CROSS-REFERENCE TO A RELATED APPLICATION

[0001] This patent application claims the benefit of U.S. Provisional Patent Application No. 62/181,628, filed June 18, 2015, which is incorporated by reference.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY

SPONSORED RESEARCH

[0002] This work was supported by The Eunice Kennedy Shriver National Institute of Child Health and Human Development of the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

[0003] Macroautophagy, also known as autophagy, is an evolutionary conserved process for cellular degradation and recycling of cytosolic contents to maintain cellular homeostasis. A double layer membrane (the phagophore) forms around the material to be digested (the 'cargo') to produce an autophagosome. The autophagosome subsequently fuses with a lysosome to form an autolysosome. The autolysosome degrades the cargo contents, thereby providing the cell with nutrients from which it can produce energy and build new

components. Autophagy substrates are generally cellular organelles, long-lived proteins and aggregate-prone proteins. Due to its functionality to clear cytosolic contents, this process has been shown to be a promising approach for treatment of diseases characterized by the formation of intracellular aggregates, such as aging of the brain and neurodegeneration.

[0004] Upon activation of autophagy, the structure of the phagophore and then the autophagosome membrane is formed by the recruitment of Atgl2-Atg5 complex and LC3. The cytosolic form of LC3 (LC3-I) is converted into the membrane-bound form (LC3-II) to form the matured autophagosome,. The autophagosome then fuses with a lysosome, resulting in an autolysosome, which degrades unwanted cellular components. Although this system maintains cellular homeostasis and occurs upon cell survival or cell death, specific mechanisms of how cell survival or cell death is induced have not yet been identified.

Autophagy-induced cell death is classified as type II cell death and it is irrelevant to caspase-dependent apoptosis. [0005] Autophagy is associated with various pathological processes such as neurodegenerative diseases, cancer and melanogenesis. Huntington's disease is a

neurodegenerative disorder caused by the aggregation of mutant huntingtin (mHtt) protein. In the case of Parkinson's disease or Alzheimer's disease, fibrils and amyloid plaques are accumulated, and autophagy may provide a potential solution for the degradation of these components.

[0006] However, currently known small molecules which up-regulate autophagy in mammalian brains such as rapamycin are specific mTOR inhibitors. TOR proteins are known to control several cellular processes besides autophagy in organisms from yeast to human. Since rapamycin is toxic to cultured cells, mice, and humans, long-term use of these mTOR-dependent small molecule autophagy inducers may contribute to complications. Moreover, autophagy in the central nervous system is also known to be regulated differently from autophagy in non-neuronal cells and the induction thereof in neuronal cells has been shown to be more difficult than in non-neuronal cells. The known autophagy inducers either fail to induce autophagy in the cortex of mouse brains or induce only mild autophagy in neurons.

[0007] Accordingly, there remains an unmet need in the art for small molecule inducers of autophagy in the central nervous system and methods of inducing autophagy in the central nervous system.

[0008] Here the identification of a family of small molecules is reported that can increase autophagy in human cells, and in doing so, selectively reduce the amount of, for example, mutant huntingtin protein in human cells. These compounds are termed autophagy modulators (or AM compounds), because they rapidly and reversibly induced autophagy in human cells derived from healthy tissues (WI38, MCF10A), from cancers (SW480, U20S, HeLa, HCT1 16, 293T), and from patients with Huntington's, Parkinson's, Alzheimer's, Niemann-Pick's, or Hutchinson-Gilford progeria. Autophagy was identified by the dose and time dependent induction of small cytoplasmic vacuoles containing various types of cargo, as well as autophagy associated proteins such as LC3-II and p62, followed by the accumulation of larger, empty, acidic vacuoles. Moreover, vacuole formation was dependent on Atg7, a gene essential for autophagy, and either bafilomycin Al or chloroquine prevented the transition from the putative autophagosomes into autolysosomes. Autophagic flux was detected by changes in LC3-II over time, and by the reduction in vacuole density and size upon addition of bafilomycin Al to cultures in which AM compounds had induced autophagy. The AM compounds were 10 to 100 times more effective than rapamycin (a known inducer of autophagy) at up-regulating autophagy and reducing the level of mutant huntingtin protein.

BRIEF SUMMARY OF THE INVENTION

[0009] The invention provides a method of decreasing an amount of a protein aggregate in a cell comprising contacting the cell with a compound of the formula:

wherein R and R are independently H, optionally substituted Ci-C ₆ alkyl, or optionally

1 2

substituted C ₆ -Cio aryl, or wherein R and R , taken together with the N to which they are attached, form a 5- or 6-membered heterocyclyl ring,

R ³ is optionally substituted C ₆ -Cio aryl or a group of the formula: R ⁴ CH=N- wherein R ⁴ is

C ₆ -Cio aryl, heteroaryl, or fused bicyclic heteroaryl,

X is CH or N,

or a tautomer thereof,

or a pharmaceutically acceptable salt thereof, in an amount sufficient to induce autophagy in the cell and cause the degradation of the protein aggregate within the cell.

[0010] The invention also provides a method of treating or preventing a condition, syndrome, or disease in a mammal in need thereof, wherein the condition, syndrome, or disease is selected from Huntington's disease, Alzheimer's disease, Parkinson's disease, Hutchinson-Gilford progeria syndrome, Niemann-Pick type C disease, spinobulbar muscular atrophy (Kennedy disease), dentatorubral-pallidoluysian atrophy (Haw-River syndrome), spinocerebellar ataxia type 1, spinocerebellar ataxia type 2, spinocerebellar ataxia type 3, spinocerebellar ataxia type 6, and spinocerebellar ataxia type 7, comprising administering to the mammal one or more compounds selected from:

wherein R ¹ and R ² are independently H, optionally substituted C]-C ₆ alkyl, or optionally

1 9

substituted C ₆ -Cio aryl, or wherein R and R , taken together with the N to which they are attached, form a 5- or 6-membered heterocyclyl ring,

R ³ is optionally substituted C ₆ -Cio aryl or a group of the formula: R ⁴ CH=N- wherein R ⁴ is

C ₆ -Cio aryl, heteroaryl, or fused bicyclic heteroaryl,

X is CH or N,

or a tautomer thereof,

or a pharmaceutically acceptable salt thereof, in an amount sufficient to treat or prevent the condition, syndrome, or disease.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

[0011] Figure 1 shows photomicrographs of U20S cells cultured with the indicated concentrations of compound #5 for either 4h or 24h.

[0012] Figure 2A shows photomicrographs of U20S cells cultured for 24 h with the indicated compound.

[0013] Figure 2B shows photomicrographs of the cells in Figure 2A after washing out of the compounds and culturing for another 24h.

[0014] Figures 3 A and 3B show electron microscopic images of U20S cells cultured for 2 h with rapamycin. Figure 3B is a higher resolution image of the cells in Figure 3A.

[0015] Figures 3C-3F show electron microscopic images of U20S cells cultured for 2 h with compound #5. Figure 3C shows a cluster of three small vacuoles in the cytoplasm that contained cargo. Figure 3D is a high resolution mage of the same vacuole shown in Figure 3C. Figure 3E shows several cytoplasmic vacuoles with cargo surrounding a mitochondrian. Figure 3F shows three cytoplasmic vacuoles with cargo, indicative of autophagosomes induced by compound #5, and a putative double membrane phagophore.

[0016] Figure 4A graphically shows cell viability of 293T cells treated with 0.2 μΜ compound #5, 0.1 μΜ compound #1 , or 0.1 μΜ compound #4. [0017] Figure 4B graphically shows cell viability of 293T cells treated with 2 μΜ compound #5, 1 μΜ compound #1 , 1 μΜ compound #4, 1 μΜ compound #2, or 1 μΜ compound #3.

[0018] Figure 4C graphically shows cell viability of U20S cells treated with 0.2 μΜ compound #5, 0.1 μΜ compound #1, or 0.1 μΜ compound #4.

[0019] Figure 4D graphically shows cell viability of U20S cells treated with 2 μΜ compound #5, 1 μΜ compound #1 , 1 μΜ compound #4, 1 μΜ compound #2, or 1 μΜ compound #3.

[0020] Figure 5 shows phase contrast photomicrographs of U20S cells cultured with the indicated compound for 2h. Rap = rapamycin

[0021] Figure 6A shows photomicrographs of U20S cells cultured with either DMS (vehicle), Bafilomycin Al , compound #5, or compound #5 + Bafilomycin Al for 24 h.

[0022] Figure 6B shows electron microscopic images through U20S cells after culturing for 24h with the indicated compounds. Panel A shows cells cultured with 1 μΜ

compound#l . Panel B is a higher resolution image of Panel A. Panel C shows cells cultured with 1 μΜ compound#l + 50 nM Bafilomycin Al . Panel D is a higher resolution image of Panel C. Panel e shows cells cultured with 2 μΜ compound#5 + 50 nM Bafilomycin Al . Panel f is a higher resolution image of Panel E.

[0023] Figure 7 shows fluorescence and phase contrast photomicrographs of acridine orange staining of U20S cells cultured with 2 μΜ compound #1 or DMSO vehicle for 24h.

[0024] Figure 8A shows Western blots obtained from cells treated with DMSO (vehicle), rapamycin, chloroquine (CQ) or compound #5 and further treated with siGL2 or siAtg7.

[0025] Figure 8B shows electron microscopic images of the cells treated with compound #5 and siGL2.

[0026] Figure 8B shows electron microscopic images of the cells treated with compound #5 and siAtg7.

[0027] Figure 9A shows Western blots obtained from U20S cells treated with the indicated concentrations of rapamycin, compound #5, compound #1, or compound #4.

[0028] Figure 9B shows Western blots obtained from different cell lines treated for 24 h with DMSO vehicle or the indicated concentrations of compound #5. Wtl cells were from a healthy adult. NPC5, NPC4, and NPC25 cells were obtained from patients with different degrees of Niemann-Pick's disease. [0029] Figure 1 OA shows Western blots obtained from U20S cells treated with DMSO vehicle, rapamycin, or compound #5, with or without Bafilomycin Al .

[0030] Figure 10B shows Western blots obtained from U20S cells treated with DMSO vehicle, rapamycin, or compound #5, with or without chloroquine.

[0031] Figure IOC shows shows Western blots obtained from U20S cells treated with

DMSO vehicle, 10 μΜ rapamycin, or 5 μΜ compound #1 and further treated with 25 μΜ chloroquine or 2 nM wortmannin.

[0032] Figure 1 1 shows Western blots obtained from U20S cells treated for the indicated times with 25 μΜ chloroquine or 2 μΜ compound #1.

[0033] Figure 12B shows Western blots obtained from Q-74 cells (HD mutant) treated with DMSO vehicle, rapamycin, compounds #5, #2, #3, #4, #1 , or wortmannin.

[0034] Figure 12A shows Western blots obtained from Q-23 cells (wild-type) treated with DMSO vehicle, rapamycin, or the indicated concentrations of compound #5.

[0035] Figure 13A shows Western blots obtained from primary lymphocytes from a Huntingtin's disease patient treated with DMSO vehicle, wortmannin, rapamycin, or the indicated concentrations of compound #5 or treated with the indicated concentrations of compound #5 + wortmannin.

[0036] Figure 13B shows Western blots obtained from primary lymphocytes from a normal patient treated with DMSO vehicle, rapamycin, or the indicated concentrations of compound #5.

[0037] Figure 14 shows photomicrographs of cells from a Niemann-Pick Type C patient (ND34733 cells) treated with DMSO vehicle, compound #5, or the indicated concentrations of compound #1.

[0038] Figure 15 shows the structures of compounds in accordance with an embodiment of the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0039] In one embodiment, the invention provides a method of decreasing an amount of protein aggregate in a cell comprising contacting the cell with a compound of the formula:

1

wherein R and R are independently H, optionally substituted Ci-C ₆ alkyl, or optionally

1 2

substituted C ₆ -Cio aryl, or wherein R and R , taken together with the N to which they are attached, form a 5- or 6-membered heterocyclyl ring,

R ³ is optionally substituted C ₆ -Cio aryl or a group of the formula: R ⁴ CH=N- wherein R ⁴ is

C ₆ -Cio aryl, heteroaryl, or fused bicyclic heteroaryl,

X is CH or N,

or a tautomer thereof,

or a pharmaceutically acceptable salt thereof, in an amount sufficient to induce autophagy in the cell and cause the degradation of the protein aggregate within the cell.

[0040] In accordance with certain embodiments, X is N.

1 2

[0041] In accordance with certain embodiments, R and R , taken together with the N to which they are attached, form morpholinyl, and R is optionally substituted C ₆ -Cjo aryl.

[0042] In accordance with s ecific embodiments, the compound is:

[0043] In accordance with certain embodiments, R ³ is R ⁴ CH=N-, R ¹ is H, and R ² is optionally substituted C -Cio aryl.

[0044] In accordance with a specific embodiment, the compound is:

[0045] In accordance with certain embodiments, X is CH.

[0046] In accordance with certain embodiments, R ³ is R ⁴ CH=N- and wherein R ¹ and R ² , taken together with the N to which they are attached, form morpholinyl.

[0047] In accordance with s ecific embodiments, the compound is:

[0048] In another embodiment, the invention provides a method of treating or preventing a condition, syndrome, or disease in a mammal in need thereof, wherein the condition, syndrome, or disease is selected from Huntington's disease, Alzheimer's disease, Parkinson's disease, Hutchinson-Gilford progeria syndrome, Niemann-Pick type C disease, spinobulbar muscular atrophy (Kennedy disease), dentatorubral-pallidoluysian atrophy (Haw-River syndrome), spinocerebellar ataxia type 1, spinocerebellar ataxia type 2, spinocerebellar ataxia type 3, spinocerebellar ataxia type 6, and spinocerebellar ataxia type 7, comprising administering to the mammal one or more compounds selected from:

wherein R 1 , R2 , R 3 , and X are as defined herein.

[0049] Referring now to terminology used generically herein, the term "alkyl" means a straight-chain or branched alkyl substituent containing from, for example, 1 to about 6 carbon atoms, preferably from 1 to about 4 carbon atoms, more preferably from 1 to 2 carbon atoms. Examples of such substituents include methyl, ethyl, propyl, isopropyl, «-butyl, sec- butyl, isobutyl, iert-butyl, pentyl, isoamyl, hexyl, and the like.

[0050] The term "aryl" refers to an unsubstituted or substituted aromatic carbocyclic substituent, as commonly understood in the art, and the term "C ₆ -Cio aryl" includes phenyl and naphthyl. It is understood that the term aryl applies to cyclic substituents that are planar and comprise 4n+2 π electrons, according to Huckel's Rule. [0051] The term "heterocyclyl," as used herein, refers to a monocyclic or bicyclic 5- or 6-membered ring system containing one or more heteroatoms selected from the group consisting of O, N, S, and combinations thereof. The heterocyclyl group can be an aliphatic heterocyclyl group. The heterocyclyl group can be a monocyclic heterocyclyl group or a bicyclic heterocyclyl group. Suitable bicyclic heterocyclyl groups include monocylic heterocyclyl rings fused to a C ₆ -Ci ₀ aryl ring, for example, dihydrobenzofuran or 1 ,2,3,4- tetrahydroquinoline, 1,2,3,4-tetrahydroisoquinoline, or indoline. Non-limiting examples of suitable heterocyclyl groups include tetrahydrofuranyl, tetrahydropyranyl,

tetrahydrothiopheneyl, pyrrolidinyl, piperidinyl, and morpholinyl. The heterocyclyl group is optionally substituted with 1, 2, 3, 4, or 5 substituents as recited herein such as with alkyl groups such as methyl groups, ethyl groups, and the like, or with aryl groups such as phenyl groups, naphthyl groups and the like, wherein the aryl groups can be further substituted with, for example halo, dihaloalkyl, trihaloalkyl, nitro, hydroxy, alkoxy, aryloxy, amino, substituted amino, alkylcarbonyl, alkoxycarbonyl, arylcarbonyl, aryloxycarbonyl, thio, alkylthio, arylthio, and the like, wherein the optional substituent can be present at any open position on the heterocyclyl group.

[0052] The phrase "pharmaceutically acceptable salt" is intended to include non-toxic salts synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two. Generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts are found in Remington 's Pharmaceutical Sciences, 18th ed., Mack Publishing Company, Easton, PA, 1990, p. 1445, and Journal of Pharmaceutical Science, 66, 2-19 (1977).

[0053] Suitable bases include inorganic bases such as alkali and alkaline earth metal bases, such as those containing metallic cations such as sodium, potassium, magnesium, calcium and the like. Non-limiting examples of suitable bases include sodium hydroxide, potassium hydroxide, sodium carbonate, and potassium carbonate. Suitable acids include inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, and the like, and organic acids such as / toluenesulfonic, methanesulfonic acid, benzenesulfonic acid, oxalic acid, -bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, maleic acid, tartaric acid, fatty acids, long chain fatty acids, and the like. Preferred pharmaceutically acceptable salts of inventive compounds having an acidic moiety include sodium and potassium salts. Preferred pharmaceutically acceptable salts of inventive compounds having a basic moiety (such as a

dimethylaminoalkyl group) include hydrochloride and hydrobromide salts. The compounds of the present invention containing an acidic or basic moiety are useful in the form of the free base or acid or in the form of a pharmaceutically acceptable salt thereof.

[0054] It should be recognized that the particular counterion forming a part of any salt of this invention is usually not of a critical nature, so long as the salt as a whole is

pharmacologically acceptable and as long as the counterion does not contribute undesired qualities to the salt as a whole.

[0055] It is further understood that the above compounds and salts may form solvates, or exist in a substantially uncomplexed form, such as the anhydrous form. As used herein, the term "solvate" refers to a molecular complex wherein the solvent molecule, such as the crystallizing solvent, is incorporated into the crystal lattice. When the solvent incorporated in the solvate is water, the molecular complex is called a hydrate. Pharmaceutically acceptable solvates include hydrates, alcoholates such as methanolates and ethanolates, acetonitrilates and the like. These compounds can also exist in polymorphic forms.

[0056] In any of the above embodiments, the compound or salt can exist in one or more tautomeric forms. The term "tautomer" as used herein includes two or more interconvertable compounds resulting from at least one formal migration of a hydrogen atom and at least one change in valency (e.g., a single bond to a double bond, a triple bond to a single bond, or vice versa). The exact ratio of the tautomers depends on several factors, including temperature, solvent, and pH. Tautomerizations (i.e., the reaction providing a tautomeric pair) may catalyzed by acid or base. Exemplary tautomerizations include keto-to-enol; amide-to-imide; lactam-to-lactim; enamine-to-imine; and enamine-to-(a different) enamine tautomerizations. In an example, when R ³ is a group of the formula: R ⁴ CH=N- and R ⁴ includes a CH group bonded to the CH of R ⁴ CH=N-, such as -CH-CH=N-, a tautomer can be represented by the formula: -C=CH-NH-.

[0057] The present invention provides a method of decreasing an amount of protein aggregate in a cell comprising contacting the cell with an amount of one or more of the inventive compounds or a pharmaceutically acceptable salt thereof sufficient to induce autophagy in the cell and cause the degradation of the protein aggregate within the cell. The contacting can be in vitro or in vivo. When the contacting is done in vitro, the contacting can be done by any suitable method, many of which are known in the art. For example, the cell can be provided in a culture medium and the inventive compound introduced into the culture medium per se, or as a solution of the compound in an appropriate solvent. In embodiments, the cell is present in a mammal. In these embodiments, the cells can be contacted by administration of the compound to the mammal. The compound can be administered acutely or chronically.

[0058] The amount of protein aggregate can be decreased by any suitable amount. For example, the amount of protein aggregate can be decreased by about 5%, e.g., about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or even about 100%), as compared to a cell that has not been contacted with an inventive compounds or a pharmaceutically acceptable salt thereof.

[0059] In embodiments, the cell has a condition, syndrome, or disease selected from Huntington's disease, Alzheimer's disease, Parkinson's disease, Hutchinson-Gilford progeria syndrome, and Niemann-Pick type C disease. In embodiments, the cell has a condition, syndrome, or disease associated with a trinucleotide repeat disorder of the trinucleotide repeat sequence CAG (cytosine-adenine-guanine), wherein the condition, syndrome, or disease is selected from Huntington's disease, spinobulbar muscular atrophy (Kennedy disease), dentatorubral-pallidoluysian atrophy (Haw-River syndrome), spinocerebellar ataxia type 1 , spinocerebellar ataxia type 2, spinocerebellar ataxia type 3, spinocerebellar ataxia type 6, and spinocerebellar ataxia type 7. In a preferred embodiment, the cell has a condition, syndrome, or disease which is Huntington's disease.

[0060] The present invention also provides a method of treating or preventing a condition, syndrome, or disease in a mammal in need thereof, wherein the condition, syndrome, or disease is selected from Huntington's disease, Alzheimer's disease, Parkinson's disease, Hutchinson-Gilford progeria syndrome, Niemann-Pick type C disease, spinobulbar muscular atrophy (Kennedy disease), dentatorubral-pallidoluysian atrophy (Haw-River syndrome), spinocerebellar ataxia type 1 , spinocerebellar ataxia type 2, spinocerebellar ataxia type 3, spinocerebellar ataxia type 6, and spinocerebellar ataxia type 7. In a preferred embodiment, the condition, syndrome, or disease is Huntington's disease. Huntington's disease (HD) is a neurodegenerative disorder caused by the aggregation of mutant huntingtin (mHtt) protein. It has been surprising found that the compounds of the invention induce LC3-II and Beclin-1 , key biomarkers for autophagy, in a dose-dependent manner with a concomitant increase in autophagic vacuoles as shown by electron microscopy. Moreover, these events were dependent on Atg7, a gene essential for autophagy. To determine whether or not this resulted from induction of autophagy, the autophagic inhibitor bafilomycin Al was included with the compounds. This resulted in a further accumulation of LC3-II.

Furthermore, changes in LC3-II over time revealed an increase in autophagic flux. These results demonstrated that the compounds induced autophagy. To determine their therapeutic potential, the compounds were tested for their ability clear mHtt in an inducible HD cell model as well as in cells from HD patients. In both cases, the compounds cleared mHtt aggregates and reduced the levels of mHtt protein. The compounds did not clear wild-type Huntingtin protein from normal cells. Moreover, the compounds were effective at 10 to 100 times lower concentrations than required for rapamycin. These results establish a significant therapeutic potential for the compounds of the invention in the treatment of HD and other neurodegenerative diseases.

[0061] The present invention is further directed to a pharmaceutical composition comprising a pharmaceutically acceptable carrier and at least one compound or salt described herein.

[0062] It is preferred that the pharmaceutically acceptable carrier be one that is chemically inert to the active compounds and one that has no detrimental side effects or toxicity under the conditions of use.

[0063] The choice of carrier will be determined in part by the particular compound of the present invention chosen, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of the

pharmaceutical composition of the present invention. The following formulations for oral, aerosol, nasal, pulmonary, parenteral, subcutaneous, intravenous, intramuscular,

intraperitoneal, intrathecal, intratumoral, topical, rectal, and vaginal administration are merely exemplary and are in no way limiting.

[0064] The pharmaceutical composition can be administered parenterally, such as intravenously, subcutaneously, intradermally, or intramuscularly. Thus, the invention provides compositions for parenteral administration that comprise a solution or suspension of the inventive compound or salt dissolved or suspended in an acceptable carrier suitable for parenteral administration, including aqueous and non-aqueous isotonic sterile injection solutions.

[0065] Overall, the requirements for effective pharmaceutical carriers for parenteral compositions are well known to those of ordinary skill in the art. See, such as Banker and Chalmers, eds., Pharmaceutics and Pharmacy Practice, J. B. Lippincott Company,

Philadelphia, pp. 238-250 (1982), and Toissel, ASHP Handbook on Injectable Drugs, 4th ed., pp. 622-630 (1986). Such solutions can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents,

solubilizers, thickening agents, stabilizers, and preservatives. The compound or salt of the present invention may be administered in a physiologically acceptable diluent in a pharmaceutical carrier, such as a sterile liquid or mixture of liquids, including water, saline, aqueous dextrose and related sugar solutions, an alcohol, such as ethanol, isopropanol, or hexadecyl alcohol, glycols, such as propylene glycol or polyethylene glycol,

dimethylsulfoxide, glycerol ketals, such as 2,2-dimethyl-l,3-dioxolane-4-methanol, ethers, such as poly(ethyleneglycol) 400, an oil, a fatty acid, a fatty acid ester or glyceride, or an acetylated fatty acid glyceride with or without the addition of a pharmaceutically acceptable surfactant, such as a soap or a detergent, suspending agent, such as pectin, carbomers, methylcellulose, hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifying agents and other pharmaceutical adjuvants.

[0066] Oils useful in parenteral formulations include petroleum, animal, vegetable, or synthetic oils. Specific examples of oils useful in such formulations include peanut, soybean, sesame, cottonseed, corn, olive, petrolatum, and mineral. Suitable fatty acids for use in parenteral formulations include oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.

[0067] Suitable soaps for use in parenteral formulations include fatty alkali metal, ammonium, and triethanolamine salts, and suitable detergents include (a) cationic detergents such as, for example, dimethyl dialkyl ammonium halides, and alkyl pyridinium halides, (b) anionic detergents such as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionic detergents such as, for example, fatty amine oxides, fatty acid alkanolamides, and polyoxyethylenepolypropylene copolymers, (d) amphoteric detergents such as, for example, alkyl-beta-aminopropionates, and 2-alkyl-imidazoline quaternary ammonium salts, and (e) mixtures thereof.

[0068] The parenteral formulations can contain preservatives and buffers. In order to minimize or eliminate irritation at the site of injection, such compositions may contain one or more nonionic surfactants having a hydrophile-lipophile balance (HLB) of from about 12 to about 17. The quantity of surfactant in such formulations will typically range from about 5 to about 15% by weight. Suitable surfactants include polyethylene sorbitan fatty acid esters, such as sorbitan monooleate and the high molecular weight adducts of ethylene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene glycol. The parenteral formulations can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.

[0069] Topical formulations, including those that are useful for transdermal drug release, are well-known to those of skill in the art and are suitable in the context of the invention for application to skin. Topically applied compositions are generally in the form of liquids, creams, pastes, lotions and gels. Topical administration includes application to the oral mucosa, which includes the oral cavity, oral epithelium, palate, gingival, and the nasal mucosa. In some embodiments, the composition contains at least one active component and a suitable vehicle or carrier. It may also contain other components, such as an anti-irritant. The carrier can be a liquid, solid or semi-solid. In embodiments, the composition is an aqueous solution. Alternatively, the composition can be a dispersion, emulsion, gel, lotion or cream vehicle for the various components. In one embodiment, the primary vehicle is water or a biocompatible solvent that is substantially neutral or that has been rendered substantially neutral. The liquid vehicle can include other materials, such as buffers, alcohols, glycerin, and mineral oils with various emulsifiers or dispersing agents as known in the art to obtain the desired pH, consistency and viscosity. It is possible that the compositions can be produced as solids, such as powders or granules. The solids can be applied directly or dissolved in water or a biocompatible solvent prior to use to form a solution that is substantially neutral or that has been rendered substantially neutral and that can then be applied to the target site. In embodiments of the invention, the vehicle for topical application to the skin can include water, buffered solutions, various alcohols, glycols such as glycerin, lipid materials such as fatty acids, mineral oils, phosphoglycerides, collagen, gelatin and silicone based materials.

[0070] Formulations suitable for oral administration can consist of (a) liquid solutions, such as a therapeutically effective amount of the inventive compound dissolved in diluents, such as water, saline, or orange juice, (b) capsules, sachets, tablets, lozenges, and troches, each containing a predetermined amount of the active ingredient, as solids or granules, (c) powders, (d) suspensions in an appropriate liquid, and (e) suitable emulsions. Liquid formulations may include diluents, such as water and alcohols, for example, ethanol, benzyl alcohol, and the polyethylene alcohols, either with or without the addition of a

pharmaceutically acceptable surfactant, suspending agent, or emulsifying agent. Capsule forms can be of the ordinary hard- or soft-shelled gelatin type containing, for example, surfactants, lubricants, and inert fillers, such as lactose, sucrose, calcium phosphate, and corn starch. Tablet forms can include one or more of lactose, sucrose, mannitol, corn starch, potato starch, alginic acid, microcrystalline cellulose, acacia, gelatin, guar gum, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, calcium stearate, zinc stearate, stearic acid, and other excipients, colorants, diluents, buffering agents, disintegrating agents, moistening agents, preservatives, flavoring agents, and pharmacologically compatible excipients. Lozenge forms can comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the like containing, in addition to the active ingredient, such excipients as are known in the art.

[0071] The compound or salt of the present invention, alone or in combination with other suitable components, can be made into aerosol formulations to be administered via inhalation. The compounds are preferably supplied in finely divided form along with a surfactant and propellant. Typical percentages of active compound are 0.01 %-20% by weight, preferably 1%-10%. The surfactant must, of course, be nontoxic, and preferably soluble in the propellant. Representative of such surfactants are the esters or partial esters of fatty acids containing from 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olestenc and oleic acids with an aliphatic polyhydnc alcohol or its cyclic anhydride. Mixed esters, such as mixed or natural glycerides may be employed. The surfactant may constitute 0.1%-20% by weight of the composition, preferably 0.25%-5%. The balance of the composition is ordinarily propellant. A earner can also be included as desired, such as lecithin for intranasal delivery. These aerosol fonnulations can be placed into acceptable pressurized propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like. They also may be fonmilated as pharmaceuticals for non-pressured

preparations, such as in a nebulizer or an atomizer. Such spray fonnulations may be used to spray mucosa.

[0072] Additionally, the compound or salt of the present invention may be made into suppositories by mixing with a variety of bases, such as emulsifying bases or water-soluble bases. Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams, or spray formulas containing, in addition to the active ingredient, such carriers as are known in the art to be appropriate.

[0073] It will be appreciated by one of ordinary skill in the art that, in addition to the aforedescribed pharmaceutical compositions, the compound or salt of the present invention may be formulated as inclusion complexes, such as cyclodextrin inclusion complexes, or liposomes. Liposomes serve to target the compounds to a particular tissue, such as lymphoid tissue or cancerous hepatic cells. Liposomes can also be used to increase the half-life of the inventive compound. Liposomes useful in the present invention include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. In these preparations, the active agent to be delivered is incorporated as part of a liposome, alone or in conjunction with a suitable chemotherapeutic agent. Thus, liposomes filled with a desired inventive compound or salt thereof, can be directed to the site of a specific tissue type, hepatic cells, for example, where the liposomes then deliver the selected compositions. Liposomes for use in the invention are formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by consideration of, for example, liposome size and stability of the liposomes in the blood stream. A variety of methods are available for preparing liposomes, as described in, for example, Szoka et al., Ann. Rev. Biophys. Bioeng., 9, 467 (1980), and U.S. Patents 4,235,871, 4,501,728, 4,837,028, and 5,019,369. For targeting to the cells of a particular tissue type, a ligand to be

incorporated into the liposome can include, for example, antibodies or fragments thereof specific for cell surface determinants of the targeted tissue type. A liposome suspension containing a compound or salt of the present invention may be administered intravenously, locally, topically, etc. in a dose that varies according to the mode of administration, the agent being delivered, and the stage of disease being treated.

[0074] The dose administered to a mammal, particularly, a human, in accordance with the present invention should be sufficient to effect the desired response. Such responses include reversal or prevention of the adverse effects of the disease for which treatment is desired or to elicit the desired benefit. One skilled in the art will recognize that dosage will depend upon a variety of factors, including the age, condition, and body weight of the human, as well as the source, particular type of the disease, and extent of the disease in the human. The size of the dose will also be determined by the route, timing and frequency of administration as well as the existence, nature, and extent of any adverse side-effects that might accompany the administration of a particular compound and the desired physiological effect. It will be appreciated by one of skill in the art that various conditions or disease states may require prolonged treatment involving multiple administrations.

[0075] Suitable doses and dosage regimens can be determined by conventional range- finding techniques known to those of ordinary skill in the art. Generally, treatment is initiated with smaller dosages that are less than the optimum dose of the compound.

Thereafter, the dosage is increased by small increments until the optimum effect under the circumstances is reached. The present inventive method typically will involve the administration of about 0.1 to about 300 mg of one or more of the compounds described above per kg body weight of the animal or mammal.

[0076] The therapeutically effective amount of the compound or compounds

administered can vary depending upon the desired effects and the factors noted above.

Typically, dosages will be between 0.01 mg/kg and 250 mg/kg of the subject's body weight, and more typically between about 0.05 mg/kg and 100 mg/kg, such as from about 0.2 to about 80 mg/kg, from about 5 to about 40 mg/kg or from about 10 to about 30 mg/kg of the subject's body weight. Thus, unit dosage forms can be formulated based upon the suitable ranges recited above and the subject's body weight. The term "unit dosage form" as used herein refers to a physically discrete unit of therapeutic agent appropriate for the subject to be treated.

[0077] Alternatively, dosages are calculated based on body surface area and from about 1 mg/m ² to about 200 mg/m ² , such as from about 5 mg/m ² to about 100 mg/m ² will be administered to the subject per day. In particular embodiments, administration of the therapeutically effective amount of the compound or compounds involves administering to the subject from about 5 mg/m ² to about 50 mg/m ² , such as from about 10 mg/m ² to about 40 mg/m per day. It is currently believed that a single dosage of the compound or compounds is suitable, however a therapeutically effective dosage can be supplied over an extended period of time or in multiple doses per day. Thus, unit dosage forms also can be calculated using a subject's body surface area based on the suitable ranges recited above and the desired dosing schedule.

[0078] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope. EXAMPLE 1

[0079] This example demonstrates that the number and size of the vacuoles is dependent on both the dose and time of exposure to AM compounds. For example, U20S cells were cultured with the indicated concentration of compound #5 for either 4 hr or 24 hr (Fig. 1). Both the size and the density of vacuoles formed increased with the concentration of compound #5 and the extent of cell culture. Phase contrast images are 20X magnification.

EXAMPLE 2

[0080] This example demonstrates that vacuole accumulation induced by autophagy modulators is reversible (Fig. IB, compounds 1, 2, 4 and 5). U20S cells were cultured for 24 hrs with the indicated compound (DMSO vehicle, 10 μΜ rapamycin, 0.1 μΜ AM compound #1 , 1 μΜ AM compound #4, 2 μΜ AM compound #5, or 2 μΜ AM compound #2). The concentrations were chosen to produce similar levels of vacuoles in 24 hrs. The culture medium was then removed, and the cells were washed with phosphate buffered saline before adding fresh medium without drug. Cells were again cultured for 24 hrs. The vacuoles were completely gone. The same result also was observed after 48 hrs in the presence of an AM compound. Phase contrast images are 20X magnification.

EXAMPLE 3

[0081] This example demonstrates that the vacuoles induced by AM compounds are indistinguishable from those induced by rapamycin (a compound known to induce autophagy) (Fig. 1C, compound 5). The small vacuoles contained cargo, as expected for autophagosomes, and the large vacuoles were empty, as expected for autolysosomes.

Electron microscopic (EM) images of thin sections through cells confirmed that AM compound #5 is more effective than rapamycin (a known autophagy inducer) at induction of vacuoles, as indicated in example 2, and revealed that the small vacuoles induced by WWL are indistinguishable from the small vacuoles induced by rapamycin. Both compounds induced formation of small vacuoles filled with cargo, but WWL also induced large vacuoles that were comparatively empty, indicative of late stage autophagy. U20S cells were cultured for 2 hours either with 10 μΜ Rapamycin (Rap) (A, B) or with 2 μΜ compound #5 (C-F). (A, B) Arrow points to single vacuole in the cytoplasm that contained cargo, indicative of early stage autophagosomes induced by rapamycin. A mitochondrian (mt) is adjacent to the vacuole. (B) is a high resolution image of the same vacuole shown in (A). (C, D) Arrows point to a cluster of three small vacuoles in the cytoplasm that contained cargo, indicative of early stage autophago somes induced by compound #5. (D) is a high resolution image of the same vacuole shown in (C). The density of cytoplasmic vacuoles was at least 10-fold greater in the compound #5 treated cells compared with rapamycin treated cells. (E) Several cytoplasmic vacuoles with cargo surrounding a mitochondrian (mt). (F) Three cytoplasmic vacuoles with cargo, indicative of autophagosomes (ap) induced by compound #5, and a putative double membrane phagophore (pp).

EXAMPLE 4

[0082] This example demonstrates that cells treated with AM compounds remain viable, despite the accumulation of vacuoles. Subconfluent monlayers of 293T and U20S cells were treated with the indicated compound. Both the cells attached to the culture flask and the cells floating in the culture medium were isolated at the times indicated, stained with trypan blue to exclude dead cells, and then counted using a hemocytometer. Both submicromolar and micromolar concentrations of the autophagic inducers had little effect on the ability of 293T cells to remain viable and proliferate. Submicromolar concentrations of AM compounds 1 , 4 or 5 had no affect on cell viability or proliferation. Higher concentrations of AM compounds did not affect viability, although they initially impeded proliferation.

EXAMPLE 5

[0083] This example demonstrates that the density of vacuoles in cells treated with both compounds appeared to be greater than the density of vacuoles in cells treated with either compound alone, suggesting that rapamycin and AM compounds are not only different chemical structures, but affect autophagy by different mechanisms that operate

synergistically. U20S cells were cultured with the indicated compounds for 2 hours (DMSO vehicle, 10 μΜ rapamycin (Rap), 0.2 μΜ compound #5, or 10 μΜ rapamycin + 0.2 μΜ compound #5 (Rap + #5). Phase contrast images are 20X. EXAMPLE 6

[0084] This example demonstrates that the number and size of vacuoles produced by AM compounds is restricted by Bafilomycin Al (Baf), a compound known to inhibit the fusion of autophagosomes with lysosomes to form autolysosomes, in a manner consistent with autophagic flux. U20S cells were cultured with either DMSO vehicle, Baf, or AM compound #5 for 24 hours (Fig. 6A). In one sample (#5 + Baf), 50 nM Bafilomycin Al was added for the final 3 hrs. Vacuoles were absent from the DMSO and Baf controls, but readily apparent in cells treated with compound #5. Addition of Baf to cells harboring a high density of vacuoles markedly reduced both the density of vacuoles and their size distribution. This result was consistent with an accumulation of autophagosomes in the presence of compound #5, followed by inhibition of autophagic flux in presence of Baf. Baf prevented early stage autophagosomes from fusing with lysosomes to form autolysosomes, but allowed

autolysomes to be recycled. Phase contrast images are 20X.

[0085] Electron microscopic images of sections through cells (Fig. 6B) confirmed the conclusion reached in figure 6A: Bafilomycin Al prevents induction and expansion of vacuoles by AM compounds. U20S cells were cultured either with 1 μΜ AM compound #1 for 24 hrs (panels A & B), or with 1 μΜ AM compound #1 for 24 hrs plus 50 nM

Bafilomycin Al for the final 2 hrs (panels C & D). Similarly, U20S cells were cultured either with 2 μΜ AM compound #5 for 24 hrs (Fig. 3, panels C - F), or with 2 μΜ compound #5 for 24 hrs plus 50 nM Bafilomycin Al for the final 2 hrs (Fig. 6B, panels E & F). Figure 6B, panels B and D are higher resolution images of the vacuoles indicated by arrows in panels A and C. Incubation with AM compounds alone resulted in the accumulation of large cytoplasmic vacuoles devoid of cargo, whereas addition of Bafilomycin Al during the final 8-12% of the incubation period resulted in accumulation of small vacuoles containing cargo.

EXAMPLE 7

[0086] This example demonstrates that the large vacuoles are acidic, consistent with the formation of autophagosomes during late stage autophagy. Acridine orange stains late stage autophagic vacuoles. When acridine orange crosses into lysosomes it becomes protonated and emits light in the red range. Acridine orange that is not in an acidic compartment emits green light. The large late stage acuoles induced by any of the autophagy modulators were stained red by acridine orange, indicative of late stage autolysosomes. For example, U20S cells cultured with 2 μΜ compound #1 for 24 hours displayed high acridine orange red fluorescence within the vacuoles, whereas cells cultured with the DMSO vehicle did not.

EXAMPLE 8

[0087] This example demonstrates that accumulation of vacuoles by AM compounds was depended upon Atg7, a gene that is essential for induction of autophagy. siRNA transfections were carried out in the presence of the DMSO vehicle, 10 μΜ rapamycin (Rap) (a known inducer of autophagy), chloroquine (CQ) (a known inhibitor of autophagy), or 2 μΜ AM compound #5. siRNA targeted against human Atg7 gene suppressed the level of Atg7 protein in 293T cells, whereas siRNA targeted against Arabidopsis thalian glabra2 (G12) gene did not (Fig. 8A). Furthermore, siAtg7 also prevented formation of vacuoles in presence of compound #5 (Fig. 8B), whereas siG12 did not (Fig. 8C)

EXAMPLE 9

[0088] This example demonstrates up-regulation of LC3-II and p62 proteins, two proteins associated with formation of autophago somes. LC3 is a microtubule-associated protein that is ubiquitous in mammals. During autophagy, autophagosomes engulf cytoplasmic components, including cytosolic proteins and organelles. Concomitantly, a cytosolic form of LC3 (LC3-I) is conjugated to phosphatidylethanolamine to form LC3- phosphatidylethanolamine (LC3-II), which is recruited to autophagosomal membranes. The p62 protein recognizes toxic cellular waste. During autophagy, p62 binds to ubiquitinated proteins that are then sequestered by autophagy. U20S cells were cultured for 24 hours with the indicated concentration of AM compound #5, #1 or #4 (Fig. 9A). Western immuno- blotting revealed a dose-dependent increase in LC3-II protein, indicative of induction of autophagy by these compounds. As expected, LC3-II up-regulation was dependent on Atg7 expression in 293T cells (data not shown).

[0089] In Figure 9B, four different human cell lines were tested using compound #5. One cell line was from a healthy adult (Wtl) and three were from patients with different degrees of Niemann-Pick's disease (NPC5, NPC4, NPC25). These cells were cultured for 24 hours with the indicated concentration of compound #5. Western immuno-blotting revealed a dose- dependent increase in the p62 and LC3-II proteins, indicative of induction of autophagy by compound #5. The same was true for AM compounds #1 , #4, #2 and #3 (data not shown). EXAMPLE 10

[0090] This example demonstrates that Bafilomycin and Chloroquine, compounds that inhibit fusing of autophagosomes with lysosomes, increase the level of LC3-II induced by AM compounds (Figs. 1 OA and 10B), whereas Wortmannin, a compound that inhibits formation of autophagosomes, decreases the level of LC3-II induced by AM compounds (Fig. IOC). These results are consistent with induction of autophagy by AM compounds.

[0091] Bafilomycin Al prevents maturation of autophagic vacuoles by inhibiting fusion between autophagosomes and lysosomes. Chloroquine accumulates inside the acidic parts of the cell, including endosomes and lysosomes. Chloroquine inhibits autophagy, because it raises the lysosomal pH, which leads to inhibition of fusion of autophagosomes with lysosomes and to degradation of lysosomal proteins. Therefore, addition of either

Bafilomycin Al or Chloroquine to cells should increase the level of LC3-II as cells accumulate autophagosomes. U20S cells were cultured in the presence of 50 nM

Bafilomycin Al (Baf) and the indicated concentration of either rapamycin (Rap) or AM compound #5. The results revealed that either Bafilomycin or Chloroquine increased the level of LC3-II from the cell's basal level of autophagy (DMSO vehicle), the rapamycin induced level, or the compound #5 induced level, consistent with a block to the conversion of autophagosomes into autolysosomes. Since a cell's capacity for autophagy is limited, it appears that compound #5 alone nearly maximized the amount of LC3-II that can be produced by U20S cells. In figure 10B, an equivalent result was observed when 25 μΜ chloroquine (CQ) was added to cells instead of Bafilomycin Al .

[0092] Wortmannin (Wtm) is a selective and irreversible inhibitor of

phosphatidylinositol 3-kinase (PI3K). Since PI3K is required to assemble phagophores, inhibition of PI3K with Wortmannin inhibits the up-regulation of LC3-II. Thus, in Figure IOC, addition of Wortmannin to U20S cells prevented accumulation of LC3-II by inhibiting formation of phagophores, whereas chloroquine (CQ) increased accumulation of LC3-II by preventing conversion of autophagosomes into autolysosomes. Accordingly, when

Wortmannin was included with either rapamycin (Rap) or compound #1 , accumulation of LC3-II was inhibited. EXAMPLE 11

[0093] This example reveals changes in the amount of LC3 over time in the presence of AM compounds that are consistent with autophagic flux. The level of LC3-II increases with accumulation of autophagosomes and then decreases as autophagosomes are converted into autolysosomes, which degrade the cargo as well as that portion of LC3-II within the autolysosome. U20S cells were treated for the indicated times in the presence of either 25 μΜ chloroquine (CQ) or 2 μΜ AM compound #5 (Fig. 1 1). The fraction of LC3-II in control cells treated with the DMSO vehicle remained constant during the first 24 hours. The fraction of LC3-II in cells treated with chloroquine increased, whereas the fraction of LC3-II in cells treated with WWL first increased and then decreased with time. These results confirmed that chloroquine prevented formation of autolysosomes, whereas compound #5 induced autophagic flux.

EXAMPLE 12

[0094] This example demonstrates that AM compounds can deplete a protein fragment containing the Huntington's disease mutation of 74 glutamine repeats from cells without depleting a protein fragment containing a non-Huntington's disease sequence of 23 glutamine repeats. PC 12 cells (rat pheochromocytoma cells) have been engineered to express green fluorescent protein (GFP) -tagged HHT-exonl under control of a doxycyclin inducible promoter [Wyttenbach et al., Hum. Mol. Genet. 2001, 19(17):1829-45]. Q-23 cells express two proteins that cross react with anti-GFP antibodies (Figure 11 A). Only the faster migrating protein cross-reacts with anti-HHT antibodies. That protein contains 23 glutamine repeats. Q-74 cells express a mutant form of HHT protein containing 74 glutamine repeats that is characteristic of the mutated protein in Huntington's disease patients (Figure 1 IB). Normal, functioning HHT protein contains repeats of 10 to 35 glutamines.

[0095] These two cells lines were cultured in the presence of doxycyclin for 48 h before adding the indicated concentration of compound 5 along with fresh medium. Cells were then cultured for another 24 h. Total protein was isolated, fractionated by gel electrophoresis, and stained with antibodies against GFP or LC3. Core histones from the same sample were stained independently. The results revealed that AM compounds #1 , #2, #3, #4 and #5, as well as rapamycin, greatly reduced the amount of GFP-tagged HTT-exonl in Q-74 cells coincident with their induction of LC3-II (Fig. 12A). In contrast, neither rapamycin nor AM compound #5 depleted the level of GFP-tagged HHT protein in the Q-23 cells (Fig. 12B). Comparison of DMSO treated cells with cells treated with the autophagy inhibitor wortmannin (Wtm) revealed that the basal level of autophagy in these cells was minimal. These results suggest that AM compounds depleted cells of the mutated fragment of huntingtin protein by inducing autophagy.

EXAMPLE 13

[0096] This example demonstrates that AM compound #5 decreased HTT in cells from a Huntington's Disease (HD) patient, but increased Huntingtin protein (HTT) in cells from a healthy person, thereby suggesting that AM compounds have the potential to be highly effective therapeutic drugs in the treatment of Huntington's disease. Primary lymphocytes from an HD patient (Coriell Cell Repositories, GM04724) and from healthy control (Coriell Cell Repositories, GM07148) were treated as indicated for 24 hours. Total proteins were isolated, fractionated by polyacrylamide gel electrophoresis, and detected by Western blotting using tubulin (TUB) or actin (ACT) as loading controls.

[0097] Induction of autophagy by AM compound #5, and to a lesser extent rapamycin (Rap), stimulated expression of HTT in the cells taken from a healthy person, as shown in Figure 12A, whereas inhibition of autophagy by addition of Wortmannin (Wtm) had no effect. In contrast, both rapamycin and compound #5 significantly reduced the amount of HTT in cells from an HD patient, but not in the presence of Wortmannin, as shown in Figure 12B. These results reveal that compound #5 increased HTT levels in lymphocytes from a normal human, whereas compound #5 reduced HTT levels in lymphocytes from an HD patient. Given the fact that patient GM04724 is heterozygous for the mutated HTT gene, only half the protein in the HD cells should be subject to elimination by autophagy. In addition, expression of HTT from the normal allele might well be accentuated. Therefore, compound #5 has the potential of alleviating HD symptoms in humans by reducing mutant HTT while concurrently elevating the level of normal HTT.

EXAMPLE 14

[0098] This example demonstrates induction of vacuole formation by AM compounds in cells from a Niemann-Pick Type C patient (ND34733 cells), in accordance with an embodiment of the invention. ND34733 cells were treated for 24 h with either the DMSO vehicle or various concentrations of each of the five AM compounds. The results were photographed under phase contrast (20X magnification) and quantified by estimating the density and size of the vacuoles that formed (Fig. 14).

— : No observable vacuoles, or less than ten vacuoles in each cell in picture

+: More than ten vacuoles in more than one cell in picture; all vacuoles should be very small ++: All or almost all cells have vacuoles and/or some of these vacuoles may be large (-20% size of nucleus or larger)

+++: All cells have vacuoles covering at least 50% of the cell, and many of these vacuoles will be large

++++: Criteria of "+++", plus at least one cell has multiple layers of vacuoles

Using this scale, the efficacy of each AM compound was determined on cells derived from patients with Hutchinson-Gilford Progeria Syndrome (Table 1), Niemann-Pick Type C disease (Table 2), Parkinson's Disease (Table 3), and

Alzheimer's Disease (Table 4).

Samples were also collected in order to extract total protein and subject it to Western immuno-blotting for p62, LC3 and actin. One example is shown in figure 9B.

EXAMPLE 15

[0099] This example demonstrates the effect on vacuole formation in primary fibroblast cell lines from a Huchinson-Gilford Progeria Syndrome patient and from the mother of the patient by compounds 1-5.

[0100] Two primary patient fibroblast cell lines. One was from a Hutchinson-Gilford Progeria Syndrome patient, and the other from the patient's mother. Both were obtained from the Coriell Cell Repository. Cells were treated every other day with compounds in Table 1 for 20 days. Following treatment, cells were photographed at 20X magnification on a phase contrast microscope. The results set forth in Table 1 are in relation to degree of vacuole formation identified in the photographs following treatment, as explained in example 14. Table 1

EXAMPLE 16

[0101] This example demonstrates the effect on induction of autophagy in primary fibroblast cell lines from a normal patient and from three Niemann-Pick Type C patients by compounds 1-5.

[0102] Four primary patient fibroblast cell lines, one from a normal patient, and the other three from Niemann-Pick Type C patients, were provided by Dr. Forbes Porter at the NIH. Patient status is based on the age-adjusted severity score as calculated by the Porter lab. Table 2 presents the data from five separate experiments. In each experiment, all cell lines were treated for 24 hours with DMSO and two different doses of the same compound, then photographed at 20X magnification on a phase contrast microscope and harvested for western blots. Results in Table 2 are in relation to degree of vacuole formation identified in the photographs following treatment, as explained in example 13.

Table 2 Disease Niemann-Pick Type C

Least severe Second-least Most severe

Patient Status Normal

disease severe disease disease

Cell line name Wtl NPC5 NPC4 NPC25

DMSO -+ -+ -+ --+

0.5 μΜ 1 ++++ ++++ ++++ ++++

2.5 μΜ 1 ++++ ++++ ++++ ++++

1 μΜ 4 ++++ ++ ++ ++

Compounds, 5 μΜ 4 ++++ ++++ ++++ ++++

24 hour 1 μΜ 5 ++ ++ ++ ++

treatment 5 μΜ 5

2 μΜ 2 +++ ++ ++ ++

10 μΜ 2 ++++ +++ +++ +++

2 μΜ 3 ++ ++ ++ ++

10μΜ 3 ++++ +++ ++++ ++++

[0103] Western blots shown in Figure 15A-15E are from the samples described in the Table 2. In each experiment, the amount of p62 and LC3-II protein were detected to determine the progress of autophagy, and actin was used as a loading control. The p62 protein serves as a useful marker for the induction of autophagy. It is a receptor for cargo destined to be degraded by autophagy, including ubiquitinated protein aggregates destined for clearance. Figures 14A-14E show photomicrographs of ND34733 cells treated for 24 h with DMSO (control) (Figure 14A), 0.2 μΜ compound 5 (Figure 14B), and 0.1 μΜ, 0.5 μΜ, and 2.5 μΜ compound 1 (Figures 14C14E). These results demonstrate a direct correlation between vacuole formation and induction of the autophagic associated proteins p62 and LC3-II.

EXAMPLE 17

[0104] This example demonstrates the effect on vacuole formation in primary fibroblast cell lines from a normal patient and from two Parkinson's disease patients by compounds 1-5

[0105] Two primary patient fibroblast cell lines from Parkinson's disease patients were treated with compounds for 24 hours. Following treatment, cells were photographed at 20X magnification on a phase contrast microscope. Results in Table 3 are in relation to degree of vacuole formation identified in the photographs following treatment, as explained in example

13.

Table 3

EXAMPLE 18

[0106] This example demonstrates the effect on vacuole formation in primary fibroblast cell lines from two Alzheimer's disease patients by compounds 1-5.

[0107] Two primary patient fibroblast cell lines from Alzheimer's disease patients (both obtained from Coriell) were treated with compounds 1-5 for 24 hours. Following treatment, cells were photographed at 20X magnification on a phase contrast microscope. Results in Table 4 are in relation to degree of vacuole formation identified in the photographs following treatment, as explained in example 13.

Table 4

[0108] SUMMARY OF EXAMPLES 1-14

[0109] Example #1 demonstrates that the number and size of the vacuoles is dependent on both the dose and time of exposure to AM compounds (Fig. 1, compound 5).

[0110] Example #2 demonstrates that vacuole accumulation induced by autophagy modulators is reversible (Fig. 2, compounds 1 , 2, 4 and 5).

[0111] Example #3 demonstrates that the vacuoles induced by AM compounds are indistinguishable from those induced by rapamycin (a compound known to induce autophagy) (Fig. 3, compound 5). The small vacuoles contained cargo, as expected for autophagosomes, and the large vacuoles were empty, as expected for autolysosomes.

[0112] Example #4 demonstrates that cells treated with AM compounds remain viable (Fig. 4, compounds 1 , 2, 3, 4 and 5). [0113] Example #5 demonstrates that induction of vacuole formation by rapamycin (a compound known to induce autophagy) and AM compounds appears synergistic (Fig. 5, compound 5), and therefore they are not only chemically dissimilar, but appear to affect autophagy by different mechanisms.

[0114] Example #6 demonstrates that the number and size of vacuoles produced by AM compounds is restricted by Bafilomycin Al, a compound known to inhibit the fusion of autophago somes with lysosomes to form autolyso somes. Addition of Bafilomycin Al to cells containing a high density of vacuoles resulted in reduction in the density of vacuoles with a greater fraction of small vacuoles containing cargo compared with the fraction of large empty vacuoles. This result is consistent with accumulation of autophagosomes in the presence of Bafilomycin with the concomitant disappearance of autolysosomes (Figs 6A and 6B, compounds 1 and 5).

[0115] Example #7 demonstrates that the large vacuoles are acidic, consistent with the formation of autophagosomes during late stage autophagy (Fig. 7, compound 1).

[0116] Example #8 demonstrates that vacuole formation requires Atg7, a gene that is essential for autophagy (Figs. 8A - 8C, compound 5).

[0117] Example #9 demonstrates that AM compounds cause up-regulation of LC3-II and p62, two proteins associated with formation of autophagosomes (Figs. 9A, 9B, compounds 1, 4 and 5).

[0118] Example #10 demonstrates that Bafilomycin and Chloroquine, compounds that inhibit fusing of autophagosomes with lysosomes, increase the level of LC3-II induced by AM compounds (Figs. 10A, 10B, compound 5), whereas Wortmannin, a compound that inhibits formation of autophagosomes, decreases the level of LC3-II induced by AM compounds (Fig. IOC, compound 1). These results are consistent with induction of autophagy by AM compounds.

[0119] Example #11 demonstrates changes in the amount of LC3 over time in the presence of AM compounds are consistent with autophagic flux (Fig. 1 1 , compound 5). The level of LC3-II increases with accumulation of autophagosomes and then decreases as autophagosomes are converted into autolysosomes, which degrade the cargo as well as that portion of LC3-II within the autolysosome.

[0120] Example #12 demonstrates that submicromolar amounts of AM compounds can reduce the level of the mutated fragment of huntingin protein in a model cell system (Fig. 12A, compounds 1 , 2, 3, 4 and 5) without reducing the level of the normal fragment of the huntingtin protein in a model cell system (Fig. 12B).

[0121] Example #13 demonstrates that submicromolar amounts of AM compounds can reduce the level of huntingin protein in cells from a patient with Huntington's disease (Fig. 13 A, compound 5). In contrast, AM compounds can increase the level of huntingtin protein in normal cells (Fig. 13B, compound 5). Since most Huntington's disease patients are heterozygous for the mutated huntingtin gene, the ability to increase the level of wild-type huntingtin protein concurrently with depleting the mutant huntingtin protein suggests that AM compounds have the potential to be highly effective therapeutic drugs in the treatment of Huntington's disease.

[0122] Example #14 demonstrates that AM compounds could also be effective in the treatment of patients with a genetically based disease/syndrome such as Huntington's, Parkinson's, Alzheimer's, Niemann-Pick's, and Hutchinson-Gilford progeria. AM

compounds stimulated vacuole formation (Fig. 14) in these cells and up-regulated both LC3- II and p62 proteins (Fig. 9B), two genes associated with induction of autophagy. The results are summarized in Tables 1 - 4.

SUMMARY OF RESULTS

[0123] ND = Not Detected; AM compounds did not reduce cellular levels of wild-type Htt

[0124] 1 - vacuole formation refers to concentration at which vacuole formation was detected by light microscopy in human U20S cells cultured for 4 hours in the presence of the indicated AM compound. [0125] 2 - MEC cell growth refers to the concentration at which cell proliferation was inhibited, an effect that was cell type dependent. 'Greater than' (>) symbol indicates the highest concentration tested.

[0126] 3 - MEC cytotoxicity refers to the concentration at which cell viability was inhibited, as measured by the number of cells that excluded trypan blue (viable cells).

[0127] 4 - MEC protein clearance refers to the concentration at which a mutated or wild- type huntingtin protein (HTT) or protein fragment (exonl-HTT) was cleared from cells. In the PC 12 rat cell model, either the 74 glutamine repeats in exon 1 from a mutated huntingtin protein (exonl-mHHT), or the 23 glutamine repeats in exon 1 from a wild-type huntingtin protein (exonl-HTT) are expressed under control of a doxycycline inducible promoter.

Primary lymphocytes derived from a Huntington's disease patient contained full length mutated Huntingtin protein (mHHT), whereas those from a patient without Huntington's disease contained full length wild-type Huntingtin protein (HTT). The molecular weight of HTT is 347 kDa, which was too large to clearly separate mHTT from HTT by gel

electrophoresis.

[0128] 5 - The Huntington's disease patient was heterozygous for the mHtt gene, suggesting that no more than a 50% reduction in huntingtin protein could be obtained under optimal conditions. Treatment of human lymphocytes with AM compound #5 decreased the level of mHTT about 2-fold in cells from this Huntington's disease patient, and it increased the level of wild-type HTT protein by 3.5-fold in cells from a non-HD patient. The combined effect would make these AM compounds highly effective in treatment of Huntington's disease patients.

[0129] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0130] The use of the terms "a" and "an" and "the" and "at least one" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term "at least one" followed by a list of one or more items (for example, "at least one of A and B") is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0131] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.