WO2011109711A1

WO2011109711A1 - Glucocorticoid drugs as smoothened agonists

Info

Publication number: WO2011109711A1
Application number: PCT/US2011/027194
Authority: WO
Inventors: Wei Chen; Lawrence Barak; Kim H. Lyerly; Wang Jiangbo; Michael Bond; Robert A. Mook, Jr.
Original assignee: Duke University
Priority date: 2010-03-05
Filing date: 2011-03-04
Publication date: 2011-09-09

Abstract

Described herein are methods of stimulating the proliferation of stem cells using glucocorticoid compounds. Also disclosed are related methods of use and treatment, for example, methods of stimulating hair growth, methods of regenerating or repairing damaged tissue, methods of treating disorders such as Parkinson's disease, and methods of promoting wound healing, comprising administration of a glucocorticoid compound or a composition comprising the compound.

Description

GLUCOCORTICOID DRUGS AS SMOOTHENED AGONISTS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 61/31 1 ,068, filed on March 5, 2010, and to U.S. Provisional Application No. 61/314,060, filed on March 15, 2010, each of which is incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under R01 CA1 13656-01 A1 awarded by the National Cancer Institute. The United States government has certain rights in the invention.

BACKGROUND

[0003] The Hedgehog signaling pathway, mediated by the Smoothened (Smo) receptor, has been shown to regulate stem cells and is a fundamental regulator of organogenesis in developing embryos and tissue integrity in mature organisms. Smo agonists may reactivate or stimulate repair mechanisms in situations where normal regenerative capacity is compromised. Some Smo agonists such as Purmorphamine have demonstrated an ability to promote human embryonic stem cell differentiation, but the preclinical development of such small molecule Smo agonists has lagged.

SUMMARY OF INVENTION

[0004] In one aspect, the invention may provide a method of stimulating the proliferation of stem cells, comprising contacting the cells with an effective amount of a compound of formula (I):

(I)

wherein:

each— - independently represents the presence or absence of a bond;

R⁶ is selected from H and halo;

R⁹ is selected from H and halo;

R is selected from H, hydroxy, oxo and halo; R ⁶ is selected from alkyl, -OR', -SR', -N(R')₂, -NH-C(0)R" and -NR-S(0)_n-R", or may be taken together with R ⁷ to form a ring;

R ⁷ is selected from hydroxy and -OC(Z)R", or may be taken together with R ⁶ to form a ring;

Y is selected from a bond, O and S;

X is selected from a bond and CH₂;

W is selected from alkyl, hydroxy, halo and -OC(0)R";

each R' is independently H, alkyl, aryl, heterocyclyl or heteroaryl;

each R" is independently alkyl;

Z is O or S; and

n is 1 or 2;

wherein when R ⁶ and R ⁷ are taken together to form a ring, they together form the group -0-C(R^a)₂-0-, wherein each R^a is independently alkyl or two R^a are taken together to form a ring.

[0005] In some embodiments, the stem cells are selected from primary neuronal precursor cells and hedgehog-responsive cells. In some embodiments, the method further comprises contacting the cells with an additional agent. In some embodiments, the additional agent is a polypeptide. In some embodiments, the polypeptide is a hedgehog protein selected from Sonic hedgehog, Indian hedgehog and Desert hedgehog. In some embodiments, the compound of formula (I) and the hedgehog protein synergistically stimulate the proliferation of stem cells. In some embodiments, the cells are in a subject. In some embodiments, the cells are contacted with the compound ex vivo.

[0006] In another aspect, the invention may provide a method of stimulating hair growth in a subject, comprising administering to the subject an effective amount of a compound of formula (I).

[0007] In another aspect, the invention may provide a method of regenerating or repairing damaged tissue in a subject, comprising administering to the subject an effective amount of a compound of formula (I). In some embodiments, the subject may have experienced myocardial infarction, a spinal cord injury, a stroke, osteoporosis, a bone fracture, a lung injury or a liver injury.

[0008] In another aspect, the invention may provide a method of treating Parkinson's disease in a subject, comprising administering to the subject an effective amount of a compound of formula (I).

[0009] In another aspect, the invention may provide a method of promoting wound healing in a subject, comprising administering to the subject an effective amount of a compound of formula (I). In some embodiments, the wound is on the dermal layers of the skin. [0010] In any of the above aspects, in some embodiments in the compound of formula

(I):

R⁶ is selected from H and halo;

R⁹ is halo;

R is selected from hydroxy and oxo;

R ⁶ is alkyl, or may be taken together with R ⁷ to form a ring; and

R ⁷ is selected from hydroxy and -OC(0)R", or may be taken together with R ⁶ to form a ring.

[0011] In any of the above aspects, a composition comprising the compound of formula (I) may be administered to the subject in a composition further comprising a

pharmaceutically acceptable carrier or diluent. In some embodiments, the composition is administered topically, orally, parenterally (e.g., intravenously, intramuscularly, or subcutaneously), via a depot or via inhalation.

[0012] In any of the above aspects, in some embodiments, In some embodiments, R⁶ is hydrogen. In some embodiments, R⁶ is halo. In some embodiments, R⁶ is fluoro. In some embodiments, R⁹ is halo. In some embodiments, R⁹ is fluoro. In some embodiments, R is hydroxy. In some embodiments, R is oxo. In some embodiments, R ⁶ is alkyl. In some embodiments, R ⁶ is methyl. In some embodiments, R ⁷ is hydroxy. In some embodiments, R ⁷ is -OC(0)CH₂CH₃. In some embodiments, the group -Y-X-W is selected from the group consisting of -CH₂-CI, -S-CH₂-F, -CH₂-OAc, -CH₂-OH, -CH₃ and -CH₂-I. In some

embodiments, R ⁶ and R ⁷ are taken together to form a ring. In some embodiments, R ⁶ and

R ⁷ together form a group selected from -0-C(CH₃)₂-0- and

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIG. 1 illustrates that glucocorticoid drugs and other compounds regulate the intracellular distribution of arr2-GFP in cells stably over-expressing Smo-633 and arr2- GFP. In B-J, representative images of three independent experiments are shown. Scale bar, 10 μηι, Cyc: cyclopamine; Pur: Purmorphamine. In K, Data were acquired in triplicate from three independent experiments and are presented as mean ± s.e.m.

[0014] FIG. 2 illustrates that Smo agonists specifically induce Smo-YFP internalization. (A-H) Smo-YFP internalization. Arrows: internalized Smo-YFP. (I-N) Vasopressin 2 receptor- GFP (V2R-GFP) internalization. Arrows: internalized V2R-GFP. (A-N) representative images of three independent experiments are shown. Scale bar, 10 μηι. [0015] FIG. 3 illustrates that Smo agonists competitively replace Bodipy-cyclopamine binding to Smo. (A) Bodipy-cyclopamine saturation binding to over-expressed Smo in

HEK293 cells. (B) Competitive binding of Bodipy-cyclopamine with Smo agonists in HEK293 cells. Data were acquired in triplicate from three independent experiments and are presented as mean ± s.e.m.

[0016] FIG. 4 illustrates the Gli-luciferase response in Shh-LIGHT2 cells treated with Smo ligands. (A) Gli-luciferase reporter activity in Shh-LIGHT2 cells in response to Smo agonists. Results are presented as mean ± s.e.m from multiple individual experiments (n>3) performed in triplicate. (B) The effects of Shh conditioned media (Shh) on Smo agonists. Results are presented as mean ± s.e.m from multiple individual experiments (n≥ 3) performed in triplicate.

[0017] FIG. 5 illustrates effects of glucocorticoids and Shh on primary neuronal granule cell precursor (GCP) proliferation. (A) Primary neuronal GCP proliferation data of Smo agonists. The right panel shows an expanded version of the boxed region of the left panel. Data were acquired in triplicate from three independent experiments, and are presented as mean ± s.e.m. (B) Shh modulation of primary neuronal GCP proliferation in response to Smo agonists. Triplicate data are presented as mean ± s.e.m. n=3. (C) Halcinonide and

dexamethasone have opposite effects on primary neuronal GCP proliferation. Dashed lines indicate the response cells to DMSO vehicle, 2% Shh and 20% Shh. Data acquired in triplicate are presented as mean ± s.e.m. with n=3.

[0018] FIG. 6 illustrates that halcinonide, fluticasone propionate, clobetasol, fluocinonide, and other glucocorticoids activate glucocorticoid receptor GFP (GR-GFP) and regulate Cyclin D2 expression and Caspase 3 degradation. (A-H) Glucocorticoids induced GR-GFP nuclear translocation. Scale bar: 5 μηι. (I) Regulation of Cyclin D2 expression and Caspase 3 degradation in primary neuronal GCPs by Smo ligands and glucocorticoids.

[0019] FIG. 7 illustrates that glucocorticoid drugs halcinonide, fluticasone propionate, clobetasol, and fluocinonide, as well as cyclopamine, SAG, and Purmorphamine, regulate the membrane distribution of 3arr2- GFP in cells stably over-expressing wild type Smo and 3arr2-GFP. In A-H, representative images of three independent experiments are shown. Cyc: cyclopamine; Pur, Purmorphamine. Scale bar: 10 μηι.

[0020] FIG. 8 illustrates signaling of Smo agonists in Smo-/- MEF cells and in NIH 3T3 cells. Data are from three individual experiments performed in triplicate and presented as mean ± s.e.m.

[0021] FIG. 9 illustrates GCP proliferative response to Smo agonists in the presence of Ru-486. (A) Ru-486 alone and GCP proliferation. (B) Effect of RU-486 on Smo agonist- stimulated GCP proliferation. Data obtained in triplicate are presented as mean ± s.e.m, n=3. DETAILED DESCRIPTION

[0022] Described herein are methods of stimulating the proliferation of stem cells using glucocorticoid compounds, which may be Smoothened agonists. Also disclosed are methods of stimulating hair growth, methods of regenerating or repairing damaged tissue, methods of treating disorders such as Parkinson's disease, and methods of promoting wound healing comprising administration of a glucocorticoid compound or a composition comprising the compound.

Definitions

[0023] "AlkyI" refers to a saturated or unsaturated hydrocarbon chain having 1 to 18 carbon atoms, suitably 1 to 12 carbon atoms, or 1 to 6 carbon atoms, or 1 to 4 carbon atoms. "Lower alkyl" refers to a saturated or unsaturated hydrocarbon chain having 1 to 4 carbon atoms. Alkyl groups may be straight or branched. In some embodiments, branched alkyl groups have one or two branches. Unsaturated alkyl groups have one or more double bonds and/or one or more triple bonds. Suitably, unsaturated alkyl groups have one or two double bonds or one triple bond. Alkyl chains may be unsubstituted or substituted with from 1 to about 4 substituents unless otherwise specified. Suitably, alkyl groups are mono-, di-, or tri-substituted. Suitable alkyl substituents include, but are not limited to, cyano, halo, hydroxy, aryl (e.g., phenyl, tolyl, alkyloxphenyl, alkyloxycarbonylphenyl, halophenyl), heterocyclyl, and heteroaryl.

[0024] "Aromatic ring" or "aryl" refers to an aromatic hydrocarbon ring system. Aromatic rings are monocyclic or fused bicyclic ring systems. Monocyclic aromatic rings contain from about 5 to about 10 carbon atoms, suitably from 5 to 7 carbon atoms, or from 5 to 6 carbon atoms in the ring. Bicyclic aromatic rings contain from 8 to 12 carbon atoms, suitably 9 or 10 carbon atoms in the ring. Aromatic rings may be unsubstituted or substituted with from 1 to about 4 substituents on the ring. Suitable aromatic ring substituents include, but are not limited to, halo, cyano, lower alkyl, heteroalkyl, haloalkyl, phenyl, phenoxy or any

combination thereof. Suitably, the aromatic ring substituents are lower alkyl, cyano, halo, or halo alkyl.

[0025] "Carbocycle" refers to a saturated or unsaturated hydrocarbon ring. Carbocycles are not aromatic. Carbocycles are monocyclic, or are fused, spiro, or bridged bicyclic ring systems. Monocyclic carbocycles contain from about 4 to about 10 carbon atoms, suitably from 4 to 7 carbon atoms, or from 5 to 6 carbon atoms in the ring. Bicyclic carbocycles contain from 8 to 12 carbon atoms, suitably from 9 to 10 carbon atoms in the ring.

Carbocycles may be unsubstituted or substituted with from 1 to about 4 substituents on the ring. Suitable carbocycle substituents include, but are not limited to, halo, cyano, lower alkyl, heteroalkyl, haloalkyl, phenyl, phenoxy or any combination thereof. Suitably, the carbocycle substituents are halo or haloalkyl. Suitable carbocycles include, but are not limited to, cyclopentyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl.

[0026] "Halo" or "halogen" refers to fluoro, chloro, bromo, or iodo.

[0027] "Haloalkyl" refers to a straight, branched, or cyclic hydrocarbon substituted with one or more halo substituents. Suitably, the haloalkyl is C Ci₂, or C C₆, or Ci-C₄. Suitable halo substituents include fluoro and chloro. One suitable haloalkyl is trifluoromethyl. "Lower haloalkyl" refers to a haloalkyl having 1 to 4 carbon atoms.

[0028] "Heteroalkyl" refers to a saturated or unsaturated chain containing carbon and at least one heteroatom, wherein no two heteroatoms are adjacent. Heteroalkyl groups contain from 1 to 18 member atoms (carbon and heteroatoms) in the chain, or 1 to 12 member atoms, or 1 to 6 member atoms, or 1 to 4 member atoms. Heteroalkyl groups may be straight or branched. Suitably, the branched heteroalkyl may have one or two branches. Unsaturated heteroalkyl have one or more double bonds and/or one or more triple bonds. Suitably, heteroalkyl groups have one or two double bonds or one triple bond. Heteroalkyl groups may be unsubstituted or substituted with from 1 to about 4 substituents unless otherwise specified. Suitable heteroalkyl substituents include halo, aryl (e.g., phenyl, tolyl, alkyloxyphenyl, alkyloxycarbonylphenyl, halophenyl), heterocyclyl, heteroaryl. For example, alkyl chains substituted with the following substituents are heteroalkyl: alkoxy (e.g., methoxy, ethoxy, propoxy, butoxy, pentoxy), aryloxy (e.g., phenoxy, chlorophenoxy, tolyloxy, methoxyphenoxy, benzyloxy, alkyloxycarbonylphenoxy, acyloxyphenoxy), acyloxy (e.g., propionyloxy, benzoyloxy, acetoxy), carbamoyloxy, carboxy, mercapto, alkylthio, acylthio, arylthio (e.g., phenylthio, chlorophenylthio, alkylphenylthio, alkoxyphenylthio, benzylthio, alkyloxycarbonylphenylthio), amino (e.g., amino, mono- and di-C^C_g alkanylamino, methylphenylamino, methylbenzylamino, C C₃ alkanylamido, carbamamido, ureido, guanidino).

[0029] "Heteroatom" refers to a nitrogen, sulfur, or oxygen atom. Groups containing more than one heteroatom may contain different heteroatoms. As used herein, halogens are not heteroatoms.

[0030] "Heterocycle" refers to a saturated or unsaturated ring containing carbon and from 1 to about 4 heteroatoms in the ring, wherein no two heteroatoms are adjacent in the ring and no carbon in the ring that has a heteroatom attached to it also has a hydroxyl, amino, or thiol group attached to it. Heterocycles are not aromatic. Heterocycles are monocyclic, or are fused or bridged bicyclic ring systems. Monocyclic heterocycles contain from about 4 to about 10 member atoms (carbon and heteroatoms), suitably from 4 to 7 member atoms, or from 5 to 6 member atoms in the ring. Bicyclic heterocycles contain from 8 to 12 member atoms, suitably 9 or 10 member atoms in the ring. Heterocycles may be unsubstituted or substituted with from 1 to about 4 substituents on the ring. Suitably, the substituents are halo or haloalkyi. Suitable heterocycle substituents include: halo, cyano, lower alkyl, heteroalkyi, haloalkyi, phenyl, phenoxy or any combination thereof. Suitable heterocycles include, but are not limited to, piperzyl, morpholinyl, tetrahydrofuranyl, tetrahydropyranyl, and piperdyl.

[0031] "Heteroaryl" refers to an aromatic ring system containing carbon and from 1 to about 4 heteroatoms in the ring. Heteroaryls are monocyclic or fused bicyclic ring systems. Monocyclic heteroaryls contain from about 5 to about 10 member atoms (carbon and heteroatoms), or from 5 to 7 member atoms, or from 5 to 6 member atoms in the ring.

Bicyclic heteroaryls contain from 8 to 12 member atoms, or 9 or 10 member atoms in the ring. Heteroaryls may be unsubstituted or substituted with from 1 to about 4 substituents on the ring. Suitable heteroaryl substituents include: halo, cyano, lower alkyl, heteroalkyi, haloalkyi, phenyl, phenoxy, or any combination thereof. Suitably, the substituents are halo, haloalkyi, or phenyl. Suitable heteroaryls include, but are not limited to, benzothienyl, benzofuranyl, thienyl, thiazolo, purinyl, pyrimidyl, pyridyl, and furanyl.

[0032] "Lower alkyl" refers to an alkyl chain comprised of 1 to 4 carbon atoms, suitably 1 to 3 carbon atoms or 1 to 2 carbon atoms. Lower alkyl groups may be saturated or unsaturated and substituted or unsubstituted. Lower alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, and t-butyl.

[0033] "Lower heteroalkyi" refers to a heteroalkyi chain comprised of 1 to 4 member atoms. Lower heteroalkyi groups may be saturated or unsaturated and substituted or unsubstituted.

[0034] "Member atom" refers to a polyvalent atom (C, O, N, or S atom) in a chain or ring system that continues the chain or ring system. For example, in benzene the six carbon atoms are member atoms and the six hydrogen atoms are not member atoms.

[0035] "Phenyl" refers to a six-membered monocyclic aromatic ring which may or may not be substituted with from about 1 to about 4 substituents. The substituents may be substituted at the ortho, meta or para position on the phenyl ring, or any combination thereof. Suitable phenyl substituents include: halo, cyano, lower alkyl, heteroalkyi, haloalkyi, phenyl, phenoxy or any combination thereof.

[0036] The above groups, whether alone or part of another substituent, may themselves optionally be substituted with one or more groups selected from themselves and the additional substituents listed below.

[0037] Halo: -F, -CI, -Br, and -I.

[0038] Hydroxy: -OH.

[0039] Ether: -OR, wherein R is an ether substituent, for example, a C_1-7 alkyl group (also referred to 15 as a Ci_-7 alkoxy group), a C₃.₂₀ heterocyclyl group (also referred to as a C3-20 heterocyclyloxy group), or a C₅.₂₀ aryl group (also referred to as a C₅.₂₀ aryloxy group), preferably a Ci_-7 alkyl group.

[0040] Nitro: -N0₂.

[0041] Cyano (nitrile, carbonitrile) : -CN.

[0042] Acyl (keto): -C(=0)R, wherein R is an acyl substituent, for example, H, a Ci_₇ alkyl group (also referred to as d-₇ alkylacyl or Ci-₇ alkanoyl), a C₃-₂₀ heterocyclyl group (also referred to as C₃-₂₀ heterocyclylacyl), or a C₅-₂o aryl group (also referred to as C₅-₂o arylacyl), preferably a Ci_₇ alkyl group. Examples of acyl groups include, but are not limited to, - C(=0)CH₃ (acetyl), -C(=0)CH₂CH₃ (propionyl), -C(=0)C(CH₃)₃ (butyryl), and -C(=0)Ph (benzoyl, phenone).

[0043] Carboxy (carboxylic acid) : -COOH.

[0044] Ester (carboxylate, carboxylic acid ester, oxycarbonyl) : -C(=0)OR, wherein R is an ester substituent, for example, a Ci_-7 alkyl group, a C₃-₂₀ heterocyclyl group, or a C5-₂₀ aryl group, preferably a Ci_-7 alkyl group. Examples of ester groups include, but are not limited to, - C(=0)OCH₃, -C(=0)OCH₂CH₃, -C(=0)OC(CH₃)₃, and -C(=0)OPh.

[0045] Amido (carbamoyl, carbamyl, aminocarbonyl, carboxamide): -C(=0)NR R², wherein R and R² are independently amino substituents, as defined for amino groups.

Examples of amido groups include, but are not limited to, -C(=0)NH₂, -C(=0)NHCH₃, - C(=0)N(CH₃)₂, -C(=0)NHCH₂CH₃, and -C(=0)N(CH₂CH₃)₂. as well as amido groups in which R and R², together with the nitrogen atom to which they are attached, form a heterocyclic structure as in, for example, piperidinocarbonyl, morpholinocarbonyl,

thiomorpholinocarbonyl, and piperazinylcarbonyl.

[0046] Amino: -NR R², wherein R and R² are independently amino substituents, for example, hydrogen, a Ci_₇ alkyl group (also referred to as Ci-₇ alkylamino or di-Ci_-7 alkylamino), a C₃.₂₀ heterocyclyl group, or a C₅-₂₀ aryl group, preferably H or a Ci_-7 alkyl group, or, in the case of a "cyclic" amino group, R and R², taken together with the nitrogen atom to which they are attached, form a heterocyclic ring having from 4 to 8 ring atoms. Examples of amino groups include, but are not limited to, -NH₂, -NHCH₃, -NHCH(CH₃)₂, -N(CH₃)₂, - N(CH₂CH₃)₂, and -NHPh. Examples of cyclic amino groups include, but are not limited to, aziridinyl, azetidinyl, pyrrolidinyl, piperidino, piperazinyl, perhydrodiazepinyl, morpholino, and thiomorpholino. In particular, the cyclic amino groups may be substituted on their ring by any of the substituents defined here, for example carboxy, carboxylate and amido.

[0047] Acylamido (acylamino) : -NR C(=0)R², wherein R is an amide substituent, for example, hydrogen, a Ci_-7 alkyl group, a C₃.₂₀ heterocyclyl group, or a C₅.₂₀ aryl group, preferably H or a C_{1 -7} alkyl group, most preferably H, and R² is an acyl substituent, for example, a C_{1 -7} alkyl group, a C₃.₂₀ heterocyclyl group, or a C₅.₂₀ aryl group, preferably a Ci_-7 alkyl group. Examples of acylamide groups include, but are not limited to, -NHC(=0)CH₃ , - NHC(=0)CH₂CH₃, and -NHC(=0)Ph. R and R² may together form a cyclic structure, as in, for example, succinimidyl, maleimidyl, and phthalimidyl.

[0048] Ureido: -N(R )CONR²R³ wherein R² and R³ are independently amino substituents, as defined for amino groups, and R is a ureido substituent, for example, hydrogen, a Ci_-7 alkyl group, a C₃.₂₀ heterocyclyl group, or a C₅.₂₀ aryl group, preferably hydrogen or a Ci_-7 alkyl group. Examples of ureido groups include, but are not limited to, -NHCONH₂, - NHCONHMe, -NHCONHEt, -NHCONMe₂, -NHCONEt₂, -NMeCONH₂, -NMeCONHMe, - NMeCONHEt, NMeCONMe₂, -NMeCONEt₂, and -NHCONHPh.

[0049] Acyloxy (reverse ester): -OC(=0)R, wherein R is an acyloxy substituent, for example, a 0_1-7 alkyl group, a C₃-₂₀ heterocyclyl group, or a C₅-₂₀ aryl group, preferably a Ci_-7 alkyl group. Examples of acyloxy groups include, but are not limited to, -OC(=0)CH₃

(acetoxy), -OC(=0)CH₂CH₃, -OC(=0)C(CH₃)₃, -OC(=0)Ph, -OC(=0)C₆H₄F, and - OC(=0)CH₂Ph.

[0050] Oxo: =0.

[0051] Thiol: -SH.

[0052] Thioether (sulfide): -SR, wherein R is a thioether substituent, for example, a Ci_-7 alkyl group (also referred to as a C _-7 alkylthio group), a C₃.₂₀ heterocyclyl group, or a C₅.₂₀ aryl group, preferably a C_1-7 alkyl group. Examples of C_{1 7} alkylthio groups include, but are not limited to, -SCH₃ and -SCH₂CH₃.

[0053] Sulfoxide (sulfinyl): -S(=0)R, wherein R is a sulfoxide substituent, for example, a Ci-7 alkyl group, a C₃.₂₀ heterocyclyl group, or a C₅.₂₀ aryl group, preferably a Ci_₇ alkyl group. Examples of sulfoxide groups include, but are not limited to, -S(=0)CH₃ and -S(=0)CH₂CH₃.

[0054] Sulfonyl (sulfone): -S(=0)₂R, wherein R is a sulfone substituent, for example, a Ci-7 alkyl group, a C₃.₂₀ heterocyclyl group, or a C₅-₂₀ aryl group, preferably a Ci-₇ alkyl group. Examples of sulfone groups include, but are not limited to, -S(=0)₂CH₃ (methanesulfonyl, mesyl), -S(=0)₂CF₃, -S(=0)₂CH₂CH₃, and 4-methylphenylsulfonyl (tosyl).

[0055] Thioamido (thiocarbamyl): -C(=S)NR R², wherein R and R² are independently amino substituents, as defined for amino groups. Examples of amido groups include, but are not limited to, -C(=S)NH₂, -C(=S)NHCH_a, -C(=S)N(CH₃)₂, and -C(=S)NHCH₂CH₃.

[0056] Sulfonamino: -NR S(=0)₂R, wherein R is an amino substituent, as defined for amino groups, and R is a sulfonamino substituent, for example, a Ci-₇ alkyl group, a C₃.₂₀ heterocyclyl group, or a C₅-₂₀ aryl group, preferably a Ci_-7 alkyl group. Examples of sulfonamino groups include, but are not limited to, -NHS(=0)₂CH₃, -NHS(=0)₂Ph and - N(CH₃)S(=0)₂C₆H₅.

[0057] In the context of treating a disorder, the term "effective amount" as used herein refers to an amount of the compound or a composition comprising the compound which is effective, upon single or multiple dose administrations to a subject, in treating a cell, or curing, alleviating, relieving or improving a symptom of the disorder. An effective amount of the compound or composition may vary according to the application. In the context of treating a disorder, an effective amount may depend on factors such as the disease state, age, sex, and weight of the individual, and the ability of the compound to elicit a desired response in the individual. In the context of stimulating the proliferation of stem cells, an effective amount of a compound or composition refers to an amount that increases the number of cells, for example, in creases the proliferation of cells and/or survival of cells. In a further example, an effective amount of a compound is an amount that produces a statistically significant increase in the number of cells (e.g., cells in a culture) as compared to a control, such as cells (e.g., a culture of cells) not treated with the compound.

Compounds

[0058] Compounds that may be used in the methods described herein include glucocorticoids, which are a class of steroid hormones that bind to the glucocorticoid receptor. The basic skeleton of a steroid is illustrated below, showing the standard stereo orientations and numbering of the carbon atoms in the structure on the left, and lUPAC recommended ring lettering of the gonane steroid nucleus on the right. In both structures, R is a side chain.

wherein:

each— - independently represents the presence or absence of a bond;

R⁶ is selected from H and halo;

R⁹ is selected from H and halo; R is selected from H, hydroxy, oxo and halo;

R ⁶ is selected from alkyl, -OR', -SR\ -N(R')₂, -NH-C(0)R" and -NR-S(0)_n-R", or may be taken together with R ⁷ to form a ring;

Y is selected from a bond, O and S;

X is selected from a bond and CH₂;

W is selected from alkyl, hydroxy, halo and -OC(0)R";

each R' is independently H, alkyl, aryl, heterocyclyl or heteroaryl;

each R" is independently alkyl;

Z is O or S; and

n is 1 or 2;

[0060] In some embodiments, in the compound of formula (I):

R⁶ is selected from H and halo;

R⁹ is halo;

R is selected from hydroxy and oxo;

R ⁶ is alkyl, or may be taken together with R ⁷ to form a ring;

[0061] In some embodiments, R⁶ is hydrogen. In some embodiments, R⁶ is halo (e.g., fluoro). In some embodiments, R⁹ is halo (e.g., fluoro). In some embodiments, R is hydroxy. In some embodiments, R is oxo. In some embodiments, R ⁶ is alkyl (e.g., methyl). In some embodiments, R ⁷ is hydroxy. In some embodiments, R ⁷ is -OC(0)CH₂CH₃. In some embodiments, R ⁶ and R ⁷ are taken together to form a ring; for example, R ⁶ and R ⁷

may together form a group selected from -0-C(CH₃)₂-0- and

In some

embodiments, the group -Y-X-W is selected from the group consisting of -CH₂-CI, -S-CH₂-F, -CH₂-OAc, -CH₂-OH, -CH₃ and -CH₂-I.

[0062] In some embodiments, a compound of formula (I) may be selected from the group consisting of:

[0063] Additional compounds may also be suitable for use in the methods described herein, for example, molecules in which modifications are made to the A and B rings. Exemplary A and B ring modifications are illustrated below.

12

wherein:

each— - independently represents the presence or absence of a bond;

each R³ is independently H, oxo, hydroxy, amino, -OR^b, -NH-C(0)-R^b, -NH-S(0)_n-R^b, and heterocyclyl (e.g., triazine);

each R⁵ is independently hydrogen (alpha or beta);

each R⁶ is independently selected from H and halo (e.g., fluoro);

each R⁹ is independently selected from H and halo (e.g., fluoro);

each A is independently NR^b, N-OH or N-OR^b;

each D is independently O, NH or NR^b;

each Q is independently H, alkyl, haloalkyl or halo;

each R^a is independently H or alkyl;

each R^b is independently alkyl;

E is CQ or N;

G is O or S;

J¹ is CQ or N;

E² is CQ or N;

G² is CQ or N;

J² is O, S or NR^b;

E³ is O, S or NR^b;

G³ is CQ or N;

J³ is CQ or N; and

each n is independently 1 or 2.

[0064] In the chemical structures shown herein, a dashed line represents either the presence or absence of a bond. Accordingly, substitution at these positions will be consistent with standard valences. For example, when the dashed line between the group R and the carbon to which it is attached is absent, the bond between the two groups is a single bond, and R may be H, hydroxy, or halo. When the dashed line between R and the carbon to which it is attached is present, resulting in a double bond, R may be oxo.

[0065] In accordance with a convention used in the art, the group:

is used in structural formulas herein to depict the bond that is the point of attachment of the moiety or substituent to the core or backbone structure.

[0066] For compounds described herein, groups and substituents thereof may be selected in accordance with permitted valence of the atoms and the substituents, such that the selections and substitutions result in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc.

[0067] Where substituent groups are specified by their conventional chemical formulae, written from left to right, they optionally encompass substituents resulting from writing the structure from right to left, e.g., -CH₂0- optionally also recites -OCH₂-.

[0068] The compounds described herein include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds may have the present structures except for the replacement of hydrogen by deuterium or tritium, or the replacement of a carbon by a ³C- or ⁴C-enriched carbon.

[0069] A compound described herein can be in the form of a salt, e.g., a

pharmaceutically acceptable salt. The term "pharmaceutically acceptable salt" includes salts of the active compounds that are prepared with relatively nontoxic acids or bases, depending on the particular substituents found on the compounds described herein. Neutral forms of the compounds may be regenerated by contacting the salt with a base or acid and isolating the parent compound in a conventional manner. The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents, but otherwise the salts are equivalent to the parent form of the compound for the purposes of this disclosure.

[0070] In addition to salt forms, the present invention may also provide compounds that are in a prodrug form. Prodrugs of the compounds described herein are those compounds that readily undergo chemical changes under physiological conditions to provide the compounds described herein. Prodrugs can be converted to the compounds of the present invention by chemical or biochemical methods in an ex vivo environment. For example, prodrugs can be slowly converted to the compounds of the present invention when placed in a transdermal patch reservoir with a suitable enzyme or chemical reagent. [0071] A compound described herein can be, for example, an enantiomerically enriched isomer of a stereoisomer described herein. Enantiomer, as used herein, refers to either of a pair of chemical compounds whose molecular structures have a mirror-image relationship to each other. For example, the compound may have an enantiomeric excess of at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%.

[0072] A preparation of a compound disclosed herein may be enriched for an isomer of the compound having a selected stereochemistry, e.g., R or S, corresponding to a selected stereocenter. For example, the compound may have a purity corresponding to a compound having a selected stereochemistry of a selected stereocenter of at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%. A compound can, for example, include a preparation of a compound disclosed herein that is enriched for a structure or structures having a selected stereochemistry, e.g., R or S, at a selected stereocenter.

[0073] In some embodiments, a preparation of a compound disclosed herein may be enriched for isomers (subject isomers) which are diastereomers of a compound described herein. Diastereomer, as used herein, refers to a stereoisomer of a compound having two or more chiral centers that is not a mirror image of another stereoisomer of the same compound. For example, the compound may have a purity corresponding to a compound having a selected diastereomer of at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%.

[0074] When no specific indication is made of the configuration at a given stereocenter in a compound described herein, any one of the configurations or a mixture of configurations is intended. For example, for the substituent R ⁶ shown in the compound of Formula (I), both the R and S configurations (also described as a and β in the context of steroids) are included.

[0075] Compounds may be prepared in racemic form or as individual enantiomers or diastereomers by either stereospecific synthesis or by resolution. The compounds may, for example, be resolved into their component enantiomers or diastereomers by standard techniques, such as the formation of stereoisomeric pairs by salt formation with an optically active base, followed by fractional crystallization and regeneration of the free acid. The compounds may also be resolved by formation of stereoisomeric esters or amides, followed by chromatographic separation and removal of the chiral auxiliary. Alternatively, the compounds may be resolved using a chiral HPLC column. The enantiomers also may be obtained from kinetic resolution of the racemate of corresponding esters using lipase enzymes.

[0076] A compound described herein can also be modified by appending appropriate functionalities to enhance selective biological properties. Such modifications are known in the art and include those that increase biological penetration into a given biological system (e.g., blood, lymphatic system, central nervous system), increase oral availability, increase solubility to allow administration by injection, alter metabolism, and/or alter rate of excretion. Examples of these modifications include, but are not limited to, esterification with

polyethylene glycols, derivatization with pivolates or fatty acid substituents, conversion to carbamates, hydroxylation of aromatic rings, and heteroatom substitution in aromatic rings.

[0077] Compounds that may be used in the methods described herein may be

commercially available, or may be synthesized using commercially available starting materials. Methods of synthesizing the compounds of the formulae herein will be evident to those of ordinary skill in the art. Synthetic chemistry transformations and protecting group methodologies (protection and deprotection) useful in synthesizing the compounds described herein are known in the art and include, for example, those such as described in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T.W. Greene and P.G.M. Wuts, Protective Groups in Organic Synthesis, 2d. Ed., John Wiley and Sons (1991 ); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995), and subsequent editions thereof.

[0078] Compounds of formula (I) described herein may be Smoothened agonists. Such compounds may activate Hedgehog signaling and promote the proliferation of stem cells, such as primary neuronal stem/precursor cells, alone and/or synergistically in the presence of Sonic Hedgehog. The term "agonist" refers to an agent which potentiates or recapitulates the bioactivity of the Hedgehog pathway, such as to activate transcription of target genes. Compounds may mimic or enhance the activity or effect of a Hedgehog protein in a

Smoothened-dependent manner. The term "agonist" as used herein refers not only to any agent that may act by directly activating the normal function of the Smoothened protein, but also to any agent that activates the Hedgehog signaling pathway, and thus inhibits the function of Patched.

Methods of Assaying Compounds

[0079] The interaction of Hedgehog ligand with the membrane protein Patched (Ptc) enables the seven transmembrane receptor Smo to activate downstream Gli transcription factors. Activated Smo shares important behaviors with canonical G protein-coupled receptors (GPCRs), including an ability to undergo GPCR kinase phosphorylation and to recruit -arrestin2 proteins for endocytosis.

[0080] Cyclopamine, a naturally occurring steroid alkaloid, inhibits the constitutive activity of Smo via a direct antagonism, preventing its phosphorylation and interaction with β- arrestin2. This observation can be exploited in high throughput, high content screens for Smo ligands. For example, when expressed in U20S cells without exogenous added Smo, arr2-GFP is distributed homogenously throughout the cytoplasm (Fig. 1 B). The over- expression of Smo or a tail-substitution mutant of Smo, Smo-633, in DMSO treated U20S cells causes a redistribution of arr2-GFP to intracellular vesicles/aggregates (Fig. 1 C). The Smo antagonist cyclopamine at 100 nM or greater concentration reverses this effect and forces arr2-GFP back into a homogeneous distribution (Fig. 1 D). Intra-vesicular

aggregation of 3arr2-GFP can be restored in the presence of 100 nM cyclopamine with 5 μΜ of the known small-molecule Smo agonists SAG or Purmorphamine (Fig. 1 E, F).

Accordingly, a Smo agonist can be identified by its ability to aggregate arr2-GFP in U20S cells in the presence of 100 nM cyclopamine in the steady state model.

[0081] It has been demonstrated that SAG induces Smo internalization (Chen (2004) Science 306(5705):2257-2260). Accordingly, in HEK293 cells expressing Smoothened- yellow fluorescent protein (Smo-YFP), the effects of compounds on Smo-YFP internalization can be examined by confocal microscopy. Induction of Smo-YFP internalization is

consistent with a role as an as Smo agonists.

[0082] Bodipy-cyclopamine has been used to assess ligand binding to Smo (Chen (2002) Proc Natl Acad Sci USA 99(22):14071 -14076). Accordingly, competitive binding assays can be performed to determine if a compound is a Smo agonist.

[0083] Sonic Hedgehog binding to Patched relieves Patched inhibition of Smo, and results in activation of the Gli transcription factor. Accordingly, Gli-luciferase reporter assays may be used as indicators of activity downstream of Smo. SAG was discovered using such a Gli reporter assay.

[0084] Finally, cerebellar GCPs differentiate into distinct types of mature neurons that comprise the most abundant neurons in the brain, and the expansion in vivo of these granule precursor cells requires Hedgehog/Smo pathway signaling. A mouse GCP proliferation assay may used to test the growth promoting effects of potential Smo agonist compounds.

[0085] Accordingly, in one aspect, the present invention may provide a method of screening compounds for Smo agonist activity.

Compositions and Routes of Administration

[0086] The compound or a pharmaceutical composition comprising the compound may be administered to a subject by any convenient route of administration, whether

systemically/peripherally or at the site of desired action, including but not limited to, oral (e.g. by ingestion); topical (including e.g. transdermal, intranasal, ocular, buccal, and sublingual); pulmonary (e.g. by inhalation or insufflation therapy using, e.g. an aerosol, e.g. through mouth or nose); rectal; vaginal; parenteral, for example, by injection, including subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid, and intrasternal; by implant of a depot, for example,

subcutaneously or intramuscularly. Additional modes of administration may include adding the compound and/or a composition comprising the compound to a food or beverage, including a water supply for an animal, to supply the compound as part of the animal's diet.

[0087] The subject may include, without limitation, a eukaryote, an animal, a vertebrate animal, a bird, a reptile, an insect, a mammal, a rodent (e.g., a guinea pig, a hamster, a rat, a mouse), canine (e.g., a dog), feline (e.g., a cat), equine (e.g., a horse), ovine (e.g., a sheep), bovine (e.g., a cow), a primate such as a monkey (e.g. marmoset, baboon) or an ape (e.g. gorilla, chimpanzee, orangutan, gibbon), or a human.

[0088] While it is possible for the compound to be administered alone, in some embodiments the compound may be presented as a pharmaceutical composition (e.g., formulation) comprising at least one compound, as defined above, together with one or more pharmaceutically-acceptable carriers, adjuvants, excipients, diluents, fillers, buffers, stabilizers, preservatives, lubricants, or other materials well known to those skilled in the art and optionally other therapeutic or prophylactic agents.

[0089] Thus, the present invention further provides pharmaceutical compositions, as defined above, and methods of making a pharmaceutical composition comprising admixing at least one compound, as defined above, together with one or more pharmaceutically acceptable carriers, excipients, buffers, adjuvants, stabilizers, or other materials, as described herein.

[0090] Suitable carriers, excipients, etc. can be found in standard pharmaceutical texts, for example Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing Company, Easton, Pa., 1990.

[0091] The formulations may conveniently be presented in unit dosage form and may be prepared by any method well known in the art of pharmacy. Such methods include the step of bringing into association the compound with the carrier which constitutes one or more accessory ingredients. In general, the formulations may be prepared by uniformly and intimately bringing into association the compound with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product.

[0092] Formulations may be in the form of liquids, solutions, suspensions, emulsions, elixirs, syrups, tablets, lozenges, granules, powders, capsules, cachets, pills, ampoules, suppositories, pessaries, ointments, gels, pastes, creams, sprays, mists, foams, lotions, oils, boluses, electuaries, or aerosols.

[0093] Formulations suitable for oral administration (e.g., by ingestion) may be presented as discrete units such as capsules, cachets or tablets, each containing a predetermined amount of the compound; as a powder or granules; as a solution or suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion; as a bolus; as an electuary; or as a paste.

[0094] A tablet may be made by conventional means, e.g., compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the compound in a free-flowing form such as a powder or granules, optionally mixed with one or more binders (e.g., povidone, gelatin, acacia, sorbitol, tragacanth, hydroxypropylmethyl cellulose); fillers or diluents (e.g., lactose, microcrystalline cellulose, calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc, silica); disintegrants (e.g., sodium starch glycolate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose); surface-active or dispersing or wetting agents (e.g., sodium lauryl sulfate); and preservatives (e.g., methyl p-hydroxybenzoate, propyl p-hydroxybenzoate, sorbic acid). Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the compound therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile. Tablets may optionally be provided with an enteric coating, to provide release in parts of the gut other than the stomach.

[0095] Formulations suitable for topical administration (e.g., transdermal, intranasal, ocular, buccal, and sublingual) may be formulated as an ointment, cream, suspension, lotion, powder, solution, past, gel, spray, aerosol, or oil. Alternatively, a formulation may comprise a patch or a dressing such as a bandage or adhesive plaster impregnated with compounds and optionally one or more excipients or diluents. In addition, a formulation may be added to a conventional bandage, e.g. to a gauze portion that contacts a wound, as an antimicrobial agent.

[0096] Formulations suitable for topical administration in the mouth include lozenges comprising the compound in a flavored basis, usually sucrose and acacia or tragacanth; pastilles comprising the compound in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the compound in a suitable liquid carrier.

[0097] Formulations suitable for topical administration to the eye also include eye drops wherein the compound is dissolved or suspended in a suitable carrier, especially an aqueous solvent for the compound.

[0098] Formulations suitable for nasal administration, wherein the carrier is a solid, include a coarse powder having a particle size, for example, in the range of about 20 to about 500 microns which is administered in the manner in which snuff is taken, i.e., by rapid inhalation through the nasal passage from a container of the powder held close up to the nose. Suitable formulations wherein the carrier is a liquid for administration as, for example, nasal spray, nasal drops, or by aerosol administration by nebulizer, include aqueous or oily solutions of the compound.

[0099] Formulations suitable for administration by inhalation include those presented as an aerosol spray from a pressurized pack, with the use of a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gases.

[00100] Formulations suitable for topical administration via the skin include ointments, creams, and emulsions. When formulated in an ointment, the compound may optionally be employed with either a paraffinic or a water-miscible ointment base. Alternatively, the compounds may be formulated in a cream with an oil-in-water cream base. If desired, the aqueous phase of the cream base may include, for example, at least about 30% w/w of a polyhydric alcohol, i.e., an alcohol having two or more hydroxyl groups such as propylene glycol, butane-1 ,3-diol, mannitol, sorbitol, glycerol and polyethylene glycol and mixtures thereof. The topical formulations may desirably include a compound which enhances absorption or penetration of the compound through the skin or other affected areas.

Examples of such dermal penetration enhancers include dimethylsulfoxide and related analogues.

[00101 ] When formulated as a topical emulsion, the oily phase may optionally comprise merely an emulsifier (otherwise known as an emulgent), or it may comprises a mixture of at least one emulsifier with a fat or an oil or with both a fat and an oil. Preferably, a hydrophilic emulsifier is included together with a lipophilic emulsifier which acts as a stabilizer. It is also preferred to include both an oil and a fat. Together, the emulsifier(s) with or without stabilizer(s) make up the so-called emulsifying wax, and the wax together with the oil and/or fat make up the so-called emulsifying ointment base which forms the oily dispersed phase of the cream formulations.

[00102] Suitable emulgents and emulsion stabilizers include Tween 60, Span 80, cetostearyl alcohol, myristyl alcohol, glyceryl monostearate and sodium lauryl sulfate. The choice of suitable oils or fats for the formulation is based on achieving the desired cosmetic properties, since the solubility of the compound in most oils likely to be used in

pharmaceutical emulsion formulations may be very low. Thus the cream should preferably be a non-greasy, non-staining and washable product with suitable consistency to avoid leakage from tubes or other containers. Straight or branched chain, mono- or dibasic alkyl esters such as diisoadipate, isocetyl stearate, propylene glycol diester of coconut fatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate or a blend of branched chain esters known as Crodamol CAP may be used, the last three being preferred esters. These may be used alone or in combination depending on the properties required. Alternatively, high melting point lipids such as white soft paraffin and/or liquid paraffin or other mineral oils can be used.

[00103] Formulations suitable for rectal administration may be presented as a suppository with a suitable base comprising, for example, cocoa butter or a salicylate.

[00104] Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the compound, such carriers as are known in the art to be appropriate.

[00105] Formulations suitable for parenteral administration (e.g., by injection, including cutaneous, subcutaneous, intramuscular, intravenous and intradermal), include aqueous and non-aqueous isotonic, pyrogen-free, sterile injection solutions which may contain antioxidants, buffers, preservatives, stabilizers, bacteriostats in addition to the compound, and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents, and liposomes or other microparticulate systems which are designed to target the compound to blood components or one or more organs. Examples of suitable isotonic vehicles for use in such formulations include Sodium Chloride Injection, Ringer's Solution, or Lactated Ringer's Injection. Typically, the concentration of the compound in the solution is from about 1 ng/ml to about 1 μg ml, although other concentrations are possible and are encompassed within the invention. The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets. Formulations may be in the form of liposomes or other microparticulate systems which are designed to target the compound to blood components or one or more organs.

[00106] It will be appreciated that appropriate dosages of the compounds, and compositions comprising the compounds, can vary from patient to patient. Determining the optimal dosage will generally involve the balancing of the level of therapeutic benefit against any risk or deleterious side effects of the treatments of the present invention. The selected dosage level will depend on a variety of factors including, but not limited to, the activity of the particular compound, the route of administration, the time of administration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds, and/or materials used in combination, and the age, sex, weight, condition, general health, and prior medical history of the patient. The amount of compound and route of administration will ultimately be at the discretion of the physician, although generally the dosage will be to achieve local concentrations at the site of action which achieve the desired effect without causing substantial harmful or deleterious side-effects. [00107] Administration in vivo can be effected in one dose, continuously or intermittently (e.g., in divided doses at appropriate intervals) throughout the course of treatment. Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the formulation used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician.

[00108] In general, a suitable dose of the compound is in the range of about 100 μg to about 250 mg per kilogram body weight of the subject per day. Where the compound is a salt, an ester, prodrug, or the like, the amount administered is calculated on the basis of the parent compound and so the actual weight to be used is increased proportionately.

[00109] Further information regarding formulation of and treatment with pharmaceutical compositions are disclosed in United States Patent 7,196,085, incorporated herein by reference.

[00110] The composition may be administered once, on a continuous basis (e.g. by an intravenous drip), or on a periodic/intermittent basis, including about once per hour, about once per two hours, about once per four hours, about once per eight hours, about once per twelve hours, about once per day, about once per two days, about once per three days, about twice per week, about once per week, and about once per month. The composition may be administered until a desired reduction of symptoms is achieved.

[00111 ] The present compounds, compositions, and methods may be administered as part of a therapeutic regimen along with other treatments appropriate for the particular injury or disease being treated. For example, in the case of Parkinson's disease, a present compound or composition may be administered in combination with L-dopa or other

Parkinson's disease medications, or in combination with a cell based neuronal

transplantation therapy for Parkinson's disease. In the case of an injury to the spinal cord, a present compound may be administered in combination with physical therapy, hydrotherapy, massage therapy, and the like. In the case of myocardial infarction, the present compound may be administered along with angioplasty, surgery, blood pressure medication, and/or as part of an exercise and diet regimen.

Methods of Use

[00112] The compounds described herein may be used in a variety of methods, such as methods of stimulating the proliferation of stem cells, methods of stimulating hair growth, methods of regenerating or repairing damaged tissue, methods of treating disorders such as Parkinson's disease, and methods of promoting wound healing. Proliferation of stem cells

[00113] A compound described herein may be useful in cell culture techniques to promote the proliferation of stem cells. For example, in vitro neuronal culture systems have proved to be fundamental and indispensable tools for the study of neural development, as well as the identification of neurotrophic factors such as nerve growth factor (NGF), ciliary trophic factors (CNTF), and brain derived neurotrophic factor (BDNF). One use of the present invention may be in cultures of neuronal stem cells, such as in the use of such cultures for the generation of new neurons and glia. In such embodiments of the present invention, the cultured cells may be contacted with a compound described herein in order to alter the rate of proliferation of neuronal stem cells in the culture and/or alter the rate of differentiation, or to maintain the integrity of a culture of certain terminally differentiated neuronal cells. In an exemplary embodiment, the present invention may be used to culture, for example, sensory neurons or, alternatively, motor neurons. Such neuronal cultures may be used as convenient assay systems as well as sources of implantable cells for therapeutic treatments. According to the present invention, large numbers of non-tumorigenic neural progenitor cells may be perpetuated in vitro and their rate of proliferation and/or differentiation may be affected by contact with a compound described herein. Generally, a method is provided comprising the steps of isolating neural progenitor cells from an animal, perpetuating these cells in vitro or in vivo, and regulating the differentiation of these cells into particular neural phenotypes, e.g., neurons and glia, by contacting the cells with a compound described herein.

[00114] Thus, in some embodiments, the present invention provides for an in vitro method for growing or culturing cells, comprising contacting the cells with one or more of the present compounds or compositions. In some instances, the cells are progenitor cells, such as neural progenitor cells. In some instances, the cells are neuronal cells or neuronal progenitor cells.

[00115] In certain instances, the present invention may provide a method for inducing differentiation in a cell, for example a progenitor cell. The method may further comprise regulating the differentiation of these cells into particular phenotypes, for example, into neural phenotypes. Thus, one or more of the present compounds or compositions may be used to promote the differentiation of a cell (either a stem cell or a non-stem cell) to a particular differentiated cell type, such as a neuronal cell type including, but not limited to, a dopaminergic neuron, a motor neuron, a serotonergic neuron, an interneuron, a sensory neuron, and the like. In another embodiment, one or more of the present compounds or compositions promotes the differentiation of a cell to a mesodermal cell type including, but not limited to, osteocytes, chondrocytes, blood cells, cells of the immune system, skeletal muscle cells, cardiac muscle cells, smooth muscle cells, cells of the kidney, and the like. In yet another embodiment, one or more of the present compounds or compositions promotes the differentiation of a cell to an endodermal cell type including, but not limited to, pancreatic cell types (such as β-islet cells), hepatocytes, cells of the lung, and cells of the

gastrointestinal tract.

[00116] By "progenitor" it is meant an oligopotent or multipotent stem cell which is able to divide without limit and, under specific conditions, may produce daughter cells which terminally differentiate such as into neurons and glia. These cells may be used for

implantation into a heterologous or autologous host. By heterologous is meant a host other than the animal from which the progenitor cells were originally derived. By autologous is meant the identical host from which the cells were originally derived.

[00117] Cells may be placed into any known culture medium capable of supporting cell growth, including MEM, DMEM, RPMI, F- 12, and the like, containing supplements which are required for cellular metabolism such as glutamine and other amino acids, vitamins, minerals and useful proteins such as transferrin and the like. Medium may also contain antibiotics to prevent contamination with yeast, bacteria and fungi such as penicillin, streptomycin, gentamicin and the like. In some cases, the medium may contain serum derived from bovine, equine, chicken and the like. Conditions for culturing may be close to physiological conditions. The pH of the culture media may be close to physiological pH, for example, between about pH 6-8, such as about pH 7-7.5, for example, about pH 7.4. Cells may be cultured at a temperature close to physiological temperature, for example, between about 30 °C- 40 ^<C, such as between about 32 ^<C-38 °C, for example, between about 35 °C-37 ^<C.

[00118] Stem cells useful in the present invention are generally known. For example, several neural crest cells have been identified, some of which are multipotent and likely represent uncommitted neural crest cells, and others of which may generate only one type of cell, such as sensory neurons, and likely represent committed progenitor cells. The role of the present compounds employed in the present method to culture such stem cells may be to regulate differentiation of the uncommitted progenitor, or to regulate further restriction of the developmental fate of a committed progenitor cell towards becoming a terminally differentiated neuronal cell. For example, the present compounds may be used in vitro to regulate the differentiation of neural crest cells into glial cells, Schwann cells, chromaffin cells, cholinergic sympathetic or parasympathetic neurons, as well as peptidergic and serotonergic neurons. The present compounds may be used alone, or may be used in combination with other neurotrophic factors which act to more particularly enhance a particular differentiation fate of the neuronal progenitor cell. In certain instances the present compounds and compositions may be used to enhance or improve the survival rate of a neuronal cell.

[00119] It has been shown that hedgehog agonists have the ability to bias the

development of a progenitor or biased cells down a particular developmental pathway (see for example, U.S. Published Patent Application No. 2005-0019801 , Wichterle et al. (2002) Cell 1 10: 385-397, and Novitch et al. (2003) Neuron 40(1 ):81 -95). Hence, in one

embodiment, the present compounds may be used to bias the development of a progenitor or biased cell down a particular developmental pathway, i.e., to a particular differentiated cell type. The compounds may be used alone, or in combination with another agent that may bias the development of a progenitor cell, such as a retinoid (e.g., retinoic acid). In some embodiments, the particular differentiated cell type is a neuronal cell type. In yet another embodiment, the neuronal cell type is selected from motor neurons, dopaminergic neurons, cholinergic neurons, interneurons, sensory neurons, serotonergic neurons, peptidergic neurons, astrocytes, and oligodendrocytes.

Hair growth

[00120] A compound described herein may be used to stimulate hair growth in a subject, i.e., for preventing, arresting, and/or reversing hair loss, as well as promoting hair growth. Alopecia, or hair loss, is a common cosmetic problem that can also cause serious negative psychological effects. Because hair loss is a widespread problem that is considered cosmetically unappealing and often causes emotional distress, there is great demand for alopecia treatments.

[00121 ] Hair growth on the scalp does not occur continuously, but rather occurs by a cycle of activity involving alternating periods of growth and rest. This cycle is divided into three main stages; anagen, catagen, and telogen. Anagen is the growth phase of the cycle and is characterized by penetration of the hair follicle deep into the dermis with rapid proliferation of cells which are differentiating to form hair. The next phase is catagen, which is a transitional stage marked by the cessation of cell division, and during which the hair follicle regresses through the dermis and hair growth ceases. The next phase, telogen, is characterized as the resting stage during which the regressed follicle contains a germ with tightly packed dermal papilla cells. At telogen, the initiation of a new anagen phase is caused by rapid cell proliferation in the germ, expansion of the dermal papilla, and elaboration of basement membrane components. When hair growth ceases, most of the hair follicles reside in telogen and anagen is not engaged, thus causing the onset of full or partial baldness. From this model it has become clear that the larger the pool of dividing stem cells that differentiate into hair cells, the more hair growth occurs.

[00122] Accordingly, methods for increasing or reducing hair growth may be carried out by potentiating or inhibiting, respectively, the proliferation of these stem cells. It is well established that the hedgehog pathway impacts the development of hair follicles and that Shh is required for development of follicles beyond the hair germ stage of hair follicle development (Chiang et al. 1999, Dev Biol. 205(l):l-9 and St. Jacques et al. 1998, Curr. Biol. 8(19):1058-68). It has been further shown that post-natally, Shh is expressed in the anagen hair bulb close to the skin surface (Gat et al., 1998, Cell 95(5):605-14; Gambardella et al. 2000, Mech Dev. 96(2):215- 8; and Oro et al. 2003, Dev Biol. 255(2) :238-48). Moreover, Shh, patched, and Gli- 1 expression is upregulated during the anagen stage and down regulated during the telogen stage of the hair cycle. (Sato et al., 1999, J Clin Invest. 104(7):855-64 and Oro et al. 2003, Dev Biol. 255(2):238-48). Infecting mice with a retroviral vector expressing Shh has been shown to induce anagen in telogenic skin (Sato et al., 1999, J Clin Invest. 104(7):855-64). Generally, Shh and pathway genes are expressed during follicle formation and upregulated during post-natal anagen stage. Thus, Shh is important in the growth and maturation of hair follicles and in inducing anagen in the adult hair follicle. For example, disrupting Shh activity, either in knockout mice lacking Shh expression or through

immunoneutralization of endogenous Shh, prevents the normal appearance of hair.

Furthermore, exogenously administered

[00123] Shh, either through local application of Shh protein or through gene therapy using a dermally applied Shh-expressing vector, promotes hair growth.

[00124] In certain embodiments, the present compounds promote, induce, or prolong the anagen stage. For example, administration of the present compounds to follicle cells in the telogen stage may induce the anagen stage in such cells. Hence, the present compounds may also be considered to inhibit, cease, or truncate the telogen stage in follicle cells, for example, in favor of the anagen stage. As such, the present compounds have the ability to regulate the hair cell cycle.

[00125] Consequently, the present invention provides a method for inducing anagen in a telogenic hair follicle, comprising administering one or more of the present compounds or compositions. In certain embodiments, the above method is an ex vivo method. In other embodiments, the one or more present compounds or compositions is administered to a patient. In certain instances, the patient is a human, who, for example, may display a hair loss or growth disorder, for example, male or female pattern baldness. In other instances, the patient is a non-human, for example, a dog or cat. In some applications, the one or more present compounds or compositions is administered orally. In other applications, the one or more present compounds or compositions is administered topically.

[00126] Since the deregulation of the hair cycle is often an underlying cause of a number of hair growth disorders, hedgehog agonists, such as one or more of the present compounds, may be used to treat such disorders. For example, in some instances, the present

compounds possess the ability to regulate hair growth, for example by promoting hair growth and/or by inhibiting or stopping hair loss. Thus, in certain embodiments, the present invention may be employed as a way of promoting the growth of human hair, e.g., to correct baldness, alopecia, or other diseases characterized by hair loss. In addition to other modes of administration described herein, present compounds may be administered topically for the treatment or prevention of hair loss or growth disorders.

[00127] In these embodiments, an "effective amount" is an amount sufficient to stimulate hair growth in a subject treated with the compound. One of skill in the art will understand that effective amounts vary based on factors including the species, sex, age, body surface area, weight, and physical condition of the mammal being treated, as well as the chosen route of administration, the use of pharmaceutical carriers, diluents, or excipients, and any cotherapy, for example, coadministration of additional agents for promoting hair growth. A physician or veterinarian uses standard techniques known in the art to determine the dosage and frequency of administration of a compound to stimulate hair growth according to the methods described herein. Treatment may be continued until the desired hair growth has occurred, and then as needed to sustain such hair growth. When the cause of hair loss is ongoing, such as hair loss due to genetics or aging, treatment can be continued indefinitely to maintain hair growth.

[00128] In some embodiments, the compound can be administered topically to a mammal in one or more areas where hair growth is desired, i.e., target areas. When administered topically, the compound may stimulate hair growth locally in the target area of administration, while causing little undesired hair growth in other areas of the body such as, for example, facial hair in women.

[00129] A compound described herein may be administered alone or in combination with one or more additional hair growth stimulants. Additional hair growth stimulants include, but are not limited to, benzalkonium chloride, benzethonium chloride, phenol, estradiol, diphenhydramine hydrochloride, chlorpheniramine maleate, chlorophyllin derivatives, cholesterol, salicylic acid, cysteine, methionine, red pepper tincture, benzyl nicotinate, D, L- menthol, peppermint oil, calcium pantothenate, panthenol, castor oil, hinokitiol, prednisolone, resorcinol, monosaccharides and esterified monosaccharides, chemical activators of protein kinase C enzymes, glycosaminoglycan chain cellular uptake inhibitors, inhibitors of glycosidase activity, glycosaminoglycanase inhibitors, esters of pyroglutamic acid, hexosaccharic acids or acylated hexosaccharic acids, aryl-substituted ethylenes, N-acylated amino acids, cyclosporins, such as cyclosporin A, potassium channel blockers, such as minoxidil, 5-a-reductase inhibitors, such as finasteride, and androgen receptor antagonists, such as cyproterone acetate. Particularly useful additional hair growth stimulants include minoxidil, finasteride, and cyclosporin A.

Regenerating or repairing damaged tissue

[00130] The compounds and methods described herein may be used in various applications in regenerative medicine. This includes, for example, the process of creating living, functional tissues to repair or replace tissue or organ function lost due to damage, or congenital defects. Exemplary applications in regenerative medicine include those associated with heart failure (myocardial infarction), neuronal injury/degeneration, wound repair, retinal damage, lung injury, liver injury, stroke, osteoporosis and bone fractures.

[00131 ] In these embodiments, an "effective amount" is an amount sufficient to promote the regeneration of cells in the tissue or to promote repair of the tissue.

Myocardial infarction

[00132] Cardiovascular diseases are a leading cause of death, resulting in almost 40% of deaths annually in the United States. Inadequate human myocardial regeneration poses a significant public health problem. It is estimated that 13 million Americans have coronary artery disease, and more than half a million experience a myocardial infarction every year. Human cardiac tissue responds to injury, e.g. myocardial infarction, with scar formation. Because the human heart is incapable of adequate muscle regeneration, survivors of a myocardial infarction typically develop heart failure, arrhythmias, thrombosis, and other complications. Heart disease also results in the loss of cardiomyocytes. Proliferation of cardiomyocytes could enhance the regenerative capacity of mammalian hearts.

[00133] In some embodiments, a compound described herein may be administered as part of a combination therapeutic with another cardiovascular agent including, for example, an anti- arrhythmic agent, an antihypertensive agent, a calcium channel blocker, a cardioplegic solution, a cardiotonic agent, a fibrinolytic agent, a sclerosing solution, a vasoconstrictor agent, a vasodilator agent, a nitric oxide donor, a potassium channel blocker, a sodium channel blocker, statins, or a natriuretic agent.

Spinal cord injury

[00134] A compound described herein can also be used to regenerate or repair tissue in a subject who has suffered a spinal cord injury. Spinal cord injury (SCI) is an insult to the spinal cord resulting in a change, either temporary or permanent, in its normal motor, sensory, or autonomic function. Both clinical and experimental studies evidence that the spinal cord suffers from primary and secondary damage after acute SCI. Primary SCI arises from mechanical disruption, transection, extradural pathology, or distraction of neural elements. This injury usually occurs with fracture and/or dislocation of the spine. However, primary SCI may occur in the absence of spinal fracture or dislocation. Penetrating injuries due to bullets or weapons may also cause primary SCI. More commonly, displaced bone fragments cause penetrating spinal cord or segmental spinal nerve injuries. Extradural pathology may also cause primary SCI. Spinal epidural hematomas or abscesses cause acute cord compression and injury. Spinal cord compression from metastatic disease is a common oncologic emergency. Longitudinal distraction with or without flexion and/or extension of the vertebral column may result in primary SCI without spinal fracture or dislocation.

[00135] The pathophysiology of secondary SCI involves a multitude of cellular and molecular events that progress over the first few days after injury. The most important cause of secondary SCI is vascular injury to the spinal cord caused by arterial disruption, arterial thrombosis, and hypoperfusion due to shock. SCI can be sustained through ischemia from damage or impingement on the spinal arteries. SCI due to ischemia can occur during surgery where aortic blood flow is temporarily stopped.

[00136] Other classifications of SCI include central cord syndrome, Brown-Sequard syndrome, anterior cord syndrome, conus medullaris syndrome and cauda equina syndrome.

[00137] SCI are classified as complete or incomplete, based on the extent of injury, according to the American Spinal Injury Association (ASIA) Impairment Scale. In complete SCI, there is no sensory and motor function preserved in the lowest sacral segments. In incomplete SCI, sensory or motor function is preserved below the level of injury including the lowest sacral segments. Incomplete cord lesions may evolve into more complete lesions. More commonly, the injury level rises one or two spinal levels during the hours to days after the initial event.

[00138] Compounds that promote the proliferation of stem cells, such as primary neuronal precursor cells, may be useful in treating spinal cord injury. Accordingly, a compound described herein may be used to treat any classification of SCI, or a symptom thereof. A compound can be used alone or in combination with another known therapy for SCI.

Stroke

[00139] Stroke is a general term for acute brain damage resulting from disease or injury of blood vessels. Stroke can be classified into at least two main categories: hemorrhagic stroke (resulting from leakage of blood outside of the normal blood vessels) and ischemic stroke (cerebral ischemia due to lack of blood supply). Some events that can cause ischemic stroke include thrombosis, embolism, and systemic hypoperfusion (with resultant ischemia and hypoxia).

[00140] Stroke generally causes neuronal death and injury in the brain by oxygen deprivation and secondary events. The area of the brain that dies as a result of the lack of blood supply or other damage is called an infarct. In some cases, the treatments described herein can be used to reduce or minimize the size of an infarct, e.g., by reducing secondary events that cause neuronal death or injury.

[00141 ] Obstruction of a cerebral artery resulting from a thrombus which has built up on the wall of a brain artery is generally called cerebral thrombosis. In cerebral embolism, the occlusive material blocking the cerebral artery arises downstream in the circulation (e.g., an embolus is carried to the cerebral artery from the heart). Because it is difficult to discern whether a stroke is caused by thrombosis or embolism, the term thromboembolism is used to cover both these types of stroke. Systemic hypoperfusion may arise as a consequence of decreased blood levels, reduced hematocrit, low blood pressure or inability of the heart to pump blood adequately.

[00142] In another example, the present compounds may provide improved recovery from tissue damage resulting from instances of ischemia and/or poor vascular flow, e.g., resulting from stroke. For example, in some instances, the present compounds may be administered immediately following an ischemic event, such as stroke. In other embodiments, the present compounds may be administered up to about 1 , 5, 10, 30, or 60 minutes, or 2, 4, 8,16, 24, or 48 hours, or 2, 4, or 8 days after an ischemic event, such as a stroke. Administration of the present compounds post- ischemia may promote regeneration of the affected tissue and/or the affected tissue's normal function. As such, the present compounds may provide improved neuroprotection for cells susceptible to damage from ischemic episodes.

[00143] Accordingly, the present invention may provide a method for treating tissues of a patient damaged by stroke, comprising administering an effective amount of a compound or composition described herein. In some instances, the compound or composition is

administered after the stroke, for example, up to about 1 , 5, 10, 30, or 60 minutes, or 2, 4, 8, 16, 24, or 48 hours, or 2, 4, or 8 days after the stroke.

[00144] The middle cerebral artery (MCA) is the cerebral blood vessel most susceptible to stroke in humans. In animals, coagulation, permanent ligation or permanent placement of an occluding thread in the artery produces a permanent focal stroke affecting the MCA territory. Transient ligation or occlusion results in transient focal stroke. Both transient and permanent focal strokes result in varying degrees of edema and infarction in the affected brain regions. In some instances, the present compounds may reduce the volumes of edema and infarction, which is a measure of their potential as anti-stroke treatment.

[00145] A direct approach to treating cerebral ischemia is to restore circulation. However, reperfusion following transient ischemia may induce additional mechanisms of tissue damage. This phenomenon is termed "reperfusion injury" and has been found to play a role in other organ systems as well, including the heart. Recently, it has been suggested that cerebral ischemic damage is mediated, to a large extent, via excitotoxic mechanisms. During ischemia, large elevations in extracellular glutamate occur, often reaching neurotoxic levels. Accordingly, the present compounds, compositions, and methods may be used as part of a treatment or prophylaxis for ischemic or epoxic damage, particularly to alleviate certain effects of reperfusion injury. Wound repair

[00146] A method which "promotes the healing of a wound" results in the wound healing more quickly as a result of the treatment than a similar wound heals in the absence of the treatment. "Promotion of wound healing" may also mean that the method regulates the proliferation and/or growth of, inter alia, keratin ocytes, or that the wound heals with less scarring, less wound contraction, less collagen deposition and more superficial surface area. In certain instances, "promotion of wound healing" may also mean that certain methods of wound healing have improved success rates, (e.g., the take rates of skin grafts,) when used together with one or more of the present compounds or compositions. As described in further detail herein, the present invention may provide a method for promoting wound healing, comprising administering to a subject one or more of the present compounds or compositions. In some applications, the one or more of the present compounds or compositions is administered orally. In other applications, the one or more present compounds or compositions is administered topically.

[00147] Complications are a constant risk with wounds that have not fully healed and remain open. Although most wounds heal quickly without treatment, some types of wounds resist healing. Wounds which cover large surface areas also remain open for extended periods of time. In one embodiment of the present invention, the present invention may be used to accelerate the healing of wounds involving epithelial tissues, such as resulting from surgery, burns, inflammation or irritation. Certain of the compounds of the present invention may also be applied prophylactically, such as in the form of a cosmetic preparation, to enhance tissue regeneration processes, e.g., of the skin, hair and/or fingernails.

[00148] Despite significant progress in reconstructive surgical techniques, scarring can be an important obstacle in regaining normal function and appearance of healed skin. This is particularly true when pathologic scarring such as keloids or hypertrophic scars of the hands or face causes functional disability or physical deformity. In the severest circumstances, such scarring may precipitate psychosocial distress and a life of economic deprivation.

Wound repair includes the stages of hemostasis, inflammation, proliferation, and remodeling. The proliferative stage involves multiplication of fibroblasts and endothelial and epithelial cells. Through the use of the present invention, the rate of proliferation of epithelial cells in and proximal to the wound may be controlled in order to accelerate closure of the wound and/or minimize the formation of scar tissue.

[00149] In one aspect of the present invention, the present compounds may be used either locally or systemically to promote the proliferation, differentiation, and/or migration of cardiomyocytes or endothelial cells or their progenitors to and within sites of injury to repair cardiovascular or peripheral vascular tissue. [00150] Full and partial thickness burns are an example of a wound type which often covers large surface areas and therefore requires prolonged periods of time to heal. As a result, life-threatening complications such as infection and loss of bodily fluids often arise. In addition, healing in burns is often disorderly, resulting in scarring and disfigurement. In some cases wound contraction due to excessive collagen deposition results in reduced mobility of muscles in the vicinity of the wound. The compositions and methods of the present invention may be used to accelerate the rate of healing of burns and to promote healing processes that result in more desirable cosmetic outcomes and less wound contraction and scarring. Severe burns which cover large areas are often treated by skin autografts taken from undamaged areas of the patient's body. The present invention may also be used in conjunction with skin grafts to improve "take" rates of the graft by accelerating growth of both the grafted skin and the patient's skin that is proximal to the graft.

[00151 ] Dermal ulcers are yet another example of wounds that may be amenable to treatment by the present invention, e.g., to cause healing of the ulcer and/or to prevent the ulcer from becoming a chronic wound. For example, one in seven individuals with diabetes develop dermal ulcers on their extremities, which are susceptible to infection. Individuals with infected diabetic ulcers often require hospitalization, intensive services, expensive antibiotics, and, in some cases, amputation. Dermal ulcers, such as those resulting from venous disease (venous stasis ulcers), excessive pressure (decubitus ulcers) and arterial ulcers also resist healing. The prior art treatments are generally limited to keeping the wound protected, free of infection and, in some cases, to restore blood flow by vascular surgery. According to the present invention, the afflicted area of skin may be treated by a therapy which includes a present compound which promotes epithelization of the wound, e.g., accelerates the rate of the healing of the skin ulcers.

[00152] The present treatment may also be effective as part of a therapeutic or prophylactic regimen for treating oral and paraoral ulcers, e.g., resulting from radiation and/or chemotherapy. Such ulcers commonly develop within days after chemotherapy or radiation therapy. These ulcers usually begin as small, painful irregularly shaped lesions usually covered by a delicate gray necrotic membrane and surrounded by inflammatory tissue. In many instances, lack of treatment results in proliferation of tissue around the periphery of the lesion on an inflammatory basis. For instance, the epithelium bordering the ulcer usually demonstrates proliferative activity, resulting in loss of continuity of surface epithelium. These lesions, because of their size and loss of epithelial integrity, lend the body to potential secondary infection. Routine ingestion of food and water becomes a very painful event and, if the ulcers proliferate throughout the alimentary canal, diarrhea usually is evident with all its complicating factors. According to the present invention, a treatment for such ulcers which includes application of a present compound may reduce the abnormal proliferation and differentiation of the affected epithelium, helping to reduce the severity of subsequent inflammatory events.

Skeletal tissue

[00153] In still another embodiment of the present invention, compositions comprising the present compounds may be used in the in vitro generation of skeletal tissue, such as from skeletogenic stem cells, as well as the in vivo treatment or prevention of skeletal tissue deficiencies. The present invention particularly contemplates the use of the present compounds to regulate the rate of chondrogenesis and/or osteogenesis. By "skeletal tissue deficiency", it is meant a deficiency in bone or other skeletal connective tissue at any site where it is desired to restore the bone or connective tissue, no matter how the deficiency originated, e.g., whether as a result of surgical intervention, removal of tumor, ulceration, implant, fracture, or other traumatic or degenerative conditions.

[00154] For instance, the present compounds and compositions may be used as part of a regimen for restoring cartilage function to a connective tissue. For example, the use of one or more of the present compounds or compositions for promoting cartilage production in vitro is contemplated. Such methods may be useful in, for example, the production of three- dimensional cartilage grafts to repair defects or lesions in cartilage tissue. Such methods may also be useful in, for example, the repair of defects or lesions in cartilage tissue which is the result of degenerative wear such as that which results in arthritis, as well as other mechanical derangements which may be caused by trauma to the tissue, such as a displacement of torn meniscus tissue, meniscectomy, a laxation of a joint by a torn ligament, misalignment of joints, bone fracture, or by hereditary disease. The present invention may also be useful for remodeling cartilage matrix, such as in plastic or reconstructive surgery, as well as periodontal surgery. The present invention may also be applied to improving a previous reparative procedure, for example, following surgical repair of a meniscus, ligament, or cartilage. Furthermore, it may prevent the onset or exacerbation of degenerative disease if applied early enough after trauma.

[00155] In one embodiment, the present invention comprises treating the afflicted connective tissue with a therapeutically sufficient amount of a present compound, for example, a compound selective for Indian hedgehog signal transduction, to regulate a cartilage repair response in the connective tissue by managing the rate of differentiation and/or proliferation of chondrocytes embedded in the tissue. Such connective tissues as articular cartilage, interarticular cartilage (menisci), costal cartilage (connecting the true ribs and the sternum), ligaments, and tendons may be particularly amenable to treatment in reconstructive and/or regenerative therapies using the present invention. As used herein, regenerative therapies may include treatment of degenerative states which have progressed to the point of which impairment of the tissue is obviously manifest, as well as preventive treatments of tissue where degeneration is in its earliest stages or imminent. In an illustrative embodiment, the present invention may be used as part of a therapeutic intervention in the treatment of cartilage of a diarthroidal joint, such as a knee, an ankle, an elbow, a hip, a wrist, a knuckle of either a finger or toe, or a tempomandibular joint. The treatment may be directed to the meniscus of the joint, to the articular cartilage of the joint, or both. To further illustrate, the present invention may be used to treat a degenerative disorder of a knee, such as which might be the result of traumatic injury (e.g., a sports injury or excessive wear) or osteoarthritis. The present compounds may be administered as an injection into the joint with, for instance, an arthroscopic needle. In some instances, the injected agent may be in the form of a hydrogel or other slow release vehicle described above in order to permit a more extended and regular contact of the agent with the treated tissue.

[00156] In still further embodiments, the present invention may be employed as part of a regimen for the generation of bone (osteogenesis) at a site in the animal where such skeletal tissue is deficient. Indian hedgehog is particularly associated with the hypertrophic

chondrocytes that are ultimately replaced by osteoblasts. For instance, administration of a compound or composition of the present invention may be employed as part of a method for regulating the rate of bone loss in a subject. For example, preparations comprising the present compounds may be employed, for example, to control endochondral ossification in the formation of a "model" for ossification. Therapeutic compositions including the present compounds may be supplemented, if required, with other osteoinductive factors, such as bone growth factors (e.g. TGF-b factors, such as the bone morphogenetic factors BMP-2 and BMP-4, as well as activin), and may also include, or be administered in combination with, an inhibitor of bone resorption such as estrogen, bisphosphonate, sodium fluoride, calcitonin, or tamoxifen, or related compounds.

Retinal damage

[00157] Levine et al. (1997) J Neurosci 17:6277 show that hedgehog proteins may regulate mitogenesis and photoreceptor differentiation in the vertebrate retina, and Ihh is a candidate factor from the pigmented epithelium to promote retinal progenitor proliferation and photoreceptor differentiation. Likewise, Jensen et al. (1997) Development 124:363 demonstrated that treatment of cultures of perinatal mouse retinal cells with the amino- terminal fragment of Sonic hedgehog protein results in an increase in the proportion of cells that incorporate bromodeoxuridine, in total cell numbers, and in rod photoreceptors, amacrine cells and Muller glial cells, suggesting that Sonic hedgehog promotes the proliferation of retinal precursor cells. Thus, the present invention may be used in the treatment or prevention of degenerative diseases of retinal cells and regulate photoreceptor differentiation.

Parkinson's Disease

[00158] Parkinson's disease (Parkinson disease or PD) is a degenerative disorder of the central nervous system that often impairs the sufferer's motor skills, speech, and other functions. It is characterized by muscle rigidity, tremor, a slowing of physical movement (bradykinesia) and, in extreme cases, a loss of physical movement (akinesia). The primary symptoms are the results of decreased stimulation of the motor cortex by the basal ganglia, normally caused by the insufficient formation and action of dopamine, which is produced in the dopaminergic neurons of the brain. Motor symptoms can include, e.g., tremor, rigidity, Akinesia/bradykinesia, postural instability, speech and swallowing disturbances. Secondary symptoms may include high level cognitive dysfunction and subtle language problems, such as slowed reaction time, executive dysfunction, dementia, and short-term memory loss. PD is both chronic and progressive. Causes for PD include, e.g., genetic mutations, toxins, and head trauma. There are other disorders that are called Parkinson-plus diseases. These include: e.g., multiple system atrophy (MSA), progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD). These Parkinson-plus diseases may progress more quickly than typical idiopathic Parkinson disease. Current treatments for PD include, e.g., Levodapa, COMT inhibitors, Dopamine agonists, MAO-B inhibitors, surgery and deep brain stimulation, and neurorehabilitation.

[00159] Hedgehog may exert trophic and survival-promoting actions on substantia nigra dopaminergic neurons. In vivo treatment or prevention with one or more of the present compounds or compositions may stimulate the dopaminergic phenotype of substantia nigra neurons and restore functional deficits induced by axotomy or dopaminergic neurotoxins, and may be used in the treatment or prevention of Parkinson's disease. Thus, in one embodiment, the present invention may provide a method of treating a subject with

Parkinson's disease, comprising administering to the subject an amount of one or more of the present compounds or compositions effective for increasing the rate of survival of dopaminergic neurons in the subject.

[00160] The following non-limiting Examples are intended to be purely illustrative, and show specific experiments that were carried out in accordance with embodiments of the invention. EXAMPLES

EXAMPLE 1. Synthesis of Fluorinated Glucocorticoid 12

12

[00161 ] To a solution of 0.0349 g (0.07 mMole) of fluticasone propionate in 2mL methylene chloride was added 0.034 mL anhydrous pyridine and 0.035 g (0.084 mMol) Dess- Martin periodinane at room temperature. After 30 min, isopropanol was added, followed by a 1 :1 solution of 5% Na₂S0₃ and saturated NaHC0₃. Methylene chloride was added and the organic layer was separated and washed two times with 1 :1 solution of 5% Na₂S0₃ / saturated NaHC0₃. The aqueous layers were combined and washed with methylene chloride and the separated methylene chloride layers were combined, washed two times with saturated NaHC0₃, dried over Na₂S0₄, filtered and concentrated. Residual pyridine was removed by addition of toluene and azeotropic distillation, and the residue purified by chromatography on silica gel using a gradient of CHCI₃ to 20% isopropanol/CHCI₃. MS(ESI) 499 m/z (M+1 ).

EXAMPLE 2. Synthesis of Fluorinated Glucocorticoids 10 and 11

Halcinonide compound 10

compound 11

[00162] Both compounds 10 and 1 1 were prepared from the same reaction.

[00163] To a mixture of 0.045 g (0.1 mMol) halcinonide and 10 mL 2-butanone was added 0.16 g (1 mMol) Nal, which was refluxed for 6 days. After cooling the residue was extracted with CHCI₃ and water. The CHCI₃ layer was dried over Na₂S0₄, filtered and concentrated. The residue was chromatographed on silica gel using a gradient of CHCI₃ - 6% isopropanol/CHCI₃. The faster eluting compound was the iodide (compound 1 1 ), MS (ESI) 547 m/z (M+1 ), followed by the halcinonide, followed by the reduced derivative

(compound 10) MS (ESI) 421 m/z (M+1 ). [00164] The following Materials and Methods were utilized in Examples 3-8.

Materials

[00165] Fluticasone propionate, halcinonide, and clobetasol were independently supplied by Prestwick Chemicals and Sigma. Fluocinonide was re-purchased from Prestwick. SAG was purchased from Axxora. Purmorphamine was purchased from Calbiochem. Cortisone, dexamethasone, prednisolone, and corticosterone were purchased from Sigma.

Cyclopamine and Bodipy-cyclopamine were purchased from Toronto Research Chemicals. Transfection and Plasmids

[00166] Cells were transfected using either Fugene 6 (Roche) or Nucleofector™ (Amaxa). Expression vectors for arr2-GFP, wild type Smo, Smo-YFP, and Gli-luciferase reporter have been described (Chen et al. (2004) Science 306(5705) :2257-2260; Taipale et al.

(2000) Λ/afure 406(6799) :1005-1009; and Chen et al. (2002) Proc Natl Acad Sci USA 99(22):14071 -14076). Vasopressin 2 receptor (V2R)-GFP was made in a similar manner as 32-adrenergic receptor-GFP, which is described (Barak LS, et al. (1997) Mol Pharmacol 51 (2):177-184). Glucocorticoid receptor (GR)-GFP was provided by Dr. Terry Hinds, Jr. from University of Toledo. Shh conditional media (Shh) was prepared by collecting the media in which HEK 293 cells were transfected with Shh-N plasmid (provided by Dr. Philip Beachy, Stanford University). Smo-633 was constructed by swapping the 154 amino acids of the Smo C-terminus (Pro 634 to Phe 787) with the 29 amino acids of V2R C-terminus (Ala 343 to Ser 371 ).

Confocal Image Acquisition

[00167] For laser fluorescence microscopy, transfected HEK293 or U20S cells were split onto collagen-coated 35-mm plastic dishes with glass bottoms (MatTek) and cultured overnight. Cells expressing GFP-tagged proteins were directly processed for Zeiss laser scanning microscope (LSM-510) as described (Chen et al. (2004) Science 306(5705) :2257- 2260).

Primary Assay-Automated High Throughput Screening

[00168] Multiple Smo mutants were made to identify the best location to attach the V2R tail, the addition of which, when phosphorylated and precisely located, causes 3arr2-GFP to bind to Smo more strongly. Smo-633 was identified by this assay because it produces a stronger signal in the U20S Smo/parr2-GFP assay than wild-type Smo but it is otherwise pharmacologically similar.

[00169] A single 15 cm plate of U20S cells stably expressing Smo-633 and 3arr2-GFP at 95%-100% cell confluency was split into two glass-bottom 384 well plates (MGB101 -1 -2-LG, Matrical) using a Multidrop 384 dispenser (Titertek Instruments). Each well contained 25 μΙ aliquots of 6000 cells in MEM (10% FBS, 50 U/ml Pen/Strep) with 100 nM cyclopamine. Plates were incubated overnight at 37^<C in 5% C0₂. The following day compounds (5 mM in DMSO) from the Prestwick Chemical Library and the positive control 5 mM SAG as well as the vehicle control DMSO were diluted 1 :200 in MEM and 6.25 μΙ of the diluted compound, SAG, or DMSO were added to each of the corresponding wells using a Biomek FX liquid handler (Beckman Coulter), for an overall 1 :1000 dilution and final concentration of 5 μΜ. The plates were returned to the incubator for 2 hours. The media was then removed and the cells were fixed in 30 μΙ PBS containing 0.5% paraformaldehyde, and a 1 :50,000 dilution of DRAQ5 dye (Biostatus) to visualize the nuclei. Plates were stored at 4°C until analysis on an ImageXpress Ultra (Molecular Devices) equipped with a 488 nM argon laser for imaging GFP and a 568 nM krypton laser for imaging DRAQ5. Plates were screened in duplicate at the rate of 5000 wells per day and analyzed using the associated software Transfluor HT, and results were visually confirmed.

Receptor Internalization Assay

[00170] Smo-YFP and V2R-GFP internalization assays were performed as previously described in Chen W, et al. (2004) Science 306(5705) :2257-2260.

Bodipy-cvclopamine Binding Analysis of Smo Agonists in Smo Over-expressing HEK293 Cells

[00171 ] HEK293 cells stably expressing wild type Smo were split at 166,000 cells per well in the center well (glass bottom, 10 mm diameter) of collagen-coated MatTek dishes, followed by overnight incubation. The cells were then washed and fixed with 4 % (v/v) formaldehyde/PBS for 20 min at room temperature. After removing the formaldehyde buffer, the cells were incubated for 2 h at room temperature in binding buffer (HBSS salt without Ca²⁺ and Mg²⁺) containing 0-50 nM Bodipy-cyclopamine for saturation binding assay, or 5 nM Bodipy-cyclopamine and compounds over a range of concentrations from 0 - 10 μΜ for competitive binding assay. Following the incubation, the cells were washed three times with the binding buffer and imaged using a Zeiss 40x oil N.A. 1 .3 plain apo objective and 488-nm excitation laser on a Zeiss LSM-510 confocal microscope. The average fluorescence intensity corresponding to each image (i.e. concentration) was determined using the accompanying histogram software. The specific binding data over baseline for compounds were normalized to the maximal binding of Bodipy-cyclopamine. The data were analyzed by GraphPad Prism using linear and nonlinear regression.

Gli-luciferase Reporter Assay

[00172] The reporter assay using Shh-light 2 cells, Smo-/- MEF, and NIH 3T3 cells were performed as described in Sinha et al. (2006) Nat Chem β/Ό/ 2(1 ):29-30. [³H1Thymidine Proliferation Assay and Western Blots of Primary Neuronal Granule Cell Precursors (GCPs)

[00173] Primary GCPs were isolated from 4-day or 8-day postnatal wild type C57BL/6 mice as previously described (Wechsler-Reya et al. (1999) Neuron 22(1 ):103-1 14; and Heine VM et al. (2009) J Clin Invest 1 19(2):267-277). Briefly, cerebella were removed from 4-day or 8-day mice, minced and digested at 37^<C in papain before being triturated in buffer containing trypsin inhibitor and BSA. Cells were then pelleted, resuspended in BSA/PBS and strained to remove debris. Strained cells were recentrifuged and resuspended in

supplemented Neurobasal Medium. The cells from 8-day mice were cultured for 48 h in the presence of compounds with/without Shh, then pulsed with [³H]Thymidine and cultured for an additional 16 h before being measured for [³H]Thymidine incorporation.

[00174] The cells from 4-day mice were cultured for 64 h in the presence of DMSO vehicle, compounds, or 2% Shh. The cells were harvested in the SDS sample buffer. The protein samples were subjected to SDS-PAGE using 4-12% Novex^® Tris-Glycine Gels (Invitrogen), transferred to nitrocellulose membranes (Bio-Rad Laboratories), blocked with 5% no-fat milk in TBS-0.2% Tween-20 for 20 min, followed by incubation with three primary antibodies: Cyclin D2 and cleaved Caspase 3 (Cell Signaling), and Actin (Santa Cruz Biotechnology, Inc). Subsequently, immunoblots were developed using HRP-conjugated antibodies (Amersham Biosciences).

[00175] Images were developed using ECL (Pierce Biotechnology) and an Alphalmager™ imager (Alpha Innotech).

Glucocorticoid Receptor (GR) Nuclear Translocation Assay

[00176] GR-GFP transfected HEK 293 cells were split onto col lag en -coated MatTek dishes and cultured overnight, followed by 1 μΜ compound treatments for 1 h and subsequent immunostaining of an endogenous nuclear membrane protein Lamin B1 . Briefly, the compound treated cells were fixed with 4% paraformaldehyde for 20 min, permeated with 0.2% Triton-X100/PBS for 10 min, pre-blocked with 5% BSA PBS for 30 min at room temperature, followed by incubation with the primary antibody Lamin B1 (Abeam) overnight at 4^<C and secondary antibody Alexa Fluor 568 (Invitrogen) for 1 h at room temperature. Images of the cells were processed for Zeiss laser scanning microscope (LSM-510) with a 488 nM argon laser for imaging GFP and a 568 nM krypton laser for imaging Alexa Fluor 568 labeled Lamin B1 . EXAMPLE 3. Fluorinated Glucocorticoid Drugs Promote Smo Intracellular Aggregation with -arrestin2-green Fluorescent Protein ( arr2-GFP)

[00177] The structures of glucocorticoid drugs, SAG, and Purmorphamine are shown in Fig. 1 A. In the primary assay, a Smo agonist is identified by its ability to aggregate arr2- GFP in U20S cells in the presence of 100 nM cyclopamine in the steady state model.

[00178] Included among the chemical libraries screened using a 384 multi-well format was the Prestwick Chemical Library containing FDA-approved drugs. It contains 68 glucocorticoids or structurally related steroid compounds including cortisone and

dexamethasone. The primary screening assay employed U20S cells, chosen for adherence, flatness, and stable expression of arr2-GFP and Smo-633, which provided better sensitivity than wild type Smo. Images of Smo-633/parr2-GFP complexes were obtained at the rate of 5000 per day using an automated confocal based plate reader (ImageExpress Ultra,

Molecular Devices). A read out of compound activity for each well was provided by analyzing the corresponding image for changes in arr2-GFP distribution that occur as a result of compound addition.

[00179] Similar to the positive controls SAG and Purmorphamine, each hit compound at 5 μΜ in the primary assay overcame the inhibition by 100 nM cyclopamine to produce intracellular arr2-GFP aggregates (Fig. 1 G-J). The U20S cells were treated with each chemical composition for 2 h at 37 ^<C.

[00180] Hit compound agonist activities in U20S cells were also confirmed using a arr2- GFP assay with wild type Smo (Fig. 7). Confocal images of 3arr2-GFP aggregation in U20S cells stably expressing Smo and 3arr2-GFP are depicted in Fig. 7, A-H. Cells were left untreated (A), treated with 100 nM cyclopamine (B), 100 nM cyclopamine and 5 μΜ SAG (C), 100 nM cyclopamine and 5 μΜ Purmorphamine (D), 100 nM cyclopamine and 5 μΜ halcinonide (E), 100 nM cyclopamine and 5 μΜ fluticasone propionate (F), 100 nM

cyclopamine and 5 μΜ clobetasol (G), or 100 nM cyclopamine and 5 μΜ fluocinonide (H) for 2 h at 37°C. In contrast to HEK293 cells, which are thicker and show aggregates at the periphery in confocal images, the flatter U20S cells show aggregates over the whole surface.

[00181 ] Using the primary assay, U20S cells stably expressing Smo-633 and 3arr2-GFP were pretreated with 100 nM cyclopamine overnight in 384 well screening plates. The cells were then treated with compounds over a range of concentrations from 0 - 10 μΜ for 2 hours. Tiff images of cell responses acquired on an ImageXpress Ultra were analyzed by the platform-accompanying software Transfluor HT to quantify the aggregates produced by the compounds. The data were analyzed by nonlinear regression and fit to a sigmoid dose response using GraphPad Prism. From the fitted curves, the EC50s for halcinonide, fluticasone propionate, clobetasol, and fluocinonide are 1 .1 ± 0.1 μΜ, 99 ± 1 .4 nM, 1 .5 ± 0.1 μΜ, and> 5 μΜ, respectively, whereas the EC50s for the positive control agonists SAG and Purmorphamine are 0.9 ± 0.1 nM and >5 μΜ, respectively (Fig. 7K and Table 1 ). In comparison to SAG, which has an efficacy of 1 .00 ± 0.08 in the primary assay, the efficacies for halcinonide, fluticasone propionate, and clobetasol are 0.99 ± 0.05, 0.89 ± 0.05, and 0.87 ± 0.05, respectively, whereas the efficacies for Purmorphamine and fluocinonide, though greater than 0.5, could not be determined at 10 μΜ due to absence of plateaus for the fitted curves (Fig. 7K and Table 1 ). Additional results for other compounds are also presented in Table 2.

Table 1 . Potency and efficacy data of Smo agonists.^a

l: if- Sss^' i s

NA m

i2

propionate

MA

Results are presented as the mean ± s.e.m of at least three experiments.

Table 2. EC50 values in primary screening assay (β-arrestin 2-GFP translocation assay)

a EC₅₀ < 1 .0 uM = +++, EC₅₀ between 1 .01 - 5.0 uM = ++, EC₅₀ > 5 uM = +. [00182] Additionally, in a test of specificity, neither SAG nor any of halcinonide, fluticasone propionate, clobetasol, or fluocinonide induced arr2-GFP aggregation with three control seven transmembrane receptors, including the human vasopressin type 2 receptor (V2R).

EXAMPLE 4. Fluorinated Glucocorticoid Drugs Promote Smo Internalization

[00183] It has been demonstrated that SAG induces Smo internalization (Chen (2004) Science 306(5705):2257-2260). In HEK293 cells expressing Smoothened-yellow fluorescent protein (Smo-YFP), the effects of SAG, Purmorphamine, halcinonide, or fluticasone propionate on Smo-YFP internalization were examined by confocal microscopy. Confocal images were taken of Smo-YFP expressing HEK293 cells left untreated (Fig. 2A, C, E, G) and treated with 2 μΜ SAG (Fig. 2B), 5 μΜ Purmorphamine (Fig. 2D), 2 μΜ halcinonide (Fig. 2F), and 2 μΜ fluticasone propionate (Fig. 2H) for 30-40 min at 37°C. Figure 2A-D shows that in HEK 293 cells, 2 μΜ SAG and 5 μΜ Purmorphamine each stimulate Smo-YFP to internalize. All of the four primary assay hit compounds from Example 1 similarly induce Smo-YFP internalization, consistent with roles as Smo agonists. Figure 2E-H shows representative results for halcinonide and fluticasone propionate.

[00184] In contrast, in control endocytosis experiments in HEK-293 cells testing specificity for Smo, neither SAG nor fluticasone propionate produced V2R internalization (Fig. 2I-N). Confocal images were taken of V2R-GFP expressed in HEK293 cells left untreated (Fig. 2I, K, M) or treated with 2 μΜ V2R agonist 1 -deamine-4-valine-D-arginine vasopressin (dvd AVP) (Fig. 2J), 2 μΜ SAG (Fig. 2L), and 2 μΜ fluticasone propionate (Fig. 2N) for 30 min at 37^<C.

EXAMPLE 5. Fluorinated Glucocorticoid Drugs Displace Bodipy-cyclopamine from Smo-overexpressing Cells

[00185] Binding assays were performed as described in the Materials and Methods above. By measuring saturation binding in HEK 293 cells stably expressing wild type Smo, the affinity (Kd) of cyclopamine for Smo was determined as 3.5 ± 0.8 nM using the program GraphPad Prism (Fig. 3A). In competition binding using the same cell line, we observed that SAG completely displaced 5 nM Bodipy-cyclopamine from Smo (defined as an efficacy of 1 .00) with an EC50 of 1 1 ± 0.5 nM (Fig. 3B and Table 1 ). As opposed to cortisone which is unable to displace Bodipy-cyclopamine from Smo up to 10 μΜ (0 efficacy), the EC50s and efficacy for displacement for halcinonide, fluticasone propionate, clobetasol, and fluocinonide are respectively 78±2.1 nM, 0.24±0.02; 58±1 .2 nM, 0.34±0.01 ; 57 ±2.6 nM, 0.24±0.02;

1000±300 nM, 0.30±0.01 (Fig. 3B and Table 1 ). We also observed that the known Smo agonist Purmorphamine displaced Bodipy-cyclopamine poorly as previously described, with an EC50 > 5 μΜ, which is less than observed for the four steroid agonists.

[00186] Data from the competition binding assays were normalized to the maximal binding of Bodipy-cyclopamine over baseline. Competition curves for each compound were initially analyzed by linear regression and those compounds that generated a line with slope not significantly different from zero (cortisone, p= 0.59; Purmorphamine, p=0.12; n=3;

alpha=0.05) were considered not able to compete with Bodipy-cyclopamine for Smo binding. The

displacement data of the remaining compounds were analyzed by fitting to a one-site competition curve using GraphPad Prism.

EXAMPLE 6. Fluorinated Glucocorticoid Drugs Activate Gli-luciferase Reporter

[00187] In a Gli assay performed in Shh-LIGHT2 cells and utilizing only the endogenous Hedgehog signaling machinery, Shh-LIGHT2 cells cultured to confluence were individually treated for 30 h by the following compounds: halcinonide, fluticasone propionate, clobetasol, fluocinonide, the positive controls Purmorphamine and SAG, and the negative control cortisone. The four fluorinated steroids along with the positive controls activated the Gli- luciferase reporter in a dose dependent manner (Fig. 4A and Table 1 for summary). The negative control cortisone had no Gli activity in Shh-LIGHT2 cells.

[00188] In addition, there doesn't appear to be a non Smo mediated pathway that would produce the same type of response (Fig. 8). Smo-/- MEF cells or NIH 3T3 cells were transfected with Gli-luciferase reporter and control renilla pRL-TK (Promega) plasmids. The cells were cultured to confluence and treated with DMSO, 5 μΜ of the indicated compounds in Fig. 8, or 20% Shh for 30 h. The statistical significance was analyzed by two-tailed

Student's f test with ^* p<0.05 (alpha=0.05) defined as significant (over DMSO). In Smo-/- MEF cells, none of the treatments resulted in a Gli-luciferase response over baseline (Fig. 8). In NIH 3T3 cells, Gli-luciferase activities were induced by halcinonide, purmorphamine, or 20% Shh (Fig. 8).

[00189] Next, it was investigated if Shh activity from conditioned media could be potentiated by the steroid Smo agonists in Shh-LIGHT2 cells. Shh-LIGHT2 cells were cultured to confluence and treated for 30 h with DMSO, 2% Shh, 5 μΜ of the indicated compounds in Fig. 4B, or 5 μΜ of the indicated compounds in Fig. 4B in the presence of 2% Shh. Gli-luciferase activity from compound treatment was measured relative to a DMSO control (activity defined as 1 ). The statistical significance was analyzed by two-tailed

Student's f test with ^* p<0.05 (alpha=0.05) defined as significant (compound + Shh over compound). [00190] Shh alone at 0.5% produced a 3.8 fold increase in Gli response. We found that Gli-luciferase activity due to the combination of Shh (0.5%) and either 5 μΜ halcinonide, fluocinonide, clobetasol, or fluticasone propionate was increased compared to Shh or compound treatment alone (Fig. 4B). Interestingly, the combination of 5 μΜ SAG or

Purmorphamine plus 0.5% Shh did not result in significant activity change compared to either SAG or Purmorphamine treatment alone.

[00191 ] Next, cyclopamine inhibition of Smo agonist signaling in Shh-LIGHT2 cells was examined. Cells were treated with compounds in the absence and presence of 3 μΜ cyclopamine as described previously. For each compound the response R was calculated as the following ratio, R = absolute value of the luminescence signal in the absence of 3 μΜ cyclopamine / luminescence signal in the presence of 3 μΜ cyclopamine. Efficacy data are normalized to the 0.5% Shh induced Gli-luciferase activity observed with 3 μΜ cyclopamine treatment (defined as 100% efficacy). Therefore, the efficacy of cyclopamine to inhibit the signaling of a particular compound is calculated as 100% x [R(compound)-1 ] / [R(0.5% Shh)- 1 ]. The efficacy of 3 μΜ cyclopamine inhibition for the 5 μΜ SAG, Purmorphamine, or the fluorinated glucocorticoids is presented relative to the 100% inhibition of Shh (Table 3).

Table 3. Cyclopamine inhibition of Smo agonist signaling in Shh-LIGHT2 cells. ^a

Shh or compound 1 rihibition by cyciopatn ine

(percent)

! 00±27

Haieinonute

Fluticasone propionate

Clobetasol ! 5±6

Fluocinonide 32±S

Purmorphamine

SAG aThe data are the mean ± s.e.m of at least three experiments.

EXAMPLE 7. Halcinonide, Fluticasone propionate, and Clobetasol Promote Mouse Cerebellar Granule Cell Precursor (GCP) Proliferation

[00192] A mouse GCP proliferation assay was used to test the growth promoting effects of Hedgehog agonist compounds. GCPs were treated for 48 h with the Smo agonists Purmorphamine or SAG (positive controls), the lead compounds halcinonide, fluticasone propionate, clobetasol, and fluocinonide, or the negative control compound cortisone. After treatment, the cells were pulsed with [³H]Thymidine ([³H]Td) and cultured for 16 h before being measured for [³H]Td incorporation. Cubic splines were fit to the data points using GraphPad Prism to highlight the responses.

[00193] SAG had approximately a 2-fold greater efficacy than Purmorphamine in promoting a GCP proliferative response. Relative to DMSO vehicle, the treatment by halcinonide resulted in a 40 to 50 fold increase in GCP proliferation that was similar to the maximal response produced by Purmorphamine (Fig. 5A). Fluticasone propionate and clobetasol had a 5-6 fold stimulatory effect and fluocinonide like cortisone had no effect.

[00194] The experiments were repeated in the presence of 5 μΜ Mifepristone (RU-486), a glucocorticoid nuclear receptor antagonist. First, primary neuronal GCPs were treated for 48 h with DMSO or Ru-486 (5 μΜ, 10 μΜ, and 25 μΜ), pulsed with [³H]Thymidine ([³H]Td), cultured for 16 h, and then assessed for [³H]Td incorporation. RU-486 inhibited GCP proliferation in a dose dependent manner. RU-486 at 5 μΜ had no effect on GCP

proliferation (Fig. 9A). Next, the effect of RU-486 on Smo agonist-stimulated GCP proliferation was measured. Primary neuronal GCPs were treated with DMSO, 12.5 μΜ halcinonide, 2.5 μΜ fluticasone propionate, 25 μΜ clobetasol, 25 μΜ fluocinonide, or 0.5 μΜ SAG in the presence and absence of 5 μΜ Ru-486. The statistical significance was analyzed by two-tailed Student's f test with ^* p<0.05 (alpha=0.05) defined as significant (over DMSO). RU-486 at 5 μΜ had no effect on Smo agonists tested. Otherwise, these experiments repeated in the presence of 5 μΜ Mifepristone (RU-486), a glucocorticoid nuclear receptor antagonist, gave similar results as discussed above (Fig. 9B).

[00195] Using [³H]Thymidine incorporation, the relationship between Shh and the Smo agonists on GCP proliferation was investigated. The cells were treated with DMSO or 2% Shh alone; or in the absence or presence of 2% Shh with one of the following compounds: 5 μΜ halcinonide, fluticasone propionate, clobetasol, fluocinonide; 5 μΜ dexamethasone, and the positive control SAG (0.008 μΜ) or Purmorphamine (0.073 μΜ). The [³H]Td incorporation data are presented as fold-change versus DMSO treatment, which was defined as 1 . The statistical significance was analyzed by two-tailed Student's f test with ^* p<0.05 (alpha=0.05) defined as significant (compound + Shh over Shh).

[00196] Shh (2%) induced a 17 fold increase of GCP proliferation as opposed to the marginal GCP proliferation response to 5 μΜ halcinonide, fluticasone propionate, clobetasol, fluocinonide, 0.073 μΜ Purmorphamine, or 0.008 μΜ SAG (Fig. 5B). Treatment of the GCP cells with 2% Shh and an agonist compound resulted in increased GCP proliferation ranging from 30 fold (fluocinonide) to 95 fold (SAG), indicating strong synergistic effects (Fig. 5B). Interestingly, the glucocorticoid receptor (GR) agonist, dexamethasone, had the tendency to inhibit the activity of 2% Shh (Fig. 5B) as previously described (Heine et al. (2009) J Clin /nvesf 1 19(2) :267-277).

[00197] To further verify the opposite effects that the Smo agonists have on proliferation compared to dexamethasone, GCP cells were treated with Shh (2% and 20%), and various concentrations of halcinonide or dexamethasone in the absence or presence of 2% Shh. Synergism between 2% Shh and halcinonide were observed in the proliferation assay, such responses were equal to or greater than the stimulatory effects produced by 20% Shh (Fig. 5C left panel). In comparison, dexamethasone inhibited Shh-activated GCP proliferation in a dose dependent manner (Fig. 5C right panel, note: the minor change in responsiveness between experiments to 2% Shh treatment and reflected as a decrease in GCP proliferation in the panel may result from batch to batch variability in Shh).

EXAMPLE 8. Halcinonide, fluticasone propionate, clobetasol, fluocinonide, and other glucocorticoids activate glucocorticoid receptor GFP (GR-GFP) and regulate Cyclin D2 expression and Caspase 3 degradation.

[00198] It has been reported that Hedgehog signaling promotes GCP proliferation through upregulation of Cyclin D2 and inhibition of proteasomal degradation of Caspase-3 whereas dexamethasone and several other GR agonists have the opposite effects by inhibiting GCP proliferation and not inhibiting GCP apoptosis (Heine et al. (2009) J Clin Invest 1 19(2):267- 277). Various glucocorticoids were assayed as to their ability to effect GR-GFP localization. GR-GFP transfected HEK293 cells were treated with 1 μΜ of the indicated compound in Fig. 6A-H for 1 h, followed by immunostaining of endogenous Lamin B1 protein (red) to visualize nuclear membrane as described in the Methods above. DMSO (Fig. 6A), SAG (Fig. 6B) and Purmorphamine (Fig. 6C) treatment did not cause translocation of GR-GFP from the cytosol to the nucleus. Halcinonide, fluticasone propionate, clobetasol, fluocinonide (Fig. 6D-H) as well as dexamethasone induced translocation of GR -GFP from the cytosol to the nucleus.

[00199] Next, the regulation of Cyclin D2 expression and Caspase 3 degradation in primary neuronal GCPs by Smo ligands and glucocorticoids was examined. Primary neuronal GCPs derived from 4- day old mice were individually treated for 64 h with DMSO, 2% Shh, 0.625 μΜ Purmorphamine; 0.5 μΜ SAG; 2.5 μΜ fluticasone propionate; and the remaining compounds as indicated in Fig. 6I at 25 μΜ. Cells were harvested in SDS sample buffer and protein samples were resolved on SDS-PAGE gels, and the corresponding immunoblots probed by antibodies against cyclin D2, cleaved Caspase 3, and actin (n=3). A representative immunoblot is shown in Fig. 6I.

[00200] Although all of the fluorinated glucocorticoids from the study possess the ability, like dexamethasone, to activate GR as assessed by a GR-GFP nuclear translocation assay (Fig. 6A-H), GCP treatment with Shh, Purmorphamine, SAG, the fluorinated halcinonide, clobetasol, and fluticasone propionate (but not the weak Smo agonist fluocinonide) increased endogenous Cyclin D2 protein expression and inhibited Caspase 3 degradation (Fig. 61). No such growth enabling responses were observed in GCPs treated with cortisone, dexamethasone, prednisolone, and corticosterone, observations consistent with recent reports (Heine et al. (2009) J Clin Invest 1 19(2):267-277; Noguchi et al. (2008) Cell Death Differ 15(10): 1582- 1592; and Gould et al. (1999) Biol Psychiatry 46(1 1 ):1472-1479) (Fig. 6I). The opposite response of dexamethasone in the GCP proliferation assay and its similar response in the nuclear receptor assay compared to the fluorinated glucocorticoid Smo agonists suggest that the signal for the GCP proliferative response is independent of glucocorticoid nuclear receptor signaling and is most probably due directly to activation of Smo.

[00201 ] In summary, compounds such as halcinonide, fluticasone propionate, clobetasol, and fluocinonide function as Smoothened agonists, having an ability to bind Smoothened, promote Smoothened internalization, activate Gli, and synergistically stimulate the

proliferation of primary neuronal-precursor cells.

[00202] All patents, publications and references cited herein are hereby fully incorporated by reference. In case of conflict between the present disclosure and incorporated patents, publications and references, the present disclosure should control.

Claims

1 . A method of stimulating the proliferation of stem cells, comprising contacting the cells with an effective amount of a compound of formula (I):

(I)

wherein:

each— - independently represents the presence or absence of a bond;

R⁶ is selected from H and halo;

R⁹ is selected from H and halo;

R is selected from H, hydroxy, oxo and halo;

Y is selected from a bond, O and S;

X is selected from a bond and CH₂;

W is selected from alkyl, hydroxy, halo and -OC(0)R";

each R' is independently H, alkyl, aryl, heterocyclyl or heteroaryl;

each R" is independently alkyl;

Z is O or S; and

n is 1 or 2;

2. The method of claim 1 , wherein the stem cells are selected from primary neuronal precursor cells and hedgehog-responsive cells.

3. The method of any of claims 1 -2, further comprising contacting the cells with an additional agent.

4. The method of claim 3, wherein the additional agent is a polypeptide.

5. The method of claim 4, wherein the polypeptide is a hedgehog protein selected from Sonic hedgehog, Indian hedgehog and Desert hedgehog.

6. The method of claim 5, wherein the compound of formula (I) and the hedgehog protein synergistically stimulate the proliferation of stem cells.

7. The method of any of claims 1 -6, wherein the cells are in a subject.

8. The method of any of claims 1 -6, where in the cells are contacted with the compound ex vivo.

9. A method of stimulating hair growth in a subject, comprising administering to the subject an effective amount of a compound of formula (I):

(I)

wherein:

each— - independently represents the presence or absence of a bond;

R⁶ is selected from H and halo;

R⁹ is selected from H and halo;

R is selected from H, hydroxy, oxo and halo;

Y is selected from a bond, O and S;

X is selected from a bond and CH₂;

W is selected from alkyl, hydroxy, halo and -OC(0)R";

each R' is independently H, alkyl, aryl, heterocyclyl or heteroaryl; each R" is independently alkyl;

Z is O or S; and

n is 1 or 2;

10. A method of regenerating or repairing damaged tissue in a subject, comprising administering to the subject an effective amount of a compound of formula (I):

I)

wherein:

each— - independently represents the presence or absence of a bond;

R⁶ is selected from H and halo;

R⁹ is selected from H and halo;

R is selected from H, hydroxy, oxo and halo;

Y is selected from a bond, O and S;

X is selected from a bond and CH₂;

W is selected from alkyl, hydroxy, halo and -OC(0)R";

each R' is independently H, alkyl, aryl, heterocyclyl or heteroaryl;

each R" is independently alkyl;

Z is O or S; and

n is 1 or 2;

1 1 . The method of claim 10, wherein the subject has experienced myocardial infarction.

The method of claim 10, wherein the subject has a spinal cord injury. The method of claim 10, wherein the subject has experienced a stroke.

14. The method of claim 10, wherein the subject has osteoporosis.

15. The method of claim 10, wherein the subject has a bone fracture.

16. The method of claim 10, wherein the subject has a lung injury.

17. The method of claim 10, wherein the subject has a liver injury.

18. The method of claim 10, wherein the subject has retinal damage.

19. A method of treating Parkinson's disease in a subject, comprising administering to the subject an effective amount of a compound of formula (I):

(I)

wherein:

each— - independently represents the presence or absence of a bond;

Ft⁶ is selected from H and halo;

Ft⁹ is selected from H and halo;

R is selected from H, hydroxy, oxo and halo;

Y is selected from a bond, O and S;

X is selected from a bond and CH₂; W is selected from alkyl, hydroxy, halo and -OC(0)R";

each R' is independently H, alkyl, aryl, heterocyclyl or heteroaryl;

each R" is independently alkyl;

Z is O or S; and

n is 1 or 2;

20. A method of promoting wound healing in a subject, comprising administering to the subject an effective amount of a compound of formula (I):

(I)

wherein:

each— - independently represents the presence or absence of a bond;

R⁶ is selected from H and halo;

R⁹ is selected from H and halo;

R is selected from H, hydroxy, oxo and halo;

Y is selected from a bond, O and S;

X is selected from a bond and CH₂;

W is selected from alkyl, hydroxy, halo and -OC(0)R";

each R' is independently H, alkyl, aryl, heterocyclyl or heteroaryl;

each R" is independently alkyl;

Z is O or S; and

n is 1 or 2; wherein when R ⁶ and R ⁷ are taken together to form a ring, they together form the group -0-C(R^a)₂-0-, wherein each R^a is independently alkyl or two R^a are taken together to form a ring.

21 . The method of claim 20, wherein the wound is on the dermal layers of the skin.

22. The method of any of claims 1 -21 , wherein, in the compound of formula (I):

R⁶ is selected from H and halo;

R⁹ is halo;

R is selected from hydroxy and oxo;

R ⁶ is alkyl, or may be taken together with R ⁷ to form a ring; and

23. The method of any of claims 9-22, wherein a composition comprising the compound of formula (I) is administered to the subject in a composition further comprising a pharmaceutically acceptable carrier or diluent.

24. The method of claim 23, wherein the composition is administered topically, orally, parenterally, via a depot or via inhalation.

25. The method of claim 23, wherein the composition is administered intravenously, intramuscularly, or subcutaneously.

26. The method of any of claims 1 -25, wherein R⁶ is hydrogen.

27. The method of any of claims 1 -25, wherein R⁶ is halo.

28. The method of any of claims 1 -25, wherein R⁶ is fluoro.

29. The method of any of claims 1 -28, wherein R⁹ is halo.

30. The method of any of claims 1 -28, wherein R⁹ is fluoro.

31 . The method of any of claims 1 -30, wherein R is hydroxy.

32. The method of any of claims 1 -30, wherein R is oxo. The method of any of claims 1 -32, wherein R ⁶ is alkyl

The method of any of claims 1 -32, wherein R ⁶ is methyl

35. The method of any of claims 1 -34, wherein R is hydroxy.

36. The method of any of claims 1 -34, wherein R ⁷ is -OC(0)CH₂CH₃.

37. The method of any of claims 1 -34, wherein R ⁶ and R ⁷ are taken together to form a ring.

The method of any of claims 1 -34, wherein R and R together form a group

selected from -0-C(CH₃)₂-0- and

39. The method of any of claims 1 -38, wherein the group -Y-X-W is selected from the group consisting of -CH₂-CI, -S-CH₂-F, -CH₂-OAc, -CH₂-OH, -CH₃ and -CH₂-I.

40. The method of any of claims 1 -25, wherein the compound is selected from the group consisting of:

55