP injection. Error bars medium (Neurobasal medium, B27 supplement without indicate standard deviation of the mean within each group of Vitamin A, non-essential amino acids, L-Glutamine, 2 ng/ml 50 8 mice. PDGF) and co-treated with various concentrations of ben FIG. 26. Benztropine treatment induces remyelination in Ztropine and the dopamine receptor (A) agonist quinpirole or vivo in the PLP induced EAE models for MS. Benztropine (B) antagonist . Error bars indicate standard does not block lymphocyte infiltration in EAE mice, but deviations of 3 replicate measurements. leads to significantly increased LFB staining, indicating the FIG. 21. Muscarinic antagonists induce differentiation of 55 presence of myelin in areas infiltrated by lymphocytes as OPCs to oligodendrocytes. OPCs were plated in differen compared to vehicle treated mice. EAE was induced in mice tiation medium (2 ng/ml PDGF) and treated with various by injecting PLP and pertussis toxin and the mice were concentrations of compounds for 6 days. Cells were fixed treated with benztropine (10 mg/kg) or vehicle controls at and immunostained for MBP Selective muscarinic antago the first appearance of EAE symptoms. Spinal cords were nists oxybutynin, atropine, ipratropium, , and 60 isolated from mice representative of the average group induced differentiation of OPCs in a dose scores during the relapse phase of EAE, Sectioned and dependent manner. ECso values for compound induced dif stained with Luxol Fast Blue and H&E (left panel) and ferentiation are indicated. Luxol Fast Blue only (right panel). Arrows point to regions FIG. 22. OPCs express muscarinic receptors and of lymphocyte infiltration. acetyl transferase. Total RNA was isolated from OPCs 65 FIG. 27. Benztropine has no effect on T-cell activation and treated with DMSO (<0.1%) or thyroid hormone 1 uM for proliferation in vitro. Total splenocytes were isolated from 6 days, or from whole rat brain. RNA was reverse tran mice and stimulated with CD3 and analyzed by flow cytom US 9,592,288 B2 13 14 etry for expression of T-cell activation markers CD69 and Error bars represent standard deviations from 3 replicate CD25 and T-cell proliferation using CFSE. (A) Unstimu ELISAs performed on samples from 5 mice in each treat lated cells. (B) DMSO treated cells. (C) Mycophenolate ment group. Suppresses the activation and proliferation of T-cells as FIG. 31. Quantification of myelination staining in the compared to DMSO. (D) Benztropine has no effect on T-cell 5 cuprizone model. (A) Luxol Fast Blue staining was per activation. (E) Mycophenolate suppresses T-cell prolifera formed on sections from the corpus callosum region of the tion. (F) Benztropine has no effect on T-cell proliferation. isolated from mice treated either with benztropine (10 The numbers represent percentage gated populations posi mg/kg) or vehicle control after 7 weeks of exposure to tive for the given marker. cuprizone. (B) Images were converted to a 256 shade grey O scale. (C) The 256 shades of grey were divided into 5 bins FIG. 28. Benztropine shows no immunosuppressive 1 of 50 shades each. Number of objects in the corpus callosum effects in vivo after induction of EAE in mice. EAE was region in each bin were counted using Image-Pro plus. (D) induced in mice by injecting PLP and pertussis toxin. Representative images of Image-Pro rendering of the quan Benztropine (10 mg/kg) and saline (vehicle control) were tification of objects in each bin. injected intraperitoneally in the therapeutic mode for 14 15 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 1.5uM, 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 20 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.33. Combination with benztropine improves efficacy number of CD8"/CD44Hi T-cells (F). Benztropine also had and allows for a reduction in the dose of FTY720 and no effect on cytokine production measured as the number of 25 interferon-?3. EAE was induced in mice using PLP and IL2 producing CD4 T-cells (G), number of IL 10 producing pertussis toxin. Benztropine (2.5 mg/kg) and FTY720 (vari CD4"T-cells (H), number of TNF-C. producing CD4"T-cells ous doses) and interferon (various does) were injected via and number of IFN-Y producing CD4 T-cells. Error bars intraperitoneal injections in the therapeutic mode at the start indicate standard deviation of the mean within each group of of EAE symptoms. (A) Clinical EAE scores for mice treated 5 mice. Representative flow cytometry scatter plots (K) 30 with FTY720 at doses of 1 mg/kg, 0.1 mg/kg and 0.01 mg/kg show similar numbers of CD4, CD8", and CD44" cells in show a dose dependent activity for FTY720. (B) Clinical spleens isolated from vehicle treated mice and benztropine EAE scores for mice treated with interferon-B at doses of treated mice. 10,000 U, 3000 U and 1000 U per mouse shows a dose FIG. 29. Benztropine shows no immunosuppressive dependent activity for interferon-B (C) Combinations of effects in vivo in normal mice. BenZitropine (10 mg/kg) and 35 benztropine (2.5 mg/kg) with FTY720 (1 mg/kg, 0.1 mg/kg saline (vehicle control) were injected intraperitoneally in and 0.01 mg/kg). (D) Combinations of benztropine (2.5 normal mice for 14 days. Total splenocytes were isolated mg/kg) with interferon-B (10,000 U, 3000 U and 1000 U per from the mice, stimulated with PMA and ionomycin and 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 48 hrs. Protein transport was 40 (BA, 2.5 mg/kg) does not show a significantly decreased blocked in cells used for cytokine analysis using monoei clinical severity as compared to mice treated with FTY720 osin. Benztropine treatment had no effect on the number of (0.01 mg/kg) or benztropine (2.5 mg/kg) alone. (F) Clinical total splenocytes (A), number of B cells (B), number of EAE scores for mice treated with interferon-?3 (IFN: 1000 CD4" T-cells (C), number of CD8" T-cells (D), number of U/mouse) in combination with benztropine (BA, 2.5 mg/kg) CD4"/CD44Hi T-cells (E) and number of CD8"/CD44Hi 45 does not show a significantly decreased clinical severity as T-cells (F). Benztropine also had no effect on cytokine compared to mice treated with interferon-B (IFN: 1000 production measured as the number of IL2 producing CD4" U/mouse) or benztropine (2.5 mg/kg) alone. Error bars T-cells (G), number of IL10 producing CD4 T-cells (H), indicate standard deviation of the mean within each group of number of TNF-Y producing CD4 T-cells and number of 8 mice. IFN-Y producing CD4 T-cells. Error bars indicate standard 50 FIG. 34. Identified compounds decrease clinical severity deviation of the mean within each group of 5 mice. in the EAE model. EAE was induced in mice by injecting FIG. 30. Benztropine does not suppress T-cell dependent PLP in complete Freund's adjuvant (CFA) and pertussis and independent immune responses. Mice were injected toxin, and the animals were scored daily for clinical severity with Keyhole Limpet Hemocyanin protein conjugated to of disease on a scale of 0-5. (A) Clinical EAE scores for 2,4,6, trinitrophenylhapten (TNP-KLH), lipopolysaccharide 55 mice treated with benztropine (10 mg/kg) in the therapeutic conjugated to 2,4,6, trinitrophenylhapten (TNP-LPS), or mode (injections starting at the first appearance of EAE TNP (2,4,6-Trinitrophenyl)-FICOLL conjugate (TNP-Fi symptoms on day 8-10) showed a significantly decreased coll) in appropriate adjuvants and treated with vehicle or clinical severity in the relapse phase of the disease as benztropine (10 mg/kg). Serum was isolated at various time compared to vehicle treated mice. (B) Clinical EAE scores points and IgG and IgM levels were measured by ELISA. 60 for mice treated with benztropine (10 mg/kg) in the prophy (A, B) Benztropine showed no effect on TNP-LPS induced lactic mode (injections starting on day 0) showed a signifi T-cell independent B-cell responses measured as serum IgM cantly decreased clinical severity in both acute and relapse and IgG levels. (C, D) Benztropine showed no effect on phase of the disease as compared to vehicle treated mice. (C) TNP-Ficol induced T-cell independent B-cell responses Benztropine showed a dose dependent efficacy in decreasing measured as serum IgM and IgG levels. (E. F.) Benztropine 65 clinical severity scores in the EAE model, with 12.5 mg/kg showed no effect on TNP-KLH induced T-cell dependent being the most effective dose and 0.1 mg/kg showing no B-cell responses measured as serum IgM and IgG levels. effect. Clinical EAE scores for mice treated with (D) trif US 9,592,288 B2 15 16 luoperazine (10 mg/kg), (E) clemastine (10 mg/kg), or (E) vides methods of treating a subject having a demyelinating salbutamol (10 mg/kg) in the therapeutic mode showed a disease by administering to a Subject a neurotransmitter significantly decreased clinical severity in the relapse phase receptor modulating agent. II. Agents that Stimulate Increased Myelination of Nerves of the disease as compared to vehicle treated mice. Error A. Neurotransmitter Receptor Modulating Agents bars indicate standard deviation of the mean within each A neurotransmitter receptor modulating agent is an agent group of 8-10 mice. that induces oligodendrocyte precursor cell (OPC) differen tiation to a mature myelinating cell fate (e.g., myelinating DETAILED DESCRIPTION OF THE oligodendrocytes) and/or stimulates increased myelination. INVENTION 10 In some embodiments, a neurotransmitter receptor modu lating agent is selected from a muscarinic receptor antago I. Introduction nist, a dopamine receptor antagonist, a histamine receptor The present invention is based, in part, on the discovery antagonist, a beta adrenergic receptor modulator, and an that compounds that modulate various classes of neurotrans- opioid receptor modulator. As shown in the Examples sec mitter receptors, such as muscarinic receptor antagonists, 15 tion below, exemplary members of each of these classes of dopamine receptor antagonists, histamine receptor antago- compounds has been shown to induce OPC differentiation to nists, beta adrenergic receptor antagonists, and opioid recep- a myelinating oligodendrocyte cell fate, and thus stimulate increased myelination. Based on the data showing this tor modulators, promote the differentiation of oligodendro activity of exemplary muscarinic receptor antagonists, dop cyte precursor cells (OPCs) 1nto a mature myelinating cell amine receptor antagonists, histamine receptor antagonists, fate (e.g., myelinating oligodendrocytes). Accordingly, in 20 beta adrenergic receptor antagonists, and opioid receptor one aspect, the present invention provides for methods of modulators, other compounds in each of these classes and inducing OPC differentiation to myelinating oligodendro- having similar pharmacological mechanisms to the exem cytes. plified compounds, such as the compounds listed in Table 1, Without being bound by a particular theory, it is believed are also predicted to be useful for inducing OPC differen that in demyelinating diseases such as multiple Sclerosis, 25 tiation to a mature myelinating cell fate (e.g., myelinating OPCs are present and able to migrate to demyelinated oligodendrocytes) and/or stimulating increased myelination. regions, suggesting that the progressive decrease in remy- In some embodiments, a compound that is identified as an elination in these diseases is not due to defects in OPC agent that stimulates increased myelination has “selective' population or recruitment, but rather is due to impaired activity for one of these classes of neurotransmitter receptors differentiation of OPCs (reviewed in Chong and Chan, J. 30 (i.e., has an agonistic or antagonistic effect against one of a Cell Biol. 188:305-312 (2010)). Accordingly, the present muscarinic receptor, a dopamine receptor, a histamine recep invention further provides for methods of stimulating tor, a beta adrenergic receptor, or an opioid receptor, or a increased myelination of nerves in a Subject in need thereof Subtype of any of these receptors, and has a weaker effect or by administering to a Subject a neurotransmitter receptor Substantially no effect against the other receptors). In some modulating agent, such as a muscarinic receptor antagonist, 35 embodiments, a compound that is identified as an agent that a dopamine receptor antagonist, a histamine receptor stimulates increased myelination has activity against two or antagonist, a beta adrenergic receptor antagonist, or an more of these classes of neurotransmitter receptors or Sub opioid receptor modulator. The present invention also pro- types of neurotransmitter receptors. TABLE 1. Neurotransmitter receptor modulating agents Neurotransmitter Receptor(s) Modulated Low Molecular Weight Compound Muscarinic Adrenergic Opioid Antitussive (-)-Norephedrine (-)-Quinpirole hydrochloride (-)-Terbuclomine (+)- (+)-BUTACLAMOL HYDROCHLORIDE (+-)- (+)-Lappaconitine (+)-Scopolamine e (+-)-Trimethoduinol (+/-)- (1r,2r)-cyclohexane-1,2-dicarboxylic acid (3,4-dihydroxyphenylamino)-2-imidazoline .DELTA.9- 11CMNPA e 1-((6,7-Dimethoxy-3-methyl-2- benzofuranyl)carbonyl)-4-methylpiperazine monohydrochloride 1-(3-bromo-5-isoxazolyl)-2-(tert butylamino) hydrochloride 1-(3-Chlorophenyl) 1-(3-Chlorophenyl)piperazine dihydrochloride 1-(3-Chlorophenyl)piperazine hydrochloride 1-(4-Hydroxyphenyl)-2-aminoethanol 1,2,9,10-tetramethoxyaporphine e 1,4-bis(2-(4-methoxynaphthalen-1- e yl)methylidenehydrazinyl-phthalazine US 9,592,288 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

1,5-Trimethylenetetrazole e 1-3-(Trifluoromethyl) e 121524-08-1 e 125-71-3 (Parent) e 127-35-5 e 17-Hydroxy-2,3-cyclopropanoandrostane e 19-Propylorvinol e 1-Benzylimidazole e 1-M-Thiq e 1-PHENYLPIPERAZINE e 1-Propanamine, 3-dibenzo[b,ethiepin-11 (6H)- e ylidene-N,N-dimethyl 2-((2-Ethoxyphenoxy)phenylmethyl)morpholine e methanesulfonate 2-(1-Piperazinyl)pyrimidine e 2-(2-Aminoethyl)pyridine e 2-(2-Aminoethyl)pyridine dihydrochloride e 2-Detpq. e 2-Methoxyidazoxan e 2-METHYL-1,3- e 2-Thiazoleethanamine e 3- e 3-Quinuclidinyl benzilate e 4-DAMP e 4-Damp methiodide e 4-DO e 4-NMPB e 5,6-Dihydroxytryptamine e 5,7-DIHYDROXYTRYPTAMINE e 5-Carboxamidotryptamine e 5-Fhdbat e 5-Hmdptime e 5-Hydroxypropafenone e 5-Methylfurmethide e 5-Methylurapidil e 6-Dta e 6-Hydroxydopamine hydrobromide e 6-Hydroxydopamine hydrochloride e 6-nitroquipazine e 76-57-3 e e 7-Ohdpat e 87-00-3 e 8-Artp e 8-OH-Dpat e A-38SO3 e A-77636 hydrochloride e e e Acebutolol hydrochloride e e Aceclidine hydrochloride e maleate e Aceroxatidine e acetaminophen e Acetylpromazine e Acide tolfenamique INN-French e e Actifed e ACTINOQUINOL SODIUM e Adobiol e e bitartrate e ADTN e Aerolone e Aerovent e Afdx 384 e A 76 e Aktamin hydrochloride e Alaproclate e Alaproclate hydrochloride e Alesion e Aleudrin e Alfenta e e Alfentanil hydrochloride e US 9,592,288 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

uZosin e ginor e golysin e e imeZine (TN) e legra e lergival e lococaine e motriptan e nespiron e e nespirone (INN) e e omide (TN) e LOSETRON HYDROCHLORIDE e oxi e oxi (TN) e pha-Ergocryptine e pha- e pha-Methylhistamine e pha-Methyl-L-dopa e alpha-Methylserotonin e Alphaprodin e e Alprenolol hydrochloride e alrestatin e Altat e e e e Amantadine hydrochloride e e e Ambenonium chloride e Amerge e Amfebutamone e amfebutamonum e hydrochloride e e e e e Amitriptyline hydrochloride e e e amosulalol hydrochloride e e e e e Amperozide hydrochloride e e e AMPHETAMINE SULFATE e e Amsulosin e Anatran e Andantol e Anemet e e e e Anplag e e Antazoline hydrochloride e Antazoline phosphate e Antergan e e Antussan e Anzemet e e e Apomorphine HCI e e e APRACLONIDINE HYDROCHLORIDE e a- e Aprofen hydrochloride e Aprofene e Ara-putp e Arbutamina e e Arbutamine hydrochloride e Arc. 239 e e e e arotinolol hydrochloride e Artane e Arterenol bittartrate e Artex e US 9,592,288 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

Astelin e e Astomin e Astramorph e AtaraX e altenolol e e e ATROPINE e Atropine iodomethylate e Atropine methyl nitrate e Atropine sulfate e Atrovent e Auteral e Avacan e Avapyrazone e Avinza e Axert e e e e AZepexole e Azepexole hydrochloride e e Banistyl e Banthine e Batebulast e Batebulast hydrochloride e BD1047 e BE 2254 e Beforal e e e Beldavrin e e e e e Benactyzine hydrochloride e benalfocin e Benextramine e Benfuran e e Benoxathian hydrochloride e e benserazide e e Bentanidol e Benzetimide e BENZETIMIDE HYDROCHLORIDE e Benzfetamine e e Benzhexol e Benzonatate e e e benztropine e e Benztropinum e e Berachin e beta-Cft e beta-ENDORPHIN e beta-Endorphin (1-31) e beta-Flupenthixol e e Betahistine dihydrochloride e Betahistine mesilate e e Betaxolol hydrochloride e e Betazole hydrochloride e e Bethanechol chloride e e MESYLATE e e Biperiden hydrochloride e Bisguanidinium phosphate e e Bisoprolol fumarate e e US 9,592,288 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

Bladderon e Bmy-7378 e Bonamine e e e BrAAM e tosylate e e Birl 15572 e Bril 26830 e Birl 37344A e Br 48553 e Br-15572 e Br3S135A e Brocadisipal e Brolamfetamine e e e e Bromopride hydrochloride e e Brompheniramine maleate e Broncaspin e Bronitin Mist e Bronkometer e Broxalterol e BTCP e Buccastem e e e Bucindolol hydrochloride e e Buclodin e Budipine e Budipine hydrochloride e e Bufotenine e e e Bunolol e Bunolol hydrochloride e e bupranolol hydrochloride e Buprenorfina INN-Spanish e e BUPRENORPHINE HYDROCHLORIDE e Buprenorphine hydrochloride solution e e Bupropion hydrochloride e e Buscapine e e Buspirone hydrochloride e BUTACLAMOL HYDROCHLORIDE e butanoic acid e Butaxamina e e e e Butorphanol tartrate e e Butoxamine (INN) e BUTOXAMINE HYDROCHLORIDE e Butylhyoscine e Butylscopolamine e C11796 e

C19H2SNOOHC e

C5976 SIGMA e e e benzoate e Calcium fusarate e e e US 9,592,288 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 carbachol e e e Carbamazepine dihydrate e Carbamylcholine e e carbetapentane e e Carbetapentane citrate e e carbidopa e e Carbidopa hydrate e e Carbidopa Monohydrate e e Carbidopa, (S)-Isomer e e Carbidopa-levodopa e e e CARBINOXAMINEMALEATE e Carebastine e e e carteolol hydrochloride e e CCRIS 3490 e CEC dihydrochloride e celliprolol e Centralvet e e Cerocral e e e CGP 20712A e CGP 20712A methanesulfonate e CGS 1206.6B e CGS 12066B dimaleate e H38O83 e EBI: 104181 e : 117275 e : 124645 e : 126213 e : 136626 e : 142179 e : 148.898 e : 159721 e e : 161127 e : 178303 e : 2O1827 e : 238.638 e : 268876 e : 334862 e : 3SOS46 e : 36796 e e e : 399928 e : 4O751 e : 431080 e : 471 632 e : 4829S e : S17861 e : 583615 e : S84626 e : 623.294 e : 648957 e : 702837 e M BL446167 e BL93361 e O RAZINE e hlorethylclonidine e hloropyramine e hloropyramine hydrochloride e C hlorpheniramine e hlorpheniramine maleate e hlorpromazine e hlorpromazine hydrochloride e hlorprothixene e ANOPRAMINE e 105105 e DS122 e D517557 e e e e US 9,592,288 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 Cimetidine hydrochloride e e Cinanserin hydrochloride e e e e e Cisapride monohydrate e e citalopram hydrobromide e L 316,243 e L316243 e -Apb e earnal e emastine e e emastine (USAN) e e LEMASTINE FUMARATE e e C lenbuterol e hydrochloride e clidinium e CLIDINIUMBROMIDE e clobempropit e Clobutinol e e Clomipramine hydrochloride e e LONIDINE HYDROCHLORIDE e LOPENTHIXOL e opiXol e oranolol e ovoxamine e ovoxamine fumarate e e cocaethylene e Cocaethyline e Cocain-chlorhydrat German e e COCAINE HYDROCHLORIDE e Cocaine muriate e e e Cogentin e e Cogentin meSylate e e Cognex e Compazine e Concerta e Concordin e Congesteze e Contristamine e e Corindolan e Corlopam e Corynanthin e e Corynanthine hydrochloride e CP 93.129 e Crispin e e CV 705 e e e e e Cyclizine hydrochloride e Cyclogyl e e e e cyproheptadine hydrochloride e e Cystospaz e d1- e DAGO e Daipin e Dalcipran e e Dalgan e Dalmee e DAMGO e e US 9,592,288 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 Dapiprazole hydrochloride Darifenacin hydrobromide Darwon D-Chlorpheniramine D-Dopa Debridat DEBRISOQUIN SULFATE deisopropyldisopyramide C Deprenalin Deprenil Deptropine citrate (INN) Deramciclane fumarate Desipramine hydrochloride Desoratadine Desoxedrine Detrol DEXBROMPHENIRAMINEMALEATE maleate Dexmedetomidinum (INN-Latin DEXPROPRANOLOL Dexpropranolol hydrochloride Dextromethorphan hydrobromide monohydrate Dextrostat Diacetylmonoxime Diacetylmorphine Diamaprit-2HCl Dicetel Dicodethal dicyclomine Dicyclomine hydrochloride Difril Dihydro-alpha-ergocryptine meSylate Dihydrocodeine bitartrate Dihydroergocornine Dihydroergocristine DIHYDROERGOCRISTINE MESYLATE Dihydroergotamine mesilate Dihydroquinidine Dihydroquinine dihydroxyphenylalanine Dilaudid Dilevalol Dilevalol hydrochloride Dimemorfan Dimemorfan (INN) Dimethindene maleate US 9,592,288 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

DIMETHYLTRYPTAMINE e e Diphemanil e e Diphenhydramine citrate e DIPEHENHYDRAMINEHYDROCHLORIDE e DIPEHENOXYLATE e HCI e dipivefrin e Dipivefrin hydrochloride e Dironyl e Ditropan e e DL-Adrenaline e |-Desoxyephedrine e e DL-DOPA e l-Narcotine e DL- e DL-threo-3,4-Dihydroxyphenylserine e DL-threo-DOPS e obutamine e hydrochloride e ocarpamine e olasetron e MESYLATE e Dolasetronum (INN-Latin e Domin e e Omperidone e Maleate e Dopabain e e opamine e Dopamine hydrochloride e opazinol e e e DOPEXAMINE HYDROCHLORIDE e e OSulepin hydrochloride e (INN) e Dothiepin e oxazosin e meSylate e oxepin e Hydrochloride e Doxepine e DOXOFYLLINE e oxylamine e Succinate e DPDPE e Dramamine e Drixoral e e e dropropizine e e e DSP 4 e DSP-4 hydrochloride e DU-29373 e dulloxetine e e e HYDROCHLORIDE e e e DuP 734 e Duremesin e 1-13 e e e e Edronax e EEDQ e e e e Efaroxan hydrochloride e Effexor e Effortilwet e Eldoral e e Eletriptan hydrobromide e US 9,592,288 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

Eltoprazine e hydrochloride e Emadine e e Emepronum e Emergil e Enantio-PAF C-16 e 1 e Endomorphin 2 e Endovalpin e ENTACAPONE e e eperisone e e Ephedrine hydrochloride e EPHEDRINE SULFATE e Ephetonine e Epibatidine e e epinephrine e Epinephrine hydrochloride e fumarate e Ergocryptine e Ergocryptine mesylate e Ergocryptine-alpha e mesylate e Ergomar e e Ergotamin e e e e e ergotamine tartrate e Esbuphon e e Escitallopram e Escitallopram oxalate e e e Esmolol hydrochloride e ethaverine e Ethaverine hydrochloride e ethopropazine e e e Ethopropazine hydrochloride e e e ETHYLKETOCYCLAZOCINE e e e e Eticlopride hydrochloride e e etillefrine hydrochloride e Etintidine e Etintidine hydrochloride e e EU-010O372 e Eupaverina e Euspirol e Evoxac e e Exaprolol hydrochloride e e Famotidina e amotidine e HCI e e e Fastin e e Femoxetinum (INN-Latin e Fencarbamide hydrochloride e Fencarol e e enfluramine e Fenistill e enoldopam e bromide e enoterol e hydrobromide e e entanyl e US 9,592,288 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

Fentora e exofenadine e FG 4963 e Finaten e Finibron e law Oxate e avoxate hydrochloride e b. 457 e esinoxan e esinoxan hydrochloride e LESTOLOL e LESTOLOL SULFATE e uanxol depot (TN) e lunarizine e lunarizine hydrochloride e uorofen e e LUPENTHIXOL DECANOATE e upentixol e LUPHENAZINE e uphenazine hydrochloride e uspirilene e e FLUVOXAMINEMALEATE e Focalin e FOMINOBEN e FONAZINE e ormoterol e e Frovatiriptan Succinate e usaric acid e e Galantamin e Galantamine e Galantamine hydrobromide e Ganglefene e Ganglerone e Gastrozepin e Gbr 12783 e GBR12935 e Geodon e e e GEPIRONE HYDROCHLORIDE e Gewatran e glafenine e e e Glauconex e GlaxoWellcome brand of acrivastine e GLYCOPYRROLATE e Gnoscopine e Gotensin e GR 1138O8 e GR-127935 e e Granisetron hydrochloride e e Guanabenz acetate e e guanethidine Sulfate e e guanfacine hydrochloride e guanidine e Guanidine bromide e Guanidine hydrochloride e Guanidine nitrate e GUANIDINIUM e Gynergen e e Hag-PC e haloperidol e Haymine e HEAT e Hemicholinium e Hemicholinium-3 e hydrochloride e US 9,592,288 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

Hhsi-difenidol e e e histamine e Histamine dihydrochloride e Histamine diphosphate e Histamine hydrochloride e Histantin e Hoe-893d e e Homatropine hydrobromide (RS) e omocodeine e Hycodan e e Hydergine e e Hydriatine e Hydroaminacrine e e e e ydromorphone hydrochloride e hydroquinidine e ydroquinidine hydrochloride e HYDROQUININE e e Hydroxyzine pamoate e yoscine hydrobromide e yoscine Methobromide e hyoscyamine e yoscyamine (D)- e Hyoscyamine Sulfate e Hyoscyamine sulfate (USP) e Hypostamine e dysco e bopamine e ibuprofen e BZM e catibant e catibant acetate e c 118551 e CI-894O6 e DAZOXAN e DAZOXAN HYDROCHLORIDE e fenprodil e ifenprodil tartrate e HEAT e ldamen e e metit dihydrobromide e midacloprid e e mipramine hydrochloride e MPROMIDINE e mpromidine hydrochloride e napetyl e e NDALPINE e e e inderal e indolophenanthridine e e NDORAMIN e Hydrochloride e nopamil e insidon e intropin e odocyanopindolol e ipratropium e e pratropium bromide monohydrate e prazochrome e ps-339 e PSAPIRONE e psapirone hydrochloride e Smelin e Soaminile e Socodeine e e US 9,592,288 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 isoetharine e isoproterenol e SOPROTERENOL HYDROCHLORIDE e soproterenol sulfate e Sospaglumic acid e Sothipendyl e e SOXSuprine hydrochloride e trop e animine e etrium tartrate e Kadian e Kerlone e e Ketanserin tartrate e e Ketogan e e KETOTIFENFUMARATE e Kinichron e e Kytril e L 657743 e L-741,626 e abetalol e hydrochloride e afutidine e L-alpha-Acetyl-N-normethadol e e appaconitine e Lazabemide e Legatrin e Leoplexamin e -Ephedrine e Lethidrone e e e Levamfetamine e e Levcromakalim e Levetimide e e LevobunololoEHC e evocabastine e HYDROCHLORIDE e e evodopa e e Levodropropizine e Levomeprazine e e e e e Levomethorphan hydrobromide e evorphanol e e Levospasme e Levsinex e LIDAMIDINE e LIDAMIDINE HYDROCHLORIDE e Lilly 53857 e - chloride e e e e isuride maleate e e e L-Noradrenaline bitartrate e LODOXAMIDE TROMETHAMINE e e Lofentanil oxalate e Longifene e Lopac0 000714 e Lopac-A-164 e Lopac-C-130 e Lopressor e oratadine e LOrcet e LotrOneX e oxapine e hydrochloride e US 9,592,288 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 Loxapine Succinate e Loxtidine e L- e LSD tantrate e HYDROCHLORIDE e LY 235959 e LY 277359 maleate e Ly-165163 e Lysergide e Lysivane e e e e madopar e Malexi e e Maprotiline hydrochloride e Marzine e Maxolon e e e Mci9042 e McIl S652 e McN-A-343 e mCPBG e m-CPBG hydrochloride e MDL-100907 e MDMA e e e MEBEVERINE HYDROCHLORIDE e Meclastine e e Meclizine hydrochloride e Meclizine Mixture With Niacin e Mecloprodine e e e Medetomidine hydrochloride e e e Medroxalol hydrochloride e e e e MEMANTINE HYDROCHLORIDE e e meperidine e e e MEPTAZINOL HYDROCHLORIDE e e Merital e e Mescomine e e e e e Mesulergine hydrochloride e e e Metabolites (street) e e metaproterenol e Metaproterenol hemisulfate e bitaritrate e Metatsin e e e e Methacholine chloride e e e Methadyl acetate e e e Methamphetamine hydrochloride e e e methalpyrilene e HYDROCHLORIDE e Metharsinat e methiothepin e Methiothepin maleate e e Metholes e e e METHOXAMINE HYDROCHLORIDE e e US 9,592,288 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 methyldopa e METHYLDOPA SESQUIHYDRATE e Methylfurmetide e methyloctatropine bromide e e Methylscopolamine e e Methysergide maleate e e e e Metoclopramide dihydrochloride e metoprolo e fumarate e e e e Mianserin hydrochloride e e e Mictonorm e Midaglizole e Midaglizole hydrochloride e e MIDODRINE HYDROCHLORIDE e Mifentidine e e e Minipress e Mintussin e Minusine e Mirapex e e e e e e MK-212 e MK-912 e M-Mptp e Mofegiline e Mofegiline hydrochloride e MALEATE e e Morphine hydrochloride e MORPHINE SULFATE e morphinesulfate e e Mosapride citrate e e e moxisyllyte hydrochloride e MPTP e Muscarin e Myonal e Myophedrine e N,3-Dimethylmorphinan e Naaxia e N-acetylaspartylglutamate e N-Acetyl-Asp-Glu e e e nafronyl e Nafronyl oxalate e e malbuphine e e Nalorphine hydrochloride e e Naloxone hydrochloride e NAN-190 hydrobromide e e NAPHAZOLINE HYDROCHLORIDE e Naphazoline nitrate e Naphthisen e e Nargoline e Narphen e Navaron e NaZasetron e NCGCOOO15261-01 e US 9,592,288 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 inchembio873-comp43 e inchembio873-comp53 e inchembio873-compé7 e Ncq 298 e e Nebracetam e Nebracetam fumarate e Nedeltran e e Nefopam hydrochloride e e NEOSTIGMINE e neostigmine bromide e Neostigmine methyl sulfate e e NIH-8805 e Nipradillol e Nisentil e e nizatioline e N-Methylspiroperidol e Noleptan e e Norephedrine e e e Norfenfloramine e Norflex e Norprolac e e Nortriptyline hydrochloride e Norzine Ampuls e NoScapalin e noscapine e Novopropoxyn e n-Propylapomorphine e e NSC10004 e e e NSC11433S e NSC28.9336 e NSC61391 e NSC61806 e e NSC69886 e NSC79303 e Nubain e nylidrin e Nylidrin hydrochloride e Octatropine e e Oils, peppermint e e e e ONDANSETRON HYDROCHLORIDE e Opana e Opcon e e

e Orphenadrine hydrochloride e e Otillonium Bromide e e Oxeladin e e Oxifedirinum e e e Oximetidine hydrochloride e Oxolamine e Oxolamine citrate e e oxotremorine methiodide e US 9,592,288 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 e Oxprenolol hydrochloride e oxybutynin e Oxybutynin chloride e e Oxycodone hydrochloride e e e oxyphenonium e OXYPHENONIUMBROMIDE e oZagrel e oZagrel hydrochloride e Palfadonna e Palladone e Palomosetron e hydrochloride e e e PAPP e Paracodin e Paracymethadol e Parasan e Parlodel e e e Paroxetine hydrochloride e Paroxetine maleate e Pataday e Paxil e PBPO e p-Chloramphetamine e Pemilaston e Pemirolast e e e Pentalgine e pentazocine e Peracon e maleate e e PERGOLIDE MESYLATE e Perhydrohistrionicotoxin e Periactin e e Pernazine e Pernovine e periphenazine e hydrochloride e FHHSD e henacetin e HENAZOCINE e hencarbamide e henindamine e heniramine e HENIRAMINEMALEATE e HENOPERIDINE e henoperidine hydrochloride e henopropamine e (OXcle e e henoxybenzamine e henoxybenzamine hydrochloride e e e hentolamine meSylate e e e PHENYLEPHRINE HYDROCHLORIDE e e PHENYLPROPANOLAMINE e HYDROCHLORIDE PHOLCODNE e hospholine iodide e Picumast e Picumast dihydrochloride e e Pilocarpine- e Pilocarpine hydrochloride e US 9,592,288 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 Pilocarpine nitrate e Pilocarpine nitrate salt e bimozide e Pinaverium e bindolol e e e Pipazethate e e e e Pirenzepine dihydrochloride e e e Piribedill hydrochloride e e Piribedil mesylate e e e PIZOTYLINE e e Plegine e 3-MPPI e Pinu 99194A e Pondimin e e Pramipexol Spanish e e e e e Prazosin hydrochloride e PRBCM e Precedex e Preclamol e e Preclamollum Latin e e 21-pivalate e e e Prestwick 144 e Prialt e Privine e Prizidio e e procaterol hydrochloride e e Prochlorperazine dimaleate e Prochlorperazine edisylate e Prochlorperazine maleate e e Procyclidine hydrochloride e Progabide e Prolixin e e PROMAZINE HYDROCHLORIDE e Promedol e e PROMETHAZINE HYDROCHLORIDE e propantheline e e Propitan e Propiverine e propiverine hydrochloride e e Propranolol hydrochloride e Proroxan INN) e Proscomide e Prothiaden e e e PROTOPINE HYDROCHLORIDE e e e Protriptyline hydrochloride e Proxicromil e Prozac e PSXeladine e Psychostyl e pyrilamine e PYRILAMINEMALEATE e Pyrroxane e Quadramet e e US 9,592,288 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

Quinagollide e e Quinidex e e e e e Quinidine sulfate e e Quinine e Quinine hydrochloride e quinine Sulfate e QUININE SULFATE DIHYDRATE e QUINPIROLE e e QUIPAZINE MALEATE e R(-)-Denopamine e R-SOS47 e e Raclopride C11 e Raclopridum Latin e e Ractopamine hydrochloride e e Ramosetron hydrochloride e e Ranitidine bismuth citrate e ranitidine hydrochloride e Rapimine e e e Reboxetine mesylate e Reboxetine mesylate hydrate e Redux e Reglan e Relaspium e e REMIFENTANIL. HYDROCHLORIDE e e REMOXIPRIDE HYDROCHLORIDE e e Renzapride hydrochloride e repirinast e e REPROTEROL HYDROCHLORIDE e e Respilene e Restenacht e Rexigen e Rexolate e e Rilmenidine phosphate e e Rimiterol Hydrobromide e e e e e Ritodrine hydrochloride e e Rizatriptan benzoate e Ro 363 e Robina e Robinu. e e e e Ropinirole hydrochloride e e Rotenolone e Roti gotine e RS 86, hydrobromide e RS 86HB e RS-2S259-197 e RU 24969 e RU-24213 e S(-)Eticlopride hydrochloride e S-2OSOO e Salbutamol e Saligren e Salmeterol e US 9,592,288 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

Salmeterol Xinafoate e Samarium Sm 153 lexidronam e e Savella e e SB 206SS3 e Sch. 23982 e Sch-23982 e Scopolamine e Scopolamine butylbromide e Scopolamine hydrobromide e Sdz 205,557 e Secoverine e SECOVERINE HYDROCHLORIDE e Selecal e e Selegiline hydrochloride e Selozok e Serc e Serotone e e e Sertraline hydrochloride e e Sgd 101-75 e Silomat e Sinemet e e SKF 38393 e SKF 38393 hydrochloride e SKF 81297 e Skif 83566 e SKF 91488 e SKF 91488 dihydrochloride e Sm-Edtmp e SMROOO449272 e Snc80 e e Solifenacin Succinate e Sordinol e e Sotalol hydrochloride e Soventol e Soventol (TN) e Soventol hydrochloride e Spasril e Spectrum OO1815 e e piriva e piriva Handihaler e piroXatrine e Q 10643 e R. S923OA e T 91 e e adol e e elazine e e STP (hallucinogen) e Strattera e Subecholine e Suberyldicholine e

Sufentani citrate e e e Sultopride hydrochloride e e SUMATRIPTAN SUCCINATE e Suplatast Tosilate e Sympatholytin e e T123 SIGMA e Tacrine e Tacrine hydrochloride e Tacrine hydrochloride hydrate e tageflar e US 9,592,288 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

alacen e Tallinolol e Calipexole e e Talwin e Talwin 50 e e citrate e Tbhpbo e TCMDC-12SSO9 e maleate e e Teletux e TEMELASTINE e Tempium e Tenamfetamine e e Terazosin hydrochloride e e TERBUTALINE HEMISULFATE e Terbutaline sulfate e e e e TERODILINE HYDROCHLORIDE e Tersiga e e Tesmilifene e TETRABENAZINE e TETRAHYDROCANNABINOL e e e e Tetraspasmin-Lefa e FMP C e Thecodinum e hephorin hydrochloride e heratuSS e hiethylperazine e maleate e e hioridazine e hydrochloride e hiothixene e honzylamine e HYDROCHLORIDE e e e Tillisolol e timolo e hemihydrate e Timolol maleate e TIOTIDINE e e Tiotropium e TIOTROPIUMBROMIDE e TIPP e e izanidine e e hydrochloride e e Tobanum e Tocodilydrin e olazoline e hydrochloride e Tolcapone e olfenamic acid e olterodine e tartrate e Torecan e e tramadol hydrochloride e tranilast e Traxanox e Traxanox sodium e e Trazodone hydrochloride e Tremblex e US 9,592,288 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

Tretoquinol e Tricodein e e trifluoperazine e Trifluoperazine dihydrochloride e e e Triflupromazine hydrochloride e e e Trimebutine maleate e e Trimethoduinol e e Trimipramine maleate e e Tripelennamine citrate e Tripelennamine hydrochloride e e Triprolidine hydrochloride e e Tropine tropate e e TROPISETRONHC e Tropyl 3,5-dichlorobenzoate e e e Tulobuterol hydrochloride e Tuscodin e e Tyr-D-Ala-Gly-N-Methyl-Phe-Gly-ol e Tyr-tic-phe-phe-OH e e e Oroxatral e Valoron e Vanoxeamine e e e Ventipulmin e Ventolin e Verton e e Viloxazine hydrochloride e Vistaril e e Vf 9153 e Way 100635 e Way-100135 e Wiin-3S428 e e Wyamine sulfate e e Xamoterol hemifumarate e e Xanomeline tartrate e e Xylamidine tosylate e e Xylazine hydrochloride e XyZall e e YOHIMBINE HYDROCHLORIDE e e ZACOPRIDE HYDROCHLORIDE e e ZATOSETRON MALEATE e Zebeta e Zelmac e Zelnorm e Zeneca ZD7114 e Zetidoline e Zimeldine e Zimeldine hydrochloride e e US 9,592,288 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 Zimelidine dihydrochloride ZINTEROL HYDROCHLORIDE Zipeprol e Ziprasidone hydrochloride e Ziprasidone meSylate e Zofran e Zyrtec

1. Muscarinic Receptor Antagonists and/or are known antagonists of a dopamine receptor. There A muscarinic receptor is a G-protein coupled acetylcho fore, in Some embodiments, the dopamine receptor antago line receptor. There are five known subtypes of muscarinic nist is a compound selected from benztropine, GBR12935, receptors: M receptors, M2 receptors, M receptors, M. trifluoperazine, and salts, prodrugs, racemic mixtures, con receptors, and Ms receptors. A muscarinic receptor antago formational and/or optical isomers, crystalline polymorphs, nist is an agent able to inhibit one or more characteristic 25 and isotopic variants thereof. Alternatively, any of the dop responses of a muscarinic receptor or receptor Subtype. As amine receptor modulators listed in Table 1 can be used to a non-limiting example, an antagonist may competitively or antagonize a dopamine receptor. Thus, in some embodi non-competitively bind to (1) a muscarinic receptor, (2) an ments, the dopamine receptor antagonist is a or (or other ) of the muscarinic receptor modulator compound listed in Table 1. The com receptor, and/or (3) a downstream signaling molecule to 30 pounds described in Table 1 are readily available. inhibit the muscarinic receptor's function. As shown in the In some embodiments, the neurotransmitter receptor Examples, benztropine, carbetapentane, clemastine, ipratro modulating agent is benztropine or a salt thereof (e.g., pium, and atropine have been shown to antagonize the benztropine mesylate). In some embodiments, the neu function of a muscarinic receptor and/or are known antago rotransmitter receptor modulating agent is trifluoperazine or nists of a muscarinic receptor. Therefore, in some embodi 35 a salt thereof (e.g., trifluoperazine hydrochloride). ments, the muscarinic receptor antagonist is a compound 3. Histamine Receptor Antagonists selected from benztropine, carbetapentane, clemastine, ipra A histamine receptor is a G-protein coupled receptor, for tropium, atropine, and salts, prodrugs, racemic mixtures, which the neurotransmitter histamine is the primary endog conformational and/or optical isomers, crystalline poly enous ligand. There are four known Subtypes of histamine morphs, and isotopic variants thereof. Alternatively, any of 40 receptors: H receptors. He receptors, H receptors, and Ha the muscarinic receptor modulators listed in Table 1 can be receptors. A histamine receptor antagonist is an agent able to used to antagonize a muscarinic receptor. Thus, in some inhibit one or more characteristic responses of a histamine embodiments, the muscarinic receptor antagonist is a mus receptor or receptor Subtype. As a non-limiting example, an carinic receptor modulator compound listed in Table 1. The antagonist may competitively or non-competitively bind to compounds described in Table 1 are readily available. 45 (1) a histamine receptor, (2) an agonist or partial agonist (or In some embodiments, the neurotransmitter receptor other ligand) of the histamine receptor, and/or (3) a down modulating agent is benztropine or a salt thereof (e.g., stream signaling molecule to inhibit the histamine receptors benztropine mesylate). In some embodiments, the neu function. As shown in the Examples, clemastine has been rotransmitter receptor modulating agent is clemastine or a shown to antagonize the function of a histamine receptor. salt thereof (e.g., clemastine fumarate). 50 Therefore, in some embodiments, the histamine receptor 2. Dopamine Receptor Antagonists antagonist is clemastine or a salt, , , A dopamine receptor is a G-protein coupled receptor, for conformational and/or optical isomer, crystalline poly which the neurotransmitter dopamine is the primary endog morph, or isotopic variant thereof. Alternatively, any of the enous ligand. There are five known Subtypes of dopamine histamine receptor modulators listed in Table 1 can be used receptors: D and Ds receptors, the D-like receptors, acti 55 to antagonize a histamine receptor. Thus, in some embodi vate , while the D, D, and D receptors, ments, the histamine receptor antagonist is a histamine the D-like receptors, inhibit adenylyl cyclase and activate receptor modulator compound listed in Table 1. The com K" channels. A dopamine receptor antagonist is an agent pounds described in Table 1 are readily available. able to inhibit one or more characteristic responses of a In some embodiments, the neurotransmitter receptor dopamine receptor or receptor Subtype. As a non-limiting 60 modulating agent is clemastine or a salt thereof (e.g., cle example, an antagonist may competitively or non-competi mastine fumarate). tively bind to (1) a dopamine receptor, (2) an agonist or 4. Beta Adrenergic Receptor Modulators partial agonist (or other ligand) of the dopamine receptor, A beta adrenergic receptor is a Subtype of the adrenergic and/or (3) a downstream signaling molecule to inhibit the receptor, a G-protein coupled receptor, for which cat dopamine receptor's function. As shown in the Examples, 65 echolamines (e.g., epinephrine and norepinephrine) are the benztropine, GBR12935, and trifluoperazine have been primary endogenous ligand. There are three known Subtypes shown to antagonize the function of a dopamine receptor of beta adrenergic receptors: B receptors, B receptors, and US 9,592,288 B2 61 62 B 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 differentiate to a mature myelinating cell fate in the absence competitively bind to (1) a beta adrenergic receptor, (2) an 5 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 tion of markers of mature myelinating oligodendrocytes. In function of a beta adrenergic receptor. A beta adrenergic 10 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 mature myelinating oligodendrocytes. Examples of markers agonize the function of a beta adrenergic receptor and/or are 15 of mature myelinating oligodendrocytes include, but are not known 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), 23'-cyclic-nucleotide 3' phos is a compound selected from pindolol, Salmeterol, Salbuta phodiesterase (CNP), GalC, O1, or O4. mol, albuterol, and salts, prodrugs, racemic mixtures, con 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 analysis) levels, followed by visualization and/or quanitifi beta adrenergic receptor modulator compound listed in 25 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 of mature 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 Hybridization: A Practical Approach (Wilkinson, D. G., meterol Xinfoate). In some embodiments, the neurotransmit 30 ed.), Oxford University Press, 1992. In some embodiments, ter receptor modulating agent is salbutamol or a salt thereof a marker of mature myelinating oligodendrocytes is detected (e.g., salbutamol hemisulfate). 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 and Practice of Immunoassay, 2nd Edition, Grove’s Dic which are the primary endogenous ligand. An opioid 35 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 A detectable moiety can be used in the assays described receptor, (2) an agonist or partial agonist (or other ligand) of 40 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 receptoragonist 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 provisions. Suitable detectable moieties include, but are not example, an agonist may activate an opioid receptor. As 45 limited to, radionuclides, fluorescent dyes (e.g., fluorescein, shown in 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 protein (GFP), phycoerythrin, etc.), autoquenched fluores pentane, Snc-80, BD-1047, and salts, prodrugs, racemic 50 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 2. Cells and Reagents ments, the opioid receptor modulator is an opioid receptor 55 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 from a mammal). to identify agents that stimulate increased myelination of 60 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 screening a plurality of agents to identify an agent that example, brain tissue, spinal cord tissue, or optic nerve increases the number of cells in a sample having a differ tissue. The tissue can be from a rodent (e.g., rat or mouse), entiated, myelinating cell fate (e.g., mature myelinating chicken, dog, cat, rabbit, cow, sheep, goat, or primate (e.g., oligodendrocyte). In some embodiments, an agent promotes 65 a monkey, a chimpanzee, or a human). In some embodi or increases OPC differentiation when it increases the per ments, OPCs are derived from fetal tissue. In some embodi centage of OPCs (e.g., in a sample comprising a plurality of ments, OPCs are derived from adult tissue. Alternatively, US 9,592,288 B2 63 64 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). “building blocks.” For example, a linear combinatorial Examples of conditions suitable for OPC differentiation 5 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. lions of chemical compounds can be synthesized through OPCs are cultured with growth factor, for example, 10 Such combinatorial mixing of chemical building blocks. PDGFC.C. As a non-limiting example, OPCs are proliferated Preparation and Screening of combinatorial chemical in culture on poly-D- 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 to, peptide libraries (see, e.g., U.S. Pat. No. 5,010, 175, PDGFC.C. For differentiation, OPCs are seeded on poly-D- 15 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 PDGFC.C. 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% CO, for 6 days. At the end (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. WO92/00091), benzodiaz cal analysis. epines (e.g., U.S. Pat. No. 5.288,514), diversomers such as 3. Candidate Agents hydantoins, and dipeptides (Hobbs et al., The agents that are screened for the ability to promote Proc. Nat. Acad. Sci. USA 90:6909-6913 (1993)), vinylo OPC differentiation can be any small chemical compound, 25 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 small compound libraries (Chen et al., J. Amer: Chem. Soc. assays of the invention, although most often compounds that 30 116:2661 (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, nucleic acid libraries (see, e.g., U.S. Pat. No. 5,539,083), which are typically run in parallel (e.g., in microtiter formats 35 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 isoprenoids, U.S. Pat. No. 5,569.588; thiazolidinones and chemica Analytika (Buchs, Switzerland) and the like. 40 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 Devices for the preparation of combinatorial libraries are molecules have a higher likelihood of having physiochemi 45 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 numerous combinatorial libraries are themselves commer were described by Lipinski et al. as follows: having more 50 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 Log P Martek Biosciences, Columbia, Md., etc.). over 5 (or MLog P 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, etrate the blood-brain barrier. In some embodiments, candi e.g., Lipinski et al., Adv Drug Delivery Res 23:3-25 (1997). 55 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 . rial chemical or peptide library containing a large number of 4. Validation potential therapeutic compounds (potential modulator or 60 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 ies' or “ligand libraries' are then screened in one or more apparent activity. In some embodiments, validation assays assays, as described herein, to identify those library mem are in vitro assays. In some embodiments, such studies are bers (particular chemical species or Subclasses) that display conducted with suitable animal models. The basic format of a desired characteristic activity. The compounds thus iden 65 Such methods involves administering a lead compound tified can serve as conventional "lead compounds” or can identified during an initial screen to an animal that serves as themselves be used as potential or actual therapeutics. a disease model for humans and then determining if the US 9,592,288 B2 65 66 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, salbutamol, trifluoperazine, or a salt thereof. In some but are not limited to, primates, mice, rats and Zebrafish. 5 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 tiple Sclerosis, idiopathic inflammatory demyelinating dis prophylactic methods. In one aspect, the present invention 10 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 Charcot-Marie-Tooth disease, acute disseminated encepha mitter receptor modulating agent as described herein and 15 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 tiple sclerosis (MS). There are several subtypes of MS, tamine receptor antagonist, a beta adrenergic receptor modu- 20 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 the subject has RRMS. In some embodiments, the subject 6 days or longer under conditions suitable for OPC differ- 25 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 or more initially be diagnosed as having one subtype of MS (e.g., biological markers of mature myelinating oligodendrocytes. RRMS), and subsequently the subtype of MS afflicting the Marker assays for detecting the presence or level of myeli subject may convert to another subtype of MS (e.g., from nating oligodendrocytes are described herein, for example in 30 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 Subject having a demyelinating disease or at risk for having prises administering to the Subject a neurotransmitter recep- 35 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 “immunomodulatory agent” refers to a disease-modifying antagonist, a dopamine receptor antagonist, a histamine 40 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 phy, Guillain-Barre Syndrome, chronic inflammatory demy having a demyelinating disease. Thus, in yet another aspect, 45 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 HTLV-associated myelopathy). risk of having a demyelinating disease. Thus, in yet another 50 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 or severity of an MS relapse and/or reduce development of ing to the Subject a neurotransmitter receptor modulating 55 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 modulatory agent is an agent that modulates an immune mitter receptor modulating agent is a compound listed in 60 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 dopamine receptor modulator compound, histamine receptor ment of MS include, but are not limited to, interferons (e.g., modulator compound, beta adrenergic receptor modulator interferon-?3, e.g., interferon beta-1a or interferon beta-1b). compound, or opioid receptor modulator compound listed in glatiramer acetate, mitoxantrone, fingolimod (FTY720), or Table 1). In some embodiments, the neurotransmitter recep- 65 monoclonal antibodies (e.g., natalizumab, rituximab, dacli tor modulating agent is benztropine, cerbetapentane, clem Zumab, or alemtuzumab). Thus, in Some embodiments, the astine, pindolol, ipratropium, atropine, GBR12935, Snc-80, method of the present invention comprises administering to US 9,592,288 B2 67 68 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 , a dopamine receptor antagonist, a histamine receptor modulator compound, or an opioid receptor modu receptor antagonist, a beta adrenergic receptor antagonist, lator compound listed in Table 1, and the immunosuppres and an opioid receptor modulator) in combination with 5 sant is fingolimod (FTY720), interferon beta-1a, interferon fingolimod (FTY720), interferon beta-1a, 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 benztropine, clemastine, salmeterol, Salbutamol, or trifluop of enhancing the therapeutic effect of an immunomodulatory 10 agent in a Subject in need thereof. It has been Surprisingly erazine, or a salt thereof, and the immunomodulatory agent found that in mouse models of demyelinating disease, is fingolimod (FTY720), interferon beta-1a, interferon beta administering a combination of a neurotransmitter receptor 1b, glatiramer acetate, mitoxantrone, natalizumab, ritux modulating agent and an immunomodulatory agent results imab, daclizumab, or alemtuzumab. In some embodiments, in a significantly larger decrease in the clinical severity of 15 the neurotransmitter receptor modulating agent is benz the demyelinating disease as compared to the decrease in the tropine and the immunomodulatory agent is fingolimod clinical severity of the demyelinating disease that can be (FTY720), interferon beta-1a, or interferon beta-1b. achieved with either the neurotransmitter receptor modulat Therapeutic doses for the immunomodulatory agents fin ing agent or the immunomodulatory agent alone. Thus, in golimod (FTY720), interferon beta-1a, interferon beta-1b, Some embodiments, the method comprises administering to glatiramer acetate, mitoxantrone, and natalizumab are a Subject an immunomodulatory agent and a neurotransmit known in the art. See, e.g., Kappos et al., N Engl J Med ter receptor modulating agent; thereby enhancing the thera 362:387-401 (2010); Cohen et al., N Engl J Med 362:402-15 peutic effect of the immunomodulatory agent in the Subject. (2010); Gottesman et al., Mult Scler 12:271-80 (2006): In Some embodiments, the Subject has a demyelinating Hurwitz et al., Clin Ther 30:1102-12 (2008); Gaindh et al., disease or is at risk of having a demyelinating disease. 25 Expert Opin Biol. Ther 8:1823-29 (2008): Koch-Henriksenet Furthermore, it has also surprisingly been found that al., Neurology 66:1056-60 (2006); Benatar, Lancet 360:1428 administering a neurotransmitter receptor modulating agent, (2008); Jacobs et al., Ann Neurol 39:285-94 (1996); Lancet in combination with an immunomodulatory agent at a dose 352:1498-1504 (1998); Johnson et al., Neurology 45:1268 that, on its own, is insufficient to be therapeutic for the 76 (1995); Johnson et al., Neurology 50:701-08 (1998); treatment of a demyelinating disease, results in a therapeutic 30 Comi et al., Ann Neurol. 69: 75-82 (2011); Calabresi Nat effect that is greater than the therapeutic effect from admin Clin Pract Neurol 3:540-1 (2007); and Polman et al., NEngl istering each agent alone. Thus, in another aspect, the JMed354:899-910 (2006); the contents of each of which are present invention provides methods of treating a subject in incorporated by reference herein in their entirety. need thereof by administering an immunomodulatory agent In some embodiments, the immunomodulatory agent and a neurotransmitter receptor modulating agent, wherein 35 (e.g., fingolimod, interferon beta-1a, interferon beta-1b. the immunomodulatory agent is administered at a subthera glatiramer acetate, mitoxantrone, or natalizumab) is admin peutic dose. In some embodiments, the Subject has a demy istered at a therapeutically effective dose. In some embodi elinating disease or is at risk of having a demyelinating ments, the immunomodulatory agent (e.g., fingolimod, inter disease. feron beta-1a, interferon beta-1b, glatiramer acetate, In some embodiments, the demyelinating disease is mul 40 mitoxantrone, or natalizumab) is administered at a subthera tiple Sclerosis, idiopathic inflammatory demyelinating dis peutic dose, e.g., at a dose that is less than about 75%, less ease, transverse myelitis, Devic's disease, progressive mul than about 70%, less than about 60%, less than about 50%, tifocal leukoencephalopathy, optic neuritis, leukodystrophy, less than about 40%, less than about 30%, less than about Guillain-Barre Syndrome, chronic inflammatory demyelinat 25%, less than about 20%, less than about 15%, less than ing polyneuropathy, autoimmune peripheral neuropathy, 45 about 10%, or less than about 5% of the dose that is Charcot-Marie-Tooth disease, acute disseminated encepha conventionally administered for the immunomodulatory lomyelitis, adrenoleukodystrophy, adrenomyeloneuropathy, agent. Leber's hereditary optic neuropathy, or HTLV-associated Fingolimod (FTY720) is conventionally administered at a myelopathy. In some embodiments, the demyelinating dis therapeutically effective dose of from about 0.5 mg per day ease is multiple Sclerosis, e.g., relapsing-remitting multiple 50 to about 1.5 mg per day (e.g., about 0.5, about 0.6, about 0.7. sclerosis (RRMS), secondary progressive multiple sclerosis about 0.8, about 0.9, about 1.0, about 1.1, about 1.2, about (SPMS), primary progressive multiple sclerosis (PPMS), or 1.3, about 1.4, or about 1.5 mg per day). See, e.g., Kappos progressive relapsing multiple sclerosis (PRMS). In some et al., N Engl J Med 362:387-401 (2010). Thus, in some embodiments, the Subject is initially diagnosed as having embodiments, a subtherapeutic dose of fingolimod is from one subtype of MS (e.g., RRMS), and subsequently the 55 about 0.005 mg per day to about 0.375 mg per day (e.g., subtype of MS afflicting the subject converts to another about 0.005, about 0.01, about 0.02, about 0.03, about 0.04, subtype of MS (e.g., from RRMS to SPMS). about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, In some embodiments, the neurotransmitter receptor about 0.1, about 0.15, about 0.2, about 0.25, about 0.3, about modulating agent is selected from a muscarinic receptor 0.35, or about 0.375 mg per day). antagonist, a dopamine receptor antagonist, a histamine 60 Interferon beta-1a is conventionally administered at a receptor antagonist, a beta adrenergic receptor modulator, therapeutically effective dose of about 30 ug per week. See and an opioid receptor modulator, and the immunomodula e.g., Jacobs et al., Ann Neurol 39:285-94 (1996). Thus, in tory agent is selected from fingolimod (FTY720), interferon Some embodiments, a Subtherapeutic dose of interferon beta-1a, interferon beta-1b, glatiramer acetate, mitoxan beta-1a is from about 0.3 ug per week to about 23 Jug per trone, natalizumab, rituximab, daclizumab, and alemtu 65 week (e.g., about 0.3, about 0.5, about 0.75, about 1, about Zumab. In some embodiments, the neurotransmitter receptor 2, about 3, about 4, about 5, about 6, about 7, about 8, about modulating agent is a muscarinic receptor modulator com 9, about 10, about 11, about 12, about 13, about 14, about 15, US 9,592,288 B2 69 70 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 Jug per week). and/or interferon beta-1b is administered at a therapeutically Interferon beta-1b 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., 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, 10 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 15 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 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-1a, or interferon beta-1b. In some embodi less than about 75%, less than about 70%, less than about 25 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 15%, 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-1a is administered at a subtherapeutic dose of neurotransmitter receptor modulating agent. In some 30 from about 0.3 ug per week to about 23 Jug 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 35 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 benztropine may salbutamol, trifluoperazine, or a salt thereof) is administered be from about 1 mg per day to about 10 mg per day (e.g., 40 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, 45 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-1a, 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 50 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-1a, 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 55 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-1a 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., 60 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-1a, or interferon beta day. 1b. In some embodiments, benztropine is administered at a In some embodiments, a neurotransmitter receptor modu therapeutically effective dose of from about 1 mg per day to lating agent (e.g., muscarinic receptor antagonist, dopamine about 10 mg per day; fingolimod is administered at a 65 receptor antagonist, histamine receptor antagonist, beta therapeutically effective dose of from about 0.5 mg per day adrenergic receptor modulator, or opioid receptor modulator to about 1.5 mg per day; interferon beta-1a is administered listed in Table 1, e.g., benztropine, clemastine, salmeterol, US 9,592,288 B2 71 72 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%, 1a, 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-1a, interferon beta-1b. IV. Pharmaceutical Compositions glatiramer acetate, mitoxantrone, or natalizumab) is admin 10 istered at a subtherapeutic dose, e.g., at a dose that is less In another aspect, the present invention provides pharma than about 75%, less than about 70%, less than about 60%, ceutical compositions for use in the treatment of a demy less than about 50%, less than about 40%, less than about elinating disease (e.g., multiple Sclerosis, idiopathic inflam 30%, less than about 25%, less than about 20%, less than matory demyelinating disease, transverse myelitis, Devic's about 15%, less than about 10%, or less than about 5% of the 15 disease, progressive multifocal leukoencephalopathy, optic dose that is conventionally administered for the immuno neuritis, leukodystrophy, Guillain-Barre Syndrome, chronic modulatory agent. In some embodiments, the neurotrans inflammatory demyelinating polyneuropathy, autoimmune mitter receptor modulating agent is benztropine or a salt peripheral neuropathy, Charcot-Marie-Tooth disease, acute thereof (e.g., benztropine mesylate) and the immunomodu disseminated encephalomyelitis, adrenoleukodystrophy, latory agent is fingolimod (FTY720), interferon beta-1a, or adrenomyeloneuropathy, Leber's hereditary optic neuropa interferon beta-1b. In some embodiments, bentropine is thy, or human T-cell lymphotropic virus (HTLV)-associated administered at a subtherapeutic dose of from about 0.01 mg myelopathy). In some embodiments, the composition com per day to about 0.75 mg per day; fingolimod is administered prises a mixture of a neurotransmitter receptor modulating at a subtherapeutic dose of from about 0.005 mg per day to agent and an immunomodulatory agent. In some embodi about 0.375 mg per day; interferon beta-1a is administered 25 ments, the pharmaceutical composition comprises a neu at a subtherapeutic dose of from about 0.3 ug per week to rotransmitter receptor modulating agent selected from a about 23 ug per week; and/or interferon beta-1b is admin muscarinic receptor antagonist, a dopamine receptor antago istered at a subtherapeutic dose of from about 2 Lig every nist, a histamine receptor antagonist, a beta adrenergic other day to about 190 ug every other day. receptor modulator, and an opioid receptor modulator and an In some embodiments, a neurotransmitter receptor modu 30 immunomodulatory agent selected from interferon beta-1a, lating agent (e.g., a muscarinic receptor antagonist, a dop interferon beta-1b, glatiramer acetate, mitoxantrone, fingoli amine receptor antagonist, a histamine receptor antagonist, mod (FTY720), natalizumab, rituximab, daclizumab, and a beta adrenergic receptor modulator, or an opioid receptor alemtuzumab. In some embodiments, the pharmaceutical modulator listed in Table 1, e.g., benztropine, clemastine, composition comprises a neurotransmitter receptor modu salmeterol, salbutamol, trifluoperazine, or a salt thereof) is 35 lating agent selected from benztropine, clemastine, salmet administered at a therapeutically effective dose and an erol, Salbutamol, trifluoperazine, and salts thereof and an immunomodulatory agent (e.g., fingolimod, interferon beta immunomodulatory agent selected from interferon beta-1a, 1a, interferon beta-1b, glatiramer acetate, mitoxantrone, or interferon beta-1b, glatiramer acetate, mitoxantrone, fingoli natalizumab) is administered at a therapeutically effective mod (FTY720), natalizumab, rituximab, daclizumab, and dose for the treatment or prevention of a demyelinating 40 alemtuzumab. In some embodiments, the pharmaceutical disease (e.g., multiple Sclerosis). In some embodiments, a composition comprises benztropine or a salt thereof and an neurotransmitter receptor modulating agent (e.g., a musca immunomodulatory agent selected from interferon beta-1a, rinic receptor antagonist, a dopamine receptor antagonist, a interferon beta-1b, and fingolimod (FTY720). histamine receptor antagonist, a beta adrenergic receptor In some embodiments, one or both of the neurotransmitter modulator, or an opioid receptor modulator listed in Table 1, 45 receptor modulating agent and the immunomodulatory agent e.g., benztropine, clemastine, Salmeterol, Salbutamol, triflu are formulated as a therapeutically effective or optimal dose. operazine, or a salt thereof) is administered at a therapeu In some embodiments, one or both of the neurotransmitter tically effective dose and an immunomodulatory agent (e.g., receptor modulating agent and the immunomodulatory agent fingolimod, interferon beta-1a, interferon beta-1b, glati are formulated as a subtherapeutic dose. In some embodi ramer acetate, mitoxantrone, or natalizumab) is adminis 50 ments, the neurotransmitter receptor modulating agent is tered at a subtherapeutic dose for the treatment or prevention formulated as a therapeutically effective or optimal dose and of a demyelinating disease (e.g., multiple Sclerosis). In some the immunomodulatory agent is formulated as a subthera embodiments, a neurotransmitter receptor modulating agent peutic dose. In some embodiments, the immunomodulatory (e.g., a muscarinic receptor antagonist, a dopamine receptor agent is formulated as a therapeutically effective or optimal antagonist, a histamine receptor antagonist, a beta adrener 55 dose and the neurotransmitter receptor modulating agent is gic receptor modulator, or an opioid receptor modulator formulated as a subtherapeutic dose. Suitable dosage ranges listed in Table 1, e.g., benztropine, clemastine, salmeterol, for therapeutically effective and subtherapeutic doses of salbutamol, trifluoperazine, or a salt thereof) is administered neurotransmitter receptor modulating agents and immuno at a subtherapeutic dose and an immunomodulatory agent modulatory agents are described above. (e.g., fingolimod, interferon beta-1a, interferon beta-1b. 60 An agent for use in any of the therapeutic methods of the glatiramer acetate, mitoxantrone, or natalizumab) is admin present invention (e.g., an agent that stimulates increased istered at a therapeutically effective dose for the treatment or myelination as described herein, or an immunomodulatory prevention of a demyelinating disease (e.g., multiple scle agent as described herein) may be in any pharmaceutically rosis). In some embodiments, a neurotransmitter receptor acceptable form, including any pharmaceutically acceptable modulating agent (e.g., a muscarinic receptor antagonist, a 65 salts, prodrugs, racemic mixtures, conformational and/or dopamine receptor antagonist, a histamine receptor antago optical isomers, crystalline polymorphs and isotopic variants nist, a beta adrenergic receptor modulator, or an opioid of the neurotransmitter receptor modulating agents. US 9,592,288 B2 73 74 A combination of a neurotransmitter receptor modulating 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 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 10 provided as a salt and can be formed with many acids, Sules, pills, powders, granules, dragees, gels, slurries, oint 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 15 tion may be a lyophilized powder in 1 mM-50 mM , neous, intramuscular), transdermal, etc., administration. 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 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 25 treated. Pharmaceutical preparations for oral use can be manufactured in a manner that is known to those of skill in 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 30 are, in particular, fillers such as Sugars, including lactose, no way limiting. Sucrose, mannitol, or ; 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 35 lulose, and/or polyvinylpyrrolidone (PVP). If desired, dis containing the therapeutic agent. Various types of Sustained 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 40 include push-fit capsules made of gelatin, as well as Soft, materials and wax-based tablets with pore-forming excipi 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 45 magnesium Stearate and, optionally, stabilizers. In soft cap (2002); and Schmidt, et al., Int. J. Pharm. 216:9 (2001)). 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 50 ages Suitable for Such administration. pared using naturally-occurring or synthetic polymers, for 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, 55 dioxide, lacquer Solutions, and Suitable organic solvents or and hydroxypropylmethylcellulose; and carboxypolymeth solvent mixtures. 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 60 tion by injection, e.g., by bolus injection or continuous clay (see, U.S. Pat. No. 6,638,521, herein incorporated by 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 tion include those described in U.S. Pat. Nos. 6,635,680; other similar oils, synthetic aliphatic acid glycerides, esters 6,624,200; 6,613,361; 6,613,358, 6,596,308; 6,589,563: 65 of higher aliphatic acids or propylene glycol, and if desired, 6,562,375; 6,548,084; 6,541,020; 6,537,579; 6,528,080 and with conventional additives such as Solubilizers, isotonic 6,524,621, each of which is hereby incorporated herein by agents, Suspending agents, emulsifying agents, stabilizers US 9,592,288 B2 75 76 and preservatives. In some embodiments, an agent of the 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, 5 in the art, especially in light of the detailed disclosure e.g., in ampules or in multi-dose containers, with an added 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 pending, stabilizing and/or dispersing agents. 10 the administered dose or dosages until a desired therapeutic Pharmaceutical formulations for parenteral administra effect is observed in the treated subject, with minimal or no tion include aqueous Solutions of the active agents in water toxic . Applicable methods for determining an soluble form. Additionally, Suspensions of the active agents appropriate dose and dosing schedule for administration of can be prepared as appropriate oily injection Suspensions. the present invention are described, for example, in Good Suitable lipophilic solvents or vehicles include fatty oils 15 man and Gilman's The Pharmacological Basis of Thera Such as Sesame oil, or synthetic fatty acid esters, such as peutics, 11th Ed., Brunton, Lazo and Parker, Eds. McGraw ethyl oleate or triglycerides, or liposomes. Aqueous injection Hill (2006), and in Remington. The Science and Practice of Suspensions can contain Substances which increase the vis Pharmacy, 21st Ed., Gennaro, Ed., Lippencott Williams & cosity of the Suspension, Such as Sodium carboxymethyl Wilkins (2003), both of which are hereby incorporated cellulose, Sorbitol, or dextran. Optionally, the Suspension 20 herein by reference. In some embodiments, one or both of a can also contain Suitable stabilizers or agents which increase neurotransmitter receptor modulating agent and an immu the solubility of the compounds to allow for the preparation nomodulary agent are formulated in a therapeutically effec of highly concentrated Solutions. Alternatively, the active tive dose. In some embodiments, one or both of a neu ingredient can be in powder form for constitution with a rotransmitter receptor modulating agent and an Suitable vehicle, e.g., sterile pyrogen-free water, before use. 25 immunomodulary agent are formulated in a subtherapeutic Systemic administration can also be by transmucosal or dose. In some embodiments, a neurotransmitter receptor transdermal means. For transmucosal or transdermal admin modulating agent is formulated in a therapeutically effective istration, penetrants appropriate to the barrier to be perme dose and an immunomodulary agent is formulated in a ated are used in the formulation. For topical administration, Subtherapeutic dose. In some embodiments, an immuno the agents are formulated into ointments, creams, salves, 30 modulary agent is formulated in a therapeutically effective powders and gels. In one embodiment, the transdermal dose and a neurotransmitter receptor modulating agent is delivery agent can be DMSO. Transdermal delivery systems formulated in a subtherapeutic dose. can include, e.g., patches. For transmucosal administration, The pharmaceutical preparation is preferably in unit dos penetrants appropriate to the barrier to be permeated are age form. In Such form the preparation is Subdivided into used in the formulation. Such penetrants are generally 35 unit doses containing appropriate quantities of the active known in the art. Exemplified transdermal delivery formu component. The unit dosage form can be a packaged prepa lations that can find use in the present invention include ration, the package containing discrete quantities of prepa those described in U.S. Pat. Nos. 6,589,549; 6,544,548: ration, such as packeted tablets, capsules, and powders in 6,517,864; 6,512,010; 6,465,006; 6,379,696; 6,312,717 and vials or ampoules. Also, the unit dosage form can be a 6,310,177, each of which are hereby incorporated herein by 40 capsule, tablet, cachet, or lozenge itself, or it can be the reference. appropriate number of any of these in packaged form. For buccal administration, the agents can take the form of Administration tablets or lozenges formulated in conventional manner. Administration of a neurotransmitter receptor modulating In addition to the formulations described previously, an agent and/or an immunomodulatory agent can be achieved agent of the present invention can also be formulated as a 45 in various ways, including oral, buccal, parenteral, including depot preparation. Such long acting formulations can be intravenous, intradermal, Subcutaneous, intramuscular, administered by implantation (for example Subcutaneously transdermal, transmucosal, intranasal, etc., administration. or intramuscularly) or by intramuscular injection. Thus, for When a neurotransmitter receptor modulating agent and an example, the agents can be formulated with Suitable poly immunomodulatory agent are co-administered, the neu meric or hydrophobic materials (for example as an emulsion 50 rotransmitter receptor modulating agent can be administered in an acceptable oil) or ion exchange resins, or as sparingly by the same or different route of administration as the soluble derivatives, for example, as a sparingly soluble salt. immunomodulatory agent. In some embodiments, a com The pharmaceutical compositions also can comprise Suit pound of the present invention (e.g., a neurotransmitter able Solid or gel phase carriers or excipients. Examples of receptor modulating agent and/or an immunomodulatory such carriers or excipients include but are not limited to 55 agent) is administered systemically. calcium carbonate, calcium phosphate, various Sugars, The dosages and frequency of administration will depend starches, cellulose derivatives, gelatin, and polymers such as upon various factors generally appreciated by those of skill polyethylene glycols. in the art, including, e.g., the severity of a Subject's disease. Pharmaceutical compositions suitable for use in the pres Generally, daily doses can range from about 0.001-100 ent invention include compositions wherein the active ingre- 60 mg/kg total body weight, from about 0.01-50 mg/kg, or from dients are contained in a therapeutically effective amount. about 0.01 mg/kg-10 mg/kg. However, doses in the range of The present invention also contemplates pharmaceutical 10-500 mg/kg per day may be effective and well tolerated. compositions comprising the neurotransmitter receptor The principal determining factor in defining the appropriate modulating compounds as described herein with an effective dose is the amount of a particular compound necessary to be amount of other therapeutic agents as combination partners, 65 therapeutically effective in a particular context. Repeated particularly those used for treating demyelinating diseases, administrations may be required in order to achieve longer Such as immunomodulary agents. An effective amount of the lasting immune tolerance. Single or multiple administrations US 9,592,288 B2 77 78 of the compositions can be carried out with the dose levels tiple Sclerosis, idiopathic inflammatory demyelinating dis and pattern being selected by the treating physician. ease, transverse myelitis, Devic's disease, progressive mul In some embodiments, a compound of the present inven tifocal leukoencephalopathy, optic neuritis, leukodystrophy, tion (e.g., a neurotransmitter receptor modulating agent Guillain-Barre Syndrome, chronic inflammatory demyelinat and/or an immunomodulatory agent) is administered to a ing polyneuropathy, autoimmune peripheral neuropathy, Charcot-Marie-Tooth disease, acute disseminated encepha subject in need thereof over an extended period of time. The lomyelitis, adrenoleukodystrophy, adrenomyeloneuropathy, methods can be carried out for at least 20 days, in some Leber's hereditary optic neuropathy, or human T-cell lym embodiments for at least 40, 60, 80 or 100 days, and in some photropic virus (HTLV)-associated myelopathy). In some embodiments for at least 150, 200, 250, 300, 350 days, 1 10 embodiments, the kit comprises a neurotransmitter receptor year or longer. In some embodiments, a compound of the modulating agent selected from a muscarinic receptor present invention (e.g., a neurotransmitter receptor modu antagonist, a dopamine receptor antagonist, a histamine lating agent and/or an immunomodulatory agent) is admin receptor antagonist, a beta adrenergic receptor modulator, istered to a subject in need thereof at or after the onset of one and an opioid receptor modulator. In some embodiments, the or more symptoms of a demyelinating disease. In some 15 kit comprises a neurotransmitter receptor modulating agent embodiments, a compound of the present invention (e.g., a selected from a compound listed in Table 1. In some neurotransmitter receptor modulating agent and/or an immu embodiments, the kit comprises a neurotransmitter receptor nomodulatory agent) is administered to a subject in need modulating agent selected from benztropine, clemastine, thereof prior to the onset of symptoms of a demyelinating salmeterol, salbutamol, trifluoperazine, and salts thereof. In disease (i.e., prophylactically). Some embodiments, the kit comprises benztropine or a salt Co-Administration thereof. In some embodiments, the methods of the present inven In some embodiments, a kit of the present invention tion comprise co-administering a neurotransmitter receptor further comprises an immunomodulatory agent. In some modulating agent and an immunomodulatory agent. Co 25 embodiments, the kit comprises a neurotransmitter receptor administered agents can be administered together or sepa modulating agent and an immunomodulatory agent for the rately, simultaneously or at different times. When adminis treatment of MS (e.g., interferon-B, e.g., interferon beta-1a tered, the neurotransmitter receptor modulating agent and or interferon beta-1b, glatiramer acetate, mitoxantrone, fin golimod (FTY720), natalizumab, rituximab, daclizumab, or the immunomodulatory agent independently can be admin 30 istered once, twice, three, four times daily or more or less alemtuzumab). In some embodiments, the kit comprises a often, as needed. In some embodiments, the agents are neurotransmitter receptor modulating agent selected from administered once daily. In some embodiments, the agents benztropine, clemastine, Salmeterol, salbutamol, trifluopera are administered at the same time or times, for instance as Zine, and salts thereof and an immunomodulatory agent 35 selected from interferon beta-1a, interferon beta-1b, glati an admixture. One or more of the agents can be administered ramer acetate, mitoxantrone, fingolimod (FTY720), natali in a Sustained-release formulation. Zumab, rituximab, daclizumab, and alemtuzumab. In some In some embodiments, co-administration includes admin embodiments, the kit comprises benztropine or a salt thereof istering one active agent within 0.5, 1, 2, 4, 6, 8, 10, 12, 16, and an immunomodulatory agent selected from interferon 20, or 24 hours of a second active agent. Co-administration 40 beta-1a, interferon beta-1b, and fingolimod (FTY720). includes administering two active agents simultaneously, In some embodiments, a kit comprises a neurotransmitter approximately simultaneously (e.g., within about 1, 5, 10. 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 45 modulating agent as described herein and an immunomodu pharmaceutical composition including both active agents latory agent in separate formulations. Suitable formulations (i.e., the neurotransmitter receptor modulating 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 50 modulating agent agent as described herein and one or more formulated separately (i.e., the neurotransmitter receptor additional therapeutic agents (e.g., an immunomodulatory modulating agent and the immunomodulatory agent are agent) independently in uniform dosage formulations administered as separate formulations). In another embodi throughout the course of treatment. In some embodiments, a ment, the active and/or adjunctive agents may be linked or kit provides a neurotransmitter receptor modulating agent as conjugated to one another. 55 described herein and one or more additional therapeutic In some embodiments, one or both of a neurotransmitter agents (e.g., an immunomodulatory agent) independently in receptor modulating agent and an immunomodulatory agent graduated dosages over the course of treatment, either can be administered prophylactically to prevent undesirable increasing or decreasing, but usually increasing to an effi recurrence of the symptoms of the demyelinating disease cacious dosage level, according to the requirements of an (e.g., to prevent or delay the recurrence of clinical attacks in 60 individual. In some embodiments, a kit comprises a neu MS), or therapeutically to achieve a desired reduction in rotransmitter receptor modulating agent as described herein symptoms of the demyelinating disease and maintain Such in a therapeutically effective dose and an immunomodula reduction in Symptoms of the demyelinating disease for a tory agent in a therapeutically effective dose. In some Sustained period of time. embodiments, a kit comprises a neurotransmitter receptor V. Kits 65 modulating agent as described herein in a therapeutically In another aspect, the present invention provides kits for effective dose and an immunomodulatory agent in a Sub use in the treatment of a demyelinating disease (e.g., mul therapeutic dose. US 9,592,288 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. 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 PDGFC.C.. 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 matory insult) was evaluated using a proteolipid protein PDGFC.C. 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% CO, 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, of MBP 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 of multiple markers of mature myeli field and >5% MBP positive cells/field. The average values 40 nating oligodendrocytes (MBP, MOG, CNP. GalC, O1, and for DMSO negative controls were consistently <0.5% MBP O4) 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 determining EC50 values in multiple replicate experiments neurological deficits ranging from impaired tail movements using the primary assay format for determining 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. ECso 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 9,592,288 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 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 10 logically active compounds, as well as a collection of ~50K treated with benztropine (10 mg/kg) in a prophylactic mode structurally diverse drug-like molecules. This led to 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 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 (ECs-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 O1 (O1) and oligodendrocyte marker O4 tiation at sites of injury that contributes to disease progres (O4)) 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, Egr1, Sox11: V. A. Swiss et al., molecules that selectively induce differentiation of OPCs at PLOS One, 6, e18088 (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 at multiple time points (FIG. 17). Maximal (T3), a known inducer of OPC differentiation (M. C. Nunes 60 induction of MBP 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., EMBO.J. 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 of OPCs to Parkinson's disease and its pharmacological activity is MBP positive oligodendrocytes following 6 days of culture thought to result from activity that decreases US 9,592,288 B2 83 84 the imbalance between dopamine and (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, P Benz 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. Simoniet 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 observed clinical severity that exceeded those observed for differentiation by benztropine (FIG. 20). Quinpirole did not the immunomodulating MS drugs FTY720 or interferon-f 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 M. M. 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 to 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 OPCs was 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-t) and (FIG. 23A). Additionally, transcript levels of cyclin D1, 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 of GST-It positive mature oligodendrocytes from ~500 to cycle progression (FIG. 23B). Activation of M and M -1 100 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 M. 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, FASEBJ, remyelination, in the context of an inflammatory environ 9, 619 (1995); M. Lopez-Ilasaca 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 of M/M 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 9,592,288 B2 85 86 which EAE had or had not been induced with PLP. In either also enhanceremyelination by exerting a protective effect on diseased or healthy animals, benztropine had no effect on 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 CD/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-ct." 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 of MS, 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 enhanceremyelination in vivo using either of two immunomodulating drugs approved for the the T-cell independent cuprizone-induced model of demy treatment of MS, interferon-?3 and FTY720 (L. Kappos et al., elination. In this model, mice are fed the copper chelatorbis 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 S1Pagonist 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-?3 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 of harvested brains with LFB (FIG.9A, B). observation may prove clinically relevant as FTY720 treat Significant demyelination was clearly observed following ment is associated with 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 the most clinically relevant model 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 of M/M 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 9,592,288 B2 87 88 resulted in enhanced functional recovery and significantly 4. The method of claim 1, wherein: decreases the dosages of the latter that are required to (i) each of the immunomodulatory agent and the musca achieve an equivalent level of efficacy. To our knowledge, rinic receptor antagonist is administered at a therapeu these results provide the first in vivo evidence supporting the tically effective dose; or (ii) the muscarinic receptor antagonist is administered at notion that a benefit can be achieved by treating MS-like a therapeutically effective dose and the immunomodu symptoms using the combination of an immunomodulator latory agent is administered at a Subtherapeutic dose; or with a remyelination enhancer. (iii) each of the immunomodulatory agent and the mus It is understood that the examples and embodiments carinic receptor antagonist is administered at a Sub described herein are for illustrative purposes only and that therapeutic dose. various modifications or changes in light thereof will be 10 5. The method of claim 1, wherein the immunomodula Suggested to persons skilled in the art and are to be included tory agent and the muscarinic receptor antagonist are admin within the spirit and purview of this application and scope of istered systemically. the appended claims. All publications, patents, and patent 6. The method of claim 1, wherein the immunomodula applications cited herein are hereby incorporated by refer tory agent and the muscarinic receptor antagonist are admin ence in their entirety for all purposes. 15 istered sequentially. 7. The method of claim 1, wherein the immunomodula tory agent and the muscarinic receptor antagonist are admin What is claimed is: istered concurrently. 1. A method of enhancing a therapeutic effect of an 8. The method of claim 1, wherein the muscarinic receptor immunomodulatory agent that is effective in treating mul antagonist is selected from atropine, benztropine, carbeta tiple Sclerosis, the method comprising: pentane, clemastine, ipratropium, oxybutynin, propiverine, administering to a human Subject having multiple Sclero and scopolamine. sis the immunomodulatory agent and a muscarinic 9. The method of claim 1, wherein the muscarinic receptor receptor antagonist; thereby enhancing the therapeutic antagonist is benztropine. effect of the immunomodulatory agent in treatment of 25 10. The method of claim 1, wherein the immunomodu multiple Sclerosis. latory agent is a monoclonal antibody. 2. The method of claim 1, wherein the muscarinic receptor 11. The method of claim 10, wherein the immunomodu antagonist is selected from atropine, benztropine, carbeta latory agent is natalizumab, rituximab, daclizumab, or alem pentane, clemastine, dicylomine, diphemanil, glycopyrro tuZumab. late, hyoscyamine, ipratropium, methantheline, methylatro 12. The method of claim 1, wherein the immunomodu pine, octatropine, oxybutynin, propantheline, propiverine, 30 latory agent is selected from an interferon-B and fingolimod. scopolamine, tiotropium, tolterodine, and salts thereof. 13. The method of claim 1, wherein the immunomodu 3. The method of claim 1, wherein the immunomodula latory agent is an S1P agonist. tory agent is fingolimod (FTY720), interferon beta-1a, inter 14. The method of claim 13, wherein the immunomodu feron beta-1b, glatiramer acetate, mitoxantrone, or natali latory agent is fingolimod. Zumab. k k k k k