TITLE OF THE INVENTION

Novel Methods of Treating or Preventing Alzheimer's Disease CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority under 35 U.S.C. § 119(e) to U.S.

Provisional Patent Application No. 61/903,200, filed November 12, 2013, which application is incorporated herein by reference in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR

DEVELOPMENT

This invention was made with government support under Contract No.

R01NS057295 awarded by the National Institutes of Health's (NIH), National Institute of Neurological Disorders and Stroke. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Alzheimer's Disease (AD) is a progressive neurodegenerative disease leading to memory loss. Particularly important is the synaptic loss observed in the hippocampus, which is the center of learning and memory. It is thought that Αβ, a neurotoxic peptide, initiates a cascade that ultimately results in synaptic dysfunction, gradual cognitive decline, and eventually neuronal death. Amyloid plaques comprising Αβ are hallmarks of pathology in neurodegenerative disorders such as AD (Braak, et al., 2011, J. Neuropathol. Exp. Neurol. 70:960).

Αβ may be generated through the proteolytic degradation of amyloid precursor protein (APP) by β- and γ-secretases, and alterations to this process may result in AD. APP is a type I transmembrane protein containing an intracellular C-terminal domain and a larger external N-terminal domain. APP is proteolytically processed through non-amyloidogenic or amyloido genie pathways. After translation, N- and O-glycosylated APP (mature APP) is trafficked to the plasma membrane where a-secretase (non-amyloidogenic) can cleave full- length APP (FL-APP) liberating a soluble N-terminal fragment (sAPPa) and a membrane tethered C-terminal fragment (APP-CTFa). Alternatively, FL-APP can be endocytosed and either recycled back to plasma membrane or localized to the early endosome, where β- secretase cleavage of FL-APP initiates amyloidogenic cleavage. This β-secretase cleavage liberates a soluble N-terminal fragment ^ΑΡΡβ) and a membrane tethered C-terminal fragment (APP-CTFP). Following this initial cleavage by either a- or β-secretase, retrograde trafficking mechanisms deliver APP-CTFs to the trans-Golgi network (TGN), where the majority of γ-secretase cleavage of APP occurs. APP-CTFs are cleaved by γ-secretase to generate, along with the APP intracellular domain (AICD), either p3 or Αβ, depending on whether the APP-CTF was derived from a- or β-secretase, respectively.

APP proteolysis is intimately associated with its subcellular localization, therefore APP trafficking plays a critical role in amyloidogenesis. Cholesterol can alter cellular membrane fluidity and trafficking of transmembrane proteins like APP. In fact, cholesterol has been shown to be an AD risk, with increasing cholesterol levels in in vitro and in vivo AD models exacerbating Αβ production.

The Hedgehog (Hh) signaling pathway is a signaling pathway that transmits information to embryonic cells required for proper development, playing a key role in animal development and being present in all bilaterians. The Hh signaling pathway takes its name from its polypeptide ligand, an intercellular signaling molecule called Hedgehog (Hh) found in fruit flies of the genus Drosophila.

Mammals have three Hedgehog homologues, Desert Hedgehog (DHh), Indian Hedgehog (IHh), and Sonic Hedgehog (SHh). The pathway is important during vertebrate embryonic development. Indeed, in knockout mice lacking components of the pathway, the brain, skeleton, musculature, gastrointestinal tract and lungs fail to develop correctly. Recent studies point to the role of Hedgehog signaling in regulating adult stem cells involved in maintenance and regeneration of adult tissues, as well as the development of some cancers, including basal cell carcinoma.

Smoothened (Smo) is a G protein-coupled receptor protein (SEQ ID NO: 1 for the human protein) encoded by the SMO gene of the Hh signaling pathway. Smo is conserved from flies to humans. Smo may function as an oncogene, with its activating mutations leading to unregulated activation of the Hh pathway and development of cancer.

There is a need in the art for novel methods of treating or preventing a neurodegenerative disease, such as AD, in a mammal in need thereof. The present invention fulfills this need.

BRIEF SUMMARY OF THE INVENTION

The invention includes a method of treating or prevent a disease or disorder selected from the group consisting of Alzheimer's Disease (AD), cerebral amyloid angiopathy and Parkinson's Disease in a mammal in need thereof. The invention further includes a method of reducing the rate of production of Αβ or the rate of γ-secretase-mediated amyloid precursor protein (APP) cleavage in a mammal in need thereof.

In certain embodiments, the method comprises administering to the mammal a therapeutically effective amount of at least one inhibitor of the Hedgehog signaling pathway.

In certain embodiments, the inhibitor comprises a Smo antagonist. In other embodiments, the antagonist is at least one selected from the group consisting of: MRT 10 (N-[[[3-benzoylamino)phenyl]amino]thioxomethyl]-3,4,5-trimet hoxy benzamide); jervine ((3p,23P)-17,23-epoxy-3-hydroxyveratraman-l 1-one); SANT-1 (N-[(3,5-dimethyl-l-phenyl- lH-pyrazol-4-yl)methylene]-4-(phenyl methyl)- 1-piperazinamine); SANT-2 (N-[3-(lH- benzimidazol-2-yl)-4-chlorophenyl]-3,4,5-triethoxy benzamide); vismodegib (2-chloro-N-(4- chloro-3-pyridin-2-ylphenyl)-4-methylsulfonylbenzamide); erismodegib (N-[6-[(2R,6S)-2,6- dimethyl-4-morpholinyl] -3-pyridinyl] -2-methyl-4 ' - (trifluoromethoxy)- [ 1,1' -biphenyl] -3- carboxamide); BMS-833923 (N-(2-methyl-5-((methylamino)methyl)phenyl)-4-((4- phenylquinazolin-2-yl)amino)benzamide); saridegib (N-((2S,3R,3aS,3'R,4a'R,6S,6a'R,

6b'S,7aR,12a'S,12b'S)-3,6,l l',12b'-tetramethyl-2',3a,3',4,4',4a',5,5',6,6',6a',6b',7,7a ,7',8', 10',12',12a',12b'-icosahydro- H,3H-spiro[furo[3,2-b]pyridine-2,9'-naphtho[2,l-a]azulen]- 3'-yl) methanesulfonamide); PF-04449913 (l-((2R,4R)-2-(lH-benzo[d]imidazol-2-yl)-l- methylpiperidin-4-yl)-3-(4-cyanophenyl)urea); LEQ-506 ((R)-2-(5-(4-(6-benzyl-4,5- dimethylpyridazin-3-yl)-2-methylpiperazin-l-yl)pyrazin-2-yl) propan-2-ol); TAK-441 (6- ethyl-N-[l-(hydroxyacetyl)piperidin-4-yl]-l-methyl-4-oxo-5-( 2-oxo-2-phenylethyl)-3-(2,2,2- trifluoroethoxy)-4,5-dihydro-lH-pyrrolo[3,2-c]pyridine-2-car boxamide); itraconazole ((2R,4S)-rel-l-(butan-2-yl)-4-{4-[4-(4-{ [(2R,4S)-2-(2,4-dichlorophenyl)-2-(lH-l,2,4-triazol· l-ylmethyl)-l,3-dioxolan-4-yl]methoxy}phenyl) piperazin-l-yl]phenyl}-4,5-dihydro-lH- l,2,4-triazol-5-one); LY2940680 (4-fluoro-N-methyl-N-(l-(4-(l-methyl-lH-pyrazol-5- yl)phthalazin-l-yl)piperidin-4-yl)-2-(trifluoromethyl)benzam ide)), a salt or solvate thereof, and any combinations thereof.

In certain embodiments, the inhibitor is administered to the mammal as part of a pharmaceutically acceptable composition.

In certain embodiments, the mammal does not present symptoms of the disease or disorder. In other embodiments, the mammal presents at least one symptom of the disease or disorder.

In certain embodiments, the inhibitor is formulated as part of an extended- release formulation. In other embodiments, the inhibitor is administered to the mammal by at least one route selected from the group consisting of inhalational, oral, rectal, vaginal, parenteral, topical, transdermal, pulmonary, intranasal, buccal, sublingual, ophthalmic, intrathecal, intravenous and intragastrical.

In certain embodiments, administration of the inhibitor decreases the rate of amyloid-beta (Αβ) production in the mammal. In other embodiments, administration of the inhibitor decreases the rate of γ-secretase-mediated APP cleavage in the mammal. In yet other embodiments, administration of the inhibitor does not substantially affect the protein level and the enzymatic activity of γ-secretase in the mammal. In yet other embodiments, administration of the inhibitor treats, stabilizes or reverses a motor neuron deficit associated with the disease or disorder in the mammal.

In certain embodiments, the mammal suffers from, is suspected of suffering from, or is likely to develop at least one disease or disorder selected from the group consisting of Alzheimer's Disease, cerebral amyloid angiopathy, and Parkinson's Disease.

In certain embodiments, the mammal is a primate. In other embodiments, the primate is human.

BRIEF DESCRIPTION OF THE DRAWINGS

For the purpose of illustrating the invention, there are depicted in the drawings certain embodiments of the present invention. However, the invention is not limited to the precise arrangements and instrumentalities of the embodiments depicted in the drawings.

Figs. 1A-1B are a set of diagrams illustrating APP metabolism (Fig. 1A) and the mammalian sonic Hedgehog signaling pathways, along with selected inhibitors thereof (Fig. 1A).

Figs. 2A-2L illustrate the finding that cyclopamine treatment alters APP metabolism. Fig. 2A: Primary rat cortical neurons treated with 5 μΜ cyclopamine for 24 hours. Endogenous FL-APP and APP-CTFs were detected using Western immunoblotting and a C-terminus APP antibody (cl/6.1). Figs. 2B-2C: FL-APP protein levels and APP- CTFs were normalized to β-actin and FL-APP, respectively. Fig. 2D: HeLa cells treated with indicated concentrations of cyclopamine for 24 hours followed by FL-APP and APP-

CTF detection in cell lysates using Western immunoblotting. Fig. 2E: Quantification of dose dependent accumulation of APP-CTFs normalized to FL-APP. Relative protein changes compared to vehicle (DMSO) control (dotted line). Fig. 2F: Western blot time course analysis of endogenous FL-APP and APP-CTFs in HeLa cells treated with 5 μΜ cyclopamine. Fig. 2G: Normalized APP-CTFs increase in a time dependent manner as compared to vehicle (DMSO) control (dotted line). Fig. 2H: Using Western blot analysis and N-terminus APP antibody (22C11), endogenous sAPP levels were monitored in supernatants of HeLa cells treated with 5 μΜ cyclopamine for 24 hours. Fig. 21: sAPP levels were normalized to protein concentrations in lysates; determined using BCA assay. Lack of change in sAPP protein levels are illustrated in comparison to vehicle control (DMSO). Fig. 2J: Endogenous FL-APP and APP-CTFs from naive HeLa cells treated with 5 μΜ

cyclopamine were compared to cells treated with 2 μΜ L-685,458 using Western immunoblot analysis and C-terminus APP antibody (cl/6.1). Lower panel represent increased exposure of APP-CTFs. Fig. 2K-2L: Cyclopamine increased APP-CTF levels (not FL-APP) compared to vehicle (DMSO) control. Values denote mean + standard errors of the means. Student's t- test was used for statistical analysis: ***p<0.005, **p<0.01, *p<0.05.

Figs. 3A-3K illustrate the finding that cyclopamine decreases γ-secretase cleavage of APP in vitro and in vivo. Fig. 3A: Secreted Αβ40 levels from HeLa cells and primary cortical neurons (DIV6) treated with 5 μΜ cyclopamine for 24 hours were measured using an ELISA and compared to vehicle (DMSO) control. Secreted Αβ40 levels were normalized to total protein in respective cell lysates. Fig. 3B: HeLa cells transiently overexpressing FL-APPGal4 treated with vehicle (DMSO) control, 2 μΜ L-685,458 or 5 μΜ cyclopamine for 24 hours. APP-CTFGal4a nd AICD-Gal4 levels analyzed using Western immunoblotting and a C-terminus APP antibody (cl/6.1). Fig. 3C: APP-AICD-Gal4 levels normalized to APP-CTF-Gal4 levels. Fig. 3D: HeLa cells transiently overexpressing AENotch-Myc treated with vehicle (DMSO) control, 2 μΜ L-685,458 or 5 μΜ cyclopamine for 24 hours. NICD and AENotch levels analyzed using Western immunoblotting, an anti- Myc and anti-NICD (Vall744) antibody. Fig. 3E: NICD levels normalized to AENotch levels. Fig. 3F: APP-CTF-Gal4 and AENotch levels normalized to β-actin. Figs. 3G-3J: representative images of rough-eye phenotype from GMR-APP-Gal4; UAS-Grim (γ-secretase reporter) flies raised on normal food, 100 nM cyclopamine or vehicle control (DMSO). Flies were scored one day after eclosion: mild (+), moderate (++), severe (+++). Fig. 3K:

Relative changes in penetrance of rough-eye phenotype in flies raised on normal food (n=168), vehicle containing food (n=72), or cyclopamine containing food (n=l 12).

Population of flies with mild, moderate or severe rough-eye phenotype is illustrated as percent of total population per experimental group. Statistical analysis in vivo experiments; a G-test: ***p<0.005, **p<0.01, *p<0.05, was performed. Student' s t- test was used for statistical analysis of cell based in vitro studies: ***p<0.005, **p<0.01, *p<0.05. Values denote mean + standard errors of the means.

Figs. 4A-4C illustrate the finding that γ-secretase activity is not altered by cyclopamine treatment. Fig. 4A: HeLa cells treated with 5 μΜ cyclopamine or vehicle control (DMSO) for 24 hours. PSEN1-CTF levels from respective lysates analyzed via Western immunoblotting. Fig. 4B: PSENl-CTFs levels normalized to β-actin. Fig. 4C: Fluorometric γ-secretase activity assay. Fluorescence intensity over time using total membranes isolated from naive HeLa cells treated with cyclopamine (20 μΜ), L-685,458 (20 μΜ) or vehicle control (DMSO). Graph represents relative changes in fluorescence as percent activity of control (membranes treated with DMSO) over time. Values denote mean + standard errors of the means. Student's i-test was used for statistical analysis: ***p<0.005, **p<0.01, *p<0.05.

Figs. 5A-5G illustrate the finding that cyclopamine induces subcellular accumulation of APP-CTF. Confocal 3-D analysis of HeLa cells treated with vehicle control (DMSO) (Fig. 5A), or 5 μΜ cyclopamine (Fig. 5B), for 0, 6, or 24 hours. APP was detected using an antibody against the C-terminus of APP (A8717). Scale bar, 10 μιη. Fig. 5C:

Number of APP puncta normalized to ROIs. ROI=10xl0 mm, 20-30 ROIs per treatment analyzed. Puncta were defined as 2X the intensity of background in cytosolic non-punctate region, and objects were restricted to 0.2-2.0 μιη skeletal diameter. Confocal 3-D analysis of naive HeLa cells treated with vehicle control (DMSO) (Fig. 5D), or 5 μΜ cyclopamine (Fig. 5E), for 24 hours. Cells were stained using (left panel) C-terminus APP (A8717) and (middle panel) N-terminus APP (22C11) antibodies. Right panel denotes merged channels. Scale bar, 10 μιη. Fig. 5F: Cell surface FL-APP levels in HeLa cells treated with 5 μΜ

cyclopamine or vehicle (DMSO) for 24 hours and then biotinylated. Biotinylated, surface FL-APP levels were measured by Western blot with a C-terminus APP antibody (cl/6.1). Top panel is total FL-APP from whole cell lysate, middle panel is biotinylated surface FL- APP purified with NeutrAvidin-coated resin. Fig. 5G: Surface FL-APP levels normalized to total FL-APP as percent of vehicle (DMSO) control. Values denote mean + standard errors of the means. Student's t-test was used for statistical analysis: ***p<0.005, **p<0.01, */?<0.05.

Fig. 6 is a set of panels illustrating endogenous APP-CTF distribution in vehicle treated HeLa cells. Confocal 3-D analysis of HeLa cells treated with vehicle control (DMSO) for 24 hours. Cells were stained for APP-CTFs using an APP C-terminal antibody (A8717) and antibodies (EEA1, MP6R, LAMP1, TGN46, LC3) for subcellular markers (middle column). Right-hand columns are the merged images. Scale bar, 10 μιη. Fig. 7 is a set of panels illustrating the finding that cyclopamine alters retrograde trafficking of APP-CTFs. Confocal 3-D analysis of HeLa cells treated with 5 μΜ cyclopamine for 24 hours. Cells were stained for APP-CTFs using an APP C-terminal antibody (A8717) and antibodies (EEA1, MP6R, LAMP1, TGN46, LC3) for subcellular markers (middle column). Right-hand columns are the merged images. Scale bar, 10 μιη.

Figs. 8A-8J illustrate the quantification of subcellular marker total intensity and APP-CTF subcellular localization in HeLa cells. Figs. 8A-8E: Single cells in each image were masked off, background intensities subtracted from each channel and sum intensity of each cell measured and normalized to volume. Also, Manders' coefficients were calculated for each cell independently, 26-51 cells were analyzed per experimental group.

Figs. 8F-8J: Manders' coefficients for co-localization. Dot plot diagrams represent raw data points, while horizontal line represents the means of Manders' coefficients of subcellular marker and APP-CTF colocalization. Student's t-test was used for statistical analysis:

***/?<0.005, **/?<0.01, */?<0.05.

Figs. 9A-9B are a set of panel illustrating immunofluorescence of APP-CTFs and MVBs in HeLa cells. Confocal 3-D analysis of HeLa cells treated with vehicle control (DMSO) (Fig. 9A), or 5 μΜ cyclopamine (Fig. 9B), for 24 hours. Cells were co-stained using (top panel) C-terminus APP (A8717) and an MVB ESCRT-I marker, TsglOl. In middle panel a MVB ESCRT-III marker, Chmp2a-GFP, was overexpressed and cells were stained with A8717. Bottom panel denotes all merged channels. Scale bar, 10 μιη. As expected, APP-CTFs are in close association with MVB markers, TsglOl and Chmp2a-GFP upon cyclopamine treatment.

Figs. 10A-10H illustrate the finding that cyclopamine increases APP-CTF levels in lysosome-enriched compartments. HeLa cells were treated with vehicle (DMSO) (Fig. 10A), or 5 μΜ cyclopamine (Fig. 10B), for 24 hours, then collected and homogenized. Post- nuclear fractions were subject OptiPrep step gradient fractionation. Fractions 2-13 (50 -10 gradient) were subject to Western blot analysis with APP C-terminal (cl/6.1), PSEN1-CTF, EEA1, LAMP 1, and TGN46 antibodies. Figs. 10C-10H: Densitometry of each fraction as percent of combined total (fractions 2- 13) densitometry for each respective protein.

Figs. 11 A-l IE illustrate the finding that cyclopamine leads to moderate decrease in lysosomal maturation and significantly attenuates APP-CTF rate of lysosomal degradation. Fig. 11 A: Western immunoblot analysis of FL-APP and APP-CTFs using a C- terminus APP antibody (cl/6.1). For FL-APP and APP-CTF analysis, naive HeLacells were pre-treated with 5 μΜ cyclopamine of vehicle (DMSO) for 24 hours, then exposed to 50 μg/ml cycloheximide for indicated times. Figs. 1 lB-11C: FL-APP and APP-CTF protein levels were normalized to β-actin first. The graphs represent protein levels as percent remaining of total protein at time 0 hour. The lines represent the linear least squares fit where the slope of the line is the rate of protein degradation. Fig. 1 ID: Naive HeLa cells treated with vehicle (DMSO) or 5 μΜ cyclopamine for 24 hours followed by mature and immature cathepsin D detection in cell lysates via Western immunoblotting. Fig. HE: Bar diagram represents ratio of mature to immature cathepsin D protein quantification normalized to β- actin. Values denote mean + standard errors of the means. Student's i-test was used for statistical analysis: ***/?<0.005, **p<0.01, */?<0.05.

Figs. 12A-12B are a set of model representations of APP-CTF retrograde trafficking and lysosomal localization upon cyclopamine exposure. Trafficking and cleavage of FL-APP and APP-CTFs in normal conditions (Fig. 12A), compared to cyclopamine treatment (Fig. 12B), FL-APP proteolysis and production of APP-CTFs occurs at the plasma membrane (a-secretase) and early endosomes (β-secretase). APP-CTFs are then trafficked, via the retrograde pathway, to trans-Golgi network (TGN) for subsequent γ-secretase cleavage and Αβ generation. Alternatively, APP-CTFs are trafficked to late

endosomes/multivesicular bodies thus destined for lysosomal degradation. Fig. 12B:

Cyclopamine treatment favors the lysosomal degradation trafficking pathway (bold arrows) of APP-CTFs thereby preventing γ-secretase proteolysis of APP-CTFs and Αβ generation.

Figs. 13A-13B are a set of graphs and images illustrating the finding that accumulation of APP-CTFs is Smo- and cyclopamine-dependent.

Fig. 14 is a set of graphs and images illustrating the finding that cyclopamine rescues AD-like pathology in a Drosophila model of AD.

Fig. 15 is a set of images and graph that illustrate the finding that cyclopamine and LY2940680 suppress external phenotypes in AD Drosophila flies.

Figs. 16A-16B are a set of graphs that illustrate the finding that cyclopamine rescues the memory deficit observed in AD Drosophila flies. Fig. 16A illustrates the data obtained for AD Drosophila flies that were treated with DMSO, and Fig. 16B illustrates the data obtained for AD Drosophila flies that were treated with cyclopamine.

Figs. 17A-17B illustrate the finding that the Hedgehog agonist SAG does not rescue cyclopamine-induced APP-CTF accumulation, as demonstrated by gel analysis (Fig. 17A) and normalized protein quantitation (Fig. 17B).

Figs. 18A-18C illustrate the finding that the Smo antagonist GDC0449 does not alter APP proteolysis, as demonstrated by gel analysis (Fig. 18B) and normalized protein quantitation (Fig. 18C).

Figs. 19A-19C illustrate the finding that the Smo knockdown using shRNA alters APP proteolysis, as demonstrated by gel analysis (Fig. 19 A) and normalized protein quantitation (Fig. 19C).

Figs. 20A-20D is a series of images and graphs that illustrate that inhibition of SHhN with the Hedgehog pathway antagonist 5E1 alters APP metabolism (Figs. 20A-20B) while inhibition of Glil/2 with GANT61 does not (Figs. 20C-20D).

Figs. 21A-21E illustrate the finding that Smoothened and Patched loss of function reduces γ-secretase cleavage of APP-C99-Gal4 in vivo. Figs. 21A-21D are a series of images for Drosophila flies that that were obtained by crossing γ-secretase reported flies with w- control, Smo, ptch and Hh ^lof alleles. Fig. 21E is a bar graph that illustrates % rough eye phenotype for distinct groups of Drosophila flies.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates in certain aspects to the unexpected discovery that inhibitors of the Hedgehog signaling pathway alter APP metabolism and decrease production of Αβ in a subject. In certain embodiments, the inhibitor comprises a Smoothened (Smo) antagonist. In other embodiments, the inhibitors useful within the invention are used to treat or prevent Alzheimer's Disease (AD) in the subject.

As demonstrated herein, pharmacological and genetic manipulation of Smo (a SHh signaling component) leads to an increase in generation of APP-CTFs and a decrease in generation of Αβ and AICD. While γ-secretase cleavage of APP is significantly altered upon treatment with cyclopamine (an exemplary Smo antagonist), protein levels and the enzymatic activity of γ-secretase remained unaffected. The in vivo results indicate a motor neuron rescue effect upon cyclopamine treatment. Taken together, these results suggest that Smo modulates γ-secretase-mediated APP cleavage, and Smo antagonists decrease neurotoxic Αβ concentrations and ameliorate motor neuron deficits observed in AD pathology. The results disclosed herein strongly suggests that Smo is a novel regulator of APP metabolism, and Smo antagonists rescue the effects of neurotoxic Αβ peptide observed in AD models.

As demonstrated herein, novel effects on APP trafficking and lysosomal maturation induced by treatment with cyclopamine were characterized. Specifically, an accumulation of APP-CTFs in lysosomes and a decrease in Αβ and AICD generation were observed. After translation, APP is trafficked to the plasma membrane via the secretory pathway. APP can then be cleaved by a- or β-secretase at the plasma membrane or early endosome after endocytosis, respectively. These APP-CTFs are then retrogradely trafficked to the trans-Golgi network (TGN) for γ-secretase cleavage and Αβ generation. APP retrograde trafficking is highly regulated because APP proteolysis is dynamic and can lead to rapid changes in Αβ production. A consequence of decreased APP-CTF trafficking to the TGN is the decrease in γ-secretase mediated cleavage of APP-CTFs and the concomitant decrease in Αβ and AICD generation. Hence, modulating APP retrograde trafficking independent of secretase activity can have novel implications for therapeutic avenues to treat AD.

Cyclopamine is a naturally occurring phytosterol isolated from the corn lily plant. The animal sterol, cholesterol, promotes amyloidogenic processing and increases Αβ generation, while cyclopamine exhibits the opposite effects on Αβ generation. Cyclopamine treatment decreases γ-secretase mediated proteolysis of APP without inhibiting γ-secretase activity directly. However, cyclopamine treatment leads to the accumulation of APP-CTFs derived from a- and β-secretase equally, because the observed cyclopamine effects were downstream of APP-CTF generation at the plasma membrane (a-secretase) or early endosome (β-secretase).

Cyclopamine decreased APP-CTF retrograde trafficking to the TGN in HeLa cells. γ-Secretase cleavage of endocytic APP-CTFs and Αβ generation occurs at the TGN. Since cyclopamine does not inhibit γ-secretase activity, the changes in APP-CTF levels are not due to changes in activity. In certain embodiments, these changes are due to APP-CTF retrograde trafficking, thus preventing colocalization with γ-secretase. Without wishing to be limited by any theory, the observed decrease localization of APP-CTFs at the TGN suggests that altered trafficking is the mechanism by which cyclopamine decreases Αβ generation (Figs. 12A-12B). Alternatively, after APP-CTF endocytosis, these fragments can be trafficked to the late endosome/MVB and then to the lysosome for degradation.

As demonstrated herein, cyclopamine increases APP-CTF trafficking to lysosomes. This increased trafficking to lysosomes could result in increased protein degradation and decreased APP-CTF levels. However, increased APP-CTF levels, and attenuated lysosomal degradation of APP-CTFs, were observed upon cyclopamine treatment. The accumulation of APP-CTFs was completely reversible upon washout of cyclopamine. Similar changes in APP processing were observed in HeLa cells and neurons.

Upon reaching the plasma membrane, FLAPP sheds the sAPP ectodomain. Lack of change in FL-APP and sAPP levels indicates cyclopamine does not alter the APP biosynthetic pathway. Decreased surface FL-APP suggests enhanced endocytosis after shedding, which can explain the increase in early endosome immunofluorescence intensity. The change in Αβ, AICD, and APP-CTF levels in the absence of changes in FL-APP and sAPP levels imply the effects of cyclopamine on APP metabolism are specific. The lack of change in FL-APP and sAPP levels upon cyclopamine treatment suggests that it is a good candidate for AD therapy as it induces APP-CTF sequestration in lysosomes resulting in modest decreases in Αβ levels, while not affecting FL-APP levels and sAPP generation. However, without wishing to be limited by any theory, the chronic accumulation of APP- CTFs in the lysosome may be detrimental to protein homeostasis.

Both APP and Notch require primary cleavage at the plasma membrane for downstream endocytosis and retrograde trafficking to the TGN for γ-secretase and

AICD/NICD generation. Cyclopamine' s effects may be specific to APP-CTFs, as the exact same changes were not observed in exogenous AENotch processing. Decreased NICD levels similar to the decrease in AICD levels were observed. However, increased AENotch levels as for exogenous APP-CTF-Gal4 and endogenous APP-CTFs were not observed.

A balance between retrograde and lysosomal degradation trafficking pathways ensures proper distribution of APP and Notch holoproteins and their metabolites. Since these metabolites are involved in regulating cell death/survival and synaptic plasticity, in certain embodiments, complete ablation of production of these metabolites is detrimental. The modest but significant decrease observed in Αβ, AICD, and NICD levels suggests cyclopamine does not have the negative consequences that γ-secretase inhibition displays in some AD patients.

The invention disclosed herein should not be construed to be limited to Alzheimer's Disease. Rather, the invention disclosed herein may be applied to any neurodegenerative disease in a subject wherein therapeutic benefit is derived from decreasing the rate of Αβ production and/or decreasing the rate of γ-secretase-mediated APP cleavage in the subject, such as Alzheimer's Disease, cerebral amyloid angiopathy, and Parkinson's Disease.

Definitions

As used herein, each of the following terms has the meaning associated with it in this section.

Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, and peptide chemistry are those well known and commonly employed in the art.

As used herein, the articles "a" and "an" refer to one or to more than one (i.e. , to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

As used herein, the term "Αβ" or "Abeta" refers to amyloid-beta, which is a peptide of 36-43 amino acids that is processed from the amyloid precursor protein (APP).

As used herein, the term "about" is understood by persons of ordinary skill in the art and varies to some extent on the context in which it is used. As used herein when referring to a measurable value such as an amount, a temporal duration, and the like, the term "about" is meant to encompass variations of +20% or +10%, more preferably +5%, even more preferably +1 %, and still more preferably +0.1 % from the specified value, as such variations are appropriate to perform the disclosed methods.

As used herein, the term "AD" refers to Alzheimer' s Disease.

As used herein, the term "AICD" refers to APP intracellular cytoplasmic/C- terminal domain, which is the smaller cleavage product of APP by γ-secretase.

As used herein, the term "APP" refers to amyloid precursor protein.

As used herein, the term "APP-CTF" refers to the APP cleaved C-terminal fragment.

As used herein, the term "composition" or "pharmaceutical composition" refers to a mixture of at least one compound useful within the invention with a

pharmaceutically acceptable carrier. The pharmaceutical composition facilitates

administration of the compound to a patient. Multiple techniques of administering a compound exist in the art including, but not limited to, intravenous, oral, aerosol, parenteral, ophthalmic, pulmonary and topical administration.

As used herein, the term "cycloheximide" refers to 4-[(2R)-2-[(l _l S ^, ,3 _l S ^, ,55 ^, )-3,5- dimethyl-2-oxocyclohexyl]-2-hydroxyethyl]piperidine-2,6-dion e, or a salt or solvate thereof.

As used herein, the term "cyclopamine" refers to 11-deoxojervine, also known as (2'R,3S,3'R,3'aS,6'S,6aS,6bS,7'aR, l laS,l lbR)- l,2,3,3'a,4,4',5',6,6',6a,6b,7,7',7'a,8, l l,l la, l lb-octadecahydro-3',6', 10, l lb-tetramethyl- spiro[9H-benzo[a]fluorene-9,2'(3'H)-furo[3,2-b]pyridin]-3-ol , or a salt or solvate thereof. As used herein, the terms "effective amount," "pharmaceutically effective amount" and "therapeutically effective amount" refer to a nontoxic but sufficient amount of an agent to provide the desired biological result. That result may be reduction and/or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. An appropriate therapeutic amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation.

As used herein, the term "FL" refers to full-length.

As used herein, the term "GANT61" refers to the Glil/2-induced transcription inhibitor 2,2'-[[dihydro-2-(4-pyridinyl)-l,3(2H,4H)-pyrimidinediyl]bis (methylene)]bis[N,N- dimethyl-benzenamine, or a salt or solvate thereof.

As used herein, the term "GDC-0449" refers to the Hh inhibitor vismodegib 2- chloro-N-(4-chloro-3-(pyridin-2-yl)phenyl)-4-(methylsulfonyl )benzamide, or a salt or solvate thereof.

As used herein, the term "Hh" refers to Hedgehog.

"Instructional material," as that term is used herein, includes a publication, a recording, a diagram, or any other medium of expression that may be used to communicate the usefulness of the compounds of the present invention. In some instances, the instructional material may be part of a kit useful for effecting alleviating or treating the various diseases or disorders recited herein. Optionally, or alternately, the instructional material may describe one or more methods of alleviating the diseases or disorders in a cell or a tissue of a mammal. The instructional material of the kit may, for example, be affixed to a container that contains the compounds of the present invention or be shipped together with a container that contains the compounds. Alternatively, the instructional material may be shipped separately from the container with the intention that the recipient uses the instructional material and the compound cooperatively. For example, the instructional material is for use of a kit;

instructions for use of the compound; or instructions for use of a formulation of the compound.

As used herein, the term "L-685,458" refers to (55')-(tert- Butoxycarbonylamino)-6-phenyl-(4R)-hydroxy-(2R)-benzylhexano yl)-L-leucy-L- phenylalaninamide, or a salt or solvate thereof.

As used herein, the term "patient," "individual" or "subject" refers to a human or a non-human mammal. Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline and murine mammals. In certain embodiments, the patient, individual or subject is human. As used herein, the term "pharmaceutically acceptable" refers to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compound, and is relatively non-toxic, i.e., the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

As used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickening agent, solvent or encapsulating material, involved in carrying or transporting a compound useful within the invention within or to the patient such that it may perform its intended function. Typically, such constructs are carried or transported from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation, including the compound useful within the invention, and not injurious to the patient. Some examples of materials that may serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil;

glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; surface active agents; alginic acid;

pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations. As used herein, "pharmaceutically acceptable carrier" also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like that are compatible with the activity of the compound useful within the invention, and are physiologically acceptable to the patient. Supplementary active compounds may also be incorporated into the compositions. The "pharmaceutically acceptable carrier" may further include a pharmaceutically acceptable salt of the compound useful within the invention.

Other additional ingredients that may be included in the pharmaceutical compositions used in the practice of the present invention are known in the art and described, for example in Remington's Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, PA), which is incorporated herein by reference. As used herein, the language "pharmaceutically acceptable salt" refers to a salt of the administered compound prepared from pharmaceutically acceptable non-toxic acids and bases, including inorganic acids, inorganic bases, organic acids, inorganic bases, solvates, hydrates, and clathrates thereof. Suitable pharmaceutically acceptable acid addition salts may be prepared from an inorganic acid or from an organic acid. Examples of inorganic acids include sulfate, hydrogen sulfate, hydrochloric, hydrobromic, hydriodic, nitric, carbonic, sulfuric, and phosphoric acids (including hydrogen phosphate and dihydrogen phosphate). Appropriate organic acids may be selected from aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids, examples of which include formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, 4-hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic), methanesulfonic,

ethanesulfonic, benzenesulfonic, pantothenic, trifluoromethanesulfonic, 2- hydroxyethanesulfonic, p-toluenesulfonic, sulfanilic, cyclohexylaminosulfonic, stearic, alginic, β-hydroxybutyric, salicylic, galactaric and galacturonic acid. Suitable

pharmaceutically acceptable base addition salts of compounds of the present invention include, for example, metallic salts including alkali metal, alkaline earth metal and transition metal salts such as, for example, calcium, magnesium, potassium, sodium and zinc salts. Pharmaceutically acceptable base addition salts also include organic salts made from basic amines such as, for example, Ν,Ν'-dibenzylethylene-diamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and procaine. All of these salts may be prepared from the corresponding compound by reacting, for example, the appropriate acid or base with the compound.

As used herein, the term "polypeptide" refers to a polymer composed of amino acid residues, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof linked via peptide bonds. Synthetic polypeptides may be synthesized, for example, using an automated polypeptide synthesizer. As used herein, the term "protein" typically refers to large polypeptides. As used herein, the term "peptide" typically refers to short polypeptides. Conventional notation is used herein to represent polypeptide sequences: the left-hand end of a polypeptide sequence is the amino-terminus, and the right-hand end of a polypeptide sequence is the carboxyl-terminus.

As used herein, amino acids are represented by the full name thereof, by the three letter code corresponding thereto, or by the one-letter code corresponding thereto, as indicated below: Aspartic Acid, Asp, D; Glutamic Acid, Glu, E; Lysine, Lys, K; Arginine, Arg, R; Histidine, His, H; Tyrosine, Tyr, Y; Cysteine, Cys, C; Asparagine, Asn, N;

Glutamine, Gin, Q; Serine, Ser, S; Threonine, Thr, T; Glycine, Gly, G; Alanine, Ala, A; Valine, Val, V; Leucine, Leu, L; Isoleucine, He, I; Methionine, Met, M; Proline, Pro, P; Phenylalanine, Phe, F; and Tryptophan, Trp, W.

As used herein, the term "prevent" or "prevention" means no disorder or disease development if none had occurred, or no further disorder or disease development if there had already been development of the disorder or disease. Also considered is the ability of one to prevent some or all of the symptoms associated with the disorder or disease.

As used herein, the term "SAG" refers to the Hedgehog agonist 3-chloro-N- ((lR,4R)-4-(methylamino)cyclohexyl)-N-(3-(pyridin-4-yl)benzy l) benzo[b]thiophene-2- carboxamide, or a salt or solvate thereof.

As used herein, the term "sAPPs" refers to soluble N-terminal APP ectodomains.

As used herein, the term "Smo" refers to the signal transducer Smoothened in the Hedgehog pathway.

As used herein, the term "TGN" refers to trans-Golgi network. As used herein, the term "treatment" or "treating" is defined as the application or administration of a therapeutic agent, i.e. , a compound useful within the invention (alone or in combination with another pharmaceutical agent), to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient (e.g. , for diagnosis or ex vivo applications), who has a disease or disorder, a symptom of a disease or disorder or the potential to develop a disease or disorder, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease or disorder, the symptoms of the disease or disorder, or the potential to develop the disease or disorder. Such treatments may be specifically tailored or modified, based on knowledge obtained from the field of pharmacogenomics.

Throughout this disclosure, various aspects of the present invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the present invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.1, 5.3, 5.5, and 6. This applies regardless of the breadth of the range.

Compounds

The compounds useful within the invention may be synthesized using techniques well-known in the art of organic synthesis. In certain embodiments, the compounds useful within the invention comprises an inhibitor of the Hedgehog signaling pathway. In other embodiments, the compounds useful within the invention comprise a Smo antagonist.

In certain embodiments, non-limiting examples of compounds useful within the invention include, but are not limited to:

cyclopamine;

MRT 10 (also known as N-[[[3-benzoylamino)phenyl]amino]thioxomethyl]-3,4,5-trimeth oxy benzamide);

jervine (also known as (3p,23P)-17,23-epoxy-3-hydroxyveratraman-l l-one);

SANT-1 (also known as N-[(3,5-dimethyl-l-phenyl-lH-pyrazol-4-yl)methylene]-4-(phen yl methyl) - 1 -piperazinamine);

SANT-2 (also known as N-[3-(lH-benzimidazol-2-yl)-4-chlorophenyl]-3,4,5-triethoxy

benzamide);

GDC-0449 (also known as vismodegib, or 2-chloro-N-(4-chloro-3-pyridin-2-ylphenyl)-4- methylsulfonylbenzamide; Genentech, Roche);

LDE-225 (also known as sonidegib, erismodegib, or N-[6-[(2R,6S)-2,6-dimethyl-4- morpholinyl] -3-pyridinyl] -2-methyl-4' -(trifluoromethoxy)-[ 1 , 1 ' -biphenyl] -3- carboxamide; Novartis);

BMS-833923 (also known as XL139, or N-(2-methyl-5-((methylamino)methyl)phenyl)-4-

((4-phenylquinazolin-2-yl)amino)benzamide; Bristol-Myers Squibb / Exelixis);

IPI-926 (also known as saridegib, or N-

((2S,3R,3aS,3'R,4a'R,6S,6a'R,6b'S,7aR,12a'S,12b'S)-3,6,l l',12b'-tetramethyl-

2',3a,3',4,4',4a',5,5',6,6',6a',6b',7,7a,7',8',10',12',12 a',12b'-icosahydro-l'H,3H- spiro[furo[3,2-b]pyridine-2,9'-naphtho[2,l-a]azulen]-3'-yl) methanesulfonamide;

Infinity);

PF-04449913 (also known as l-((2R,4R)-2-(lH-benzo[d]imidazol-2-yl)-l-methylpiperidin-4- yl)-3-(4-cyanophenyl)urea; Pfizer);

LEQ-506 (also known as (R)-2-(5-(4-(6-benzyl-4,5-dimethylpyridazin-3-yl)-2- methylpiperazin-l-yl)pyrazin-2-yl)propan-2-ol; Novartis)

TAK-441 (also known as 6-ethyl-N-[l-(hydroxyacetyl)piperidin-4-yl]-l-methyl-4-oxo-5 -(2- oxo-2-phenylethyl)-3-(2,2,2-trifluoroethoxy)-4,5-dihydro-lH- pyrrolo[3,2-c]pyridine-

2-carboxamide; Millennium);

itraconazole (also known as (2R,4S)-rel-l-(butan-2-yl)-4-{4-[4-(4-{ [(2R,4S)-2-(2,4- dichlorophenyl)-2-( 1H- 1 ,2,4-triazol- 1-ylmethyl)- 1 ,3-dioxolan-4-yl]methoxy Jphenyl) piperazin-l-yl]phenyl}-4,5-dihydro-lH-l,2,4-triazol-5-one);

LY2940680 (also known as 4-fluoro-N-methyl-N-(l-(4-(l-methyl-lH-pyrazol-5- yl)phthalazin-l-yl)piperidin-4-yl)-2-(trifluoromethyl)benzam ide);

a salt thereof, and any combinations thereof.

Methods

The invention includes a method of preventing or reducing production of Αβ in a mammal in need thereof. The invention further includes a method of decreasing the rate of γ-secretase-mediated APP cleavage in a mammal in need thereof. The invention further includes a method of treating or preventing Alzheimer's Disease (AD) in a mammal in need thereof. In certain embodiments, the methods comprise administering to the mammal a therapeutically effective amount of at least one inhibitor of the Hedgehog signaling pathway.

In certain embodiments, the inhibitor comprises a Smo antagonist. In other embodiments, the antagonist is at least one selected from the group consisting of

cyclopamine; MRT 10; jervine; SANT-1; SANT-2; vismodegib; erismodegib; BMS-833923; saridegib; PF-04449913; LEQ-506; TAK-441; itraconazole; LY2940680; a salt thereof, and any combinations thereof. In other embodiments, the inhibitor is administered to the mammal as part of a pharmaceutically acceptable composition.

In certain embodiments, the mammal does not present symptoms of AD. In other embodiments, the mammal presents at least one symptom of AD.

In certain embodiments, the inhibitor is formulated as part of an extended- release formulation. In other embodiments, the inhibitor is administered to the mammal by at least one route selected from the group consisting of inhalational, oral, rectal, vaginal, parenteral, topical, transdermal, pulmonary, intranasal, buccal, sublingual, ophthalmic, intrathecal, intravenous and intragastrical.

In certain embodiments, administration of the inhibitor decreases the rate of Αβ production in the mammal. In other embodiments, administration of the inhibitor decreases the rate of γ-secretase-mediated APP cleavage in the mammal. In yet other embodiments, administration of the inhibitor does not substantially affect the protein level and the enzymatic activity of γ-secretase in the mammal. In yet other embodiments, administration of the inhibitor treats, stabilizes or reverses a motor neuron deficit associated with AD in the mammal.

In certain embodiments, the mammal is a primate. In other embodiments, the primate is human.

Combination Therapies

In certain embodiments, the compounds of the present invention are useful in the methods of present invention in combination with one or more additional compounds useful for treating the diseases or disorders contemplated within the invention. These additional compounds may comprise compounds of the present invention or compounds, e.g., commercially available compounds, known to treat, prevent, or reduce the symptoms of the diseases or disorders contemplated within the invention.

Non-limiting examples of additional compounds contemplated within the invention include: acetylcholinesterase inhibitors (such as tacrine, rivastigmine, galantamine and donepezil), NMDA receptor antagonists (such as memantine),antipsychotic drugs (which reduce aggression and psychosis in Alzheimer's Disease patients with behavioural problems), and huperzine A.

A synergistic effect may be calculated, for example, using suitable methods such as, for example, the Sigmoid-E _max equation (Holford & Scheiner, 19981, Clin.

Pharmacokinet. 6: 429-453), the equation of Loewe additivity (Loewe & Muischnek, 1926, Arch. Exp. Pathol Pharmacol. 114: 313-326) and the median-effect equation (Chou &

Talalay, 1984, Adv. Enzyme Regul. 22: 27-55). Each equation referred to above may be applied to experimental data to generate a corresponding graph to aid in assessing the effects of the drug combination. The corresponding graphs associated with the equations referred to above are the concentration-effect curve, isobologram curve and combination index curve, respectively. Administration/Dosage/Formulations

The regimen of administration may affect what constitutes an effective amount. The therapeutic formulations may be administered to the patient either prior to or after the onset of a disease or disorder. Further, several divided dosages, as well as staggered dosages may be administered daily or sequentially, or the dose may be continuously infused, or may be a bolus injection. Further, the dosages of the therapeutic formulations may be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.

Administration of the compositions useful within the present invention to a patient, preferably a mammal, more preferably a human, may be carried out using known procedures, at dosages and for periods of time effective to treat a disease or disorder in the patient. An effective amount of the therapeutic compound necessary to achieve a therapeutic effect may vary according to factors such as the state of the disease or disorder in the patient; the age, sex, and weight of the patient; and the ability of the therapeutic compound to treat a disease or disorder in the patient. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. A non-limiting example of an effective dose range for a therapeutic compound of the present invention is from about 1 and 5,000 mg/kg of body weight/per day. One of ordinary skill in the art is able to study the relevant factors and make the determination regarding the effective amount of the therapeutic compound without undue experimentation.

Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

In particular, the selected dosage level depends upon a variety of factors including the activity of the particular compound employed, the time of administration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds or materials used in combination with the compound, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well, known in the medical arts.

A medical doctor, e.g. , physician or veterinarian, having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the present invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

In particular embodiments, it is advantageous to formulate the compound in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the patients to be treated; each unit containing a predetermined quantity of therapeutic compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical vehicle. The dosage unit forms of the present invention are dictated by and directly dependent on the unique characteristics of the therapeutic compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding/formulating such a therapeutic compound for the treatment of a disease or disorder in a patient.

In certain embodiments, the compositions useful within the invention are formulated using one or more pharmaceutically acceptable excipients or carriers. In certain embodiments, the pharmaceutical compositions of the present invention comprise a therapeutically effective amount of a compound useful within the invention and a

pharmaceutically acceptable carrier.

The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents, for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol, in the composition. Prolonged absorption of the injectable compositions may be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin.

In certain embodiments, the compositions useful within the invention are administered to the patient in dosages that range from one to five times per day or more. In other embodiments, the compositions useful within the invention are administered to the patient in range of dosages that include, but are not limited to, once every day, every two, days, every three days to once a week, and once every two weeks. It is readily apparent to one skilled in the art that the frequency of administration of the various combination compositions useful within the invention varies from individual to individual depending on many factors including, but not limited to, age, disease or disorder to be treated, gender, overall health, and other factors. Thus, the invention should not be construed to be limited to any particular dosage regime and the precise dosage and composition to be administered to any patient is determined by the attending physical taking all other factors about the patient into account.

Compounds for administration may be in the range of from about 1 μg to about 10,000 mg, about 20 μg to about 9,500 mg, about 40 μg to about 9,000 mg, about 75 μg to about 8,500 mg, about 150 μg to about 7,500 mg, about 200 μg to about 7,000 mg, about 3050 μg to about 6,000 mg, about 500 μg to about 5,000 mg, about 750 μg to about 4,000 mg, about 1 mg to about 3,000 mg, about 10 mg to about 2,500 mg, about 20 mg to about 2,000 mg, about 25 mg to about 1,500 mg, about 50 mg to about 1,000 mg, about 75 mg to about 900 mg, about 100 mg to about 800 mg, about 250 mg to about 750 mg, about 300 mg to about 600 mg, about 400 mg to about 500 mg, and any and all whole or partial increments therebetween.

In some embodiments, the dose of a compound is from about 1 mg and about 2,500 mg. In some embodiments, a dose of a compound of the present invention used in compositions described herein is less than about 10,000 mg, or less than about 8,000 mg, or less than about 6,000 mg, or less than about 5,000 mg, or less than about 3,000 mg, or less than about 2,000 mg, or less than about 1,000 mg, or less than about 500 mg, or less than about 200 mg, or less than about 50 mg. Similarly, in some embodiments, a dose of a second compound (i.e. , a drug used for treating a disease or disorder) as described herein is less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 400 mg, or less than about 300 mg, or less than about 200 mg, or less than about 100 mg, or less than about 50 mg, or less than about 40 mg, or less than about 30 mg, or less than about 25 mg, or less than about 20 mg, or less than about 15 mg, or less than about 10 mg, or less than about 5 mg, or less than about 2 mg, or less than about 1 mg, or less than about 0.5 mg, and any and all whole or partial increments thereof.

In certain embodiments, the present invention is directed to a packaged pharmaceutical composition comprising a container holding a therapeutically effective amount of a compound of the present invention, alone or in combination with a second pharmaceutical agent; and instructions for using the compound to treat, prevent, or reduce one or more symptoms of a disease or disorder in a patient.

Formulations may be employed in admixtures with conventional excipients, i.e. , pharmaceutically acceptable organic or inorganic carrier substances suitable for oral, parenteral, nasal, intravenous, subcutaneous, enteral, or any other suitable mode of administration, known to the art. The pharmaceutical preparations may be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure buffers, coloring, flavoring and/or aromatic substances and the like. They may also be combined where desired with other active agents, e.g., other cognition improving agents.

The term "container" includes any receptacle for holding the pharmaceutical composition. For example, In certain embodiments, the container is the packaging that contains the pharmaceutical composition. In other embodiments, the container is not the packaging that contains the pharmaceutical composition, i.e., the container is a receptacle, such as a box or vial that contains the packaged pharmaceutical composition or unpackaged pharmaceutical composition and the instructions for use of the pharmaceutical composition. Moreover, packaging techniques are well known in the art. It should be understood that the instructions for use of the pharmaceutical composition may be contained on the packaging containing the pharmaceutical composition, and as such the instructions form an increased functional relationship to the packaged product. However, it should be understood that the instructions may contain information pertaining to the compound' s ability to perform its intended function, e.g., treating, preventing, or reducing a disease or disorder in a patient.

Routes of administration of any of the compositions of the present invention include oral, nasal, rectal, intravaginal, parenteral, buccal, sublingual or topical. The compounds for use in the invention may be formulated for administration by any suitable route, such as for oral or parenteral, for example, transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- and perivaginally), (intra)nasal and (trans)rectal), intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and topical administration.

Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, troches, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosolized formulations for inhalation, compositions and formulations for intravesical administration and the like. It should be understood that the formulations and compositions that would be useful in the present invention are not limited to the particular formulations and compositions that are described herein.

Oral Administration

For oral application, particularly suitable are tablets, dragees, liquids, drops, suppositories, or capsules, caplets and gelcaps. The compositions intended for oral use may be prepared according to any method known in the art and such compositions may contain one or more agents selected from the group consisting of inert, non-toxic pharmaceutically excipients which are suitable for the manufacture of tablets. Such excipients include, for example an inert diluent such as lactose; granulating and disintegrating agents such as cornstarch; binding agents such as starch; and lubricating agents such as magnesium stearate. The tablets may be uncoated or they may be coated by known techniques for elegance or to delay the release of the active ingredients. Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert diluent.

For oral administration, the compounds may be in the form of tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., polyvinylpyrrolidone, hydroxypropylcellulose or

hydroxypropylmethylcellulose); fillers (e.g. , cornstarch, lactose, microcrystalline cellulose or calcium phosphate); lubricants (e.g. , magnesium stearate, talc, or silica); disintegrates (e.g., sodium starch glycollate); or wetting agents (e.g., sodium lauryl sulphate). If desired, the tablets may be coated using suitable methods and coating materials such as OPADRY™ film coating systems available from Colorcon, West Point, Pa. (e.g., OPADRY™ OY Type, OYC Type, Organic Enteric OY-P Type, Aqueous Enteric OY-A Type, OY-PM Type and

OPADRY™ White, 32K 18400). Liquid preparation for oral administration may be in the form of solutions, syrups or suspensions. The liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g. , sorbitol syrup, methyl cellulose or hydrogenated edible fats); emulsifying agent (e.g. , lecithin or acacia); non-aqueous vehicles (e.g. , almond oil, oily esters or ethyl alcohol); and preservatives (e.g. , methyl or propyl p-hydroxy benzoates or sorbic acid).

Granulating techniques are well known in the pharmaceutical art for modifying starting powders or other particulate materials of an active ingredient. The powders are typically mixed with a binder material into larger permanent free-flowing agglomerates or granules referred to as a "granulation." For example, solvent-using "wet" granulation processes are generally characterized in that the powders are combined with a binder material and moistened with water or an organic solvent under conditions resulting in the formation of a wet granulated mass from which the solvent must then be evaporated.

Melt granulation generally consists in the use of materials that are solid or semi-solid at room temperature (i.e. having a relatively low softening or melting point range) to promote granulation of powdered or other materials, essentially in the absence of added water or other liquid solvents. The low melting solids, when heated to a temperature in the melting point range, liquefy to act as a binder or granulating medium. The liquefied solid spreads itself over the surface of powdered materials with which it is contacted, and on cooling, forms a solid granulated mass in which the initial materials are bound together. The resulting melt granulation may then be provided to a tablet press or be encapsulated for preparing the oral dosage form. Melt granulation improves the dissolution rate and bioavailability of a drug by forming a solid dispersion or solid solution.

U.S. Patent No. 5,169,645 discloses directly compressible wax-containing granules having improved flow properties. The granules are obtained when waxes are admixed in the melt with certain flow improving additives, followed by cooling and granulation of the admixture. In certain embodiments, only the wax itself melts in the melt combination of the wax(es) and additives(s), and in other cases both the wax(es) and the additives(s) melt.

The present invention also includes a multi-layer tablet comprising a layer providing for the delayed release of one or more compounds of the present invention, and a further layer providing for the immediate release of a medication for treatment of a disease or disorder. Using a wax/pH-sensitive polymer mix, a gastric insoluble composition may be obtained in which the active ingredient is entrapped, ensuring its delayed release.

Parenteral Administration

For parenteral administration, the compounds may be formulated for injection or infusion, for example, intravenous, intramuscular or subcutaneous injection or infusion, or for administration in a bolus dose and/or continuous infusion. Suspensions, solutions or emulsions in an oily or aqueous vehicle, optionally containing other formulatory agents such as suspending, stabilizing and/or dispersing agents may be used.

Additional Administration Forms

Additional dosage forms of this invention include dosage forms as described in U.S. Patents Nos. 6,340,475, 6,488,962, 6,451,808, 5,972,389, 5,582,837, and 5,007,790. Additional dosage forms of this invention also include dosage forms as described in U.S. Patent Applications Nos. 20030147952, 20030104062, 20030104053, 20030044466, 20030039688, and 20020051820. Additional dosage forms of this invention also include dosage forms as described in PCT Applications Nos. WO 03/35041, WO 03/35040, WO

03/35029, WO 03/35177, WO 03/35039, WO 02/96404, WO 02/32416, WO 01/97783, WO 01/56544, WO 01/32217, WO 98/55107, WO 98/11879, WO 97/47285, WO 93/18755, and WO 90/11757. Controlled Release Formulations and Drug Delivery Systems

In certain embodiments, the formulations of the present invention may be, but are not limited to, short-term, rapid-offset, as well as controlled, for example, sustained release, delayed release and pulsatile release formulations.

The term sustained release is used in its conventional sense to refer to a drug formulation that provides for gradual release of a drug over an extended period of time, and that may, although not necessarily, result in substantially constant blood levels of a drug over an extended time period. The period of time may be as long as a month or more and should be a release which is longer that the same amount of agent administered in bolus form.

For sustained release, the compounds may be formulated with a suitable polymer or hydrophobic material which provides sustained release properties to the compounds. As such, the compounds for use the method of the present invention may be administered in the form of microparticles, for example, by injection or in the form of wafers or discs by implantation.

In certain embodiments, the compounds of the present invention are administered to a patient, alone or in combination with another pharmaceutical agent, using a sustained release formulation.

The term delayed release is used herein in its conventional sense to refer to a drug formulation that provides for an initial release of the drug after some delay following drug administration and that mat, although not necessarily, includes a delay of from about 10 minutes up to about 12 hours.

The term pulsatile release is used herein in its conventional sense to refer to a drug formulation that provides release of the drug in such a way as to produce pulsed plasma profiles of the drug after drug administration.

The term immediate release is used in its conventional sense to refer to a drug formulation that provides for release of the drug immediately after drug administration.

As used herein, short-term refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes and any or all whole or partial increments thereof after drug administration after drug

administration.

As used herein, rapid-offset refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes, and any and all whole or partial increments thereof after drug administration.

Dosing

The therapeutically effective amount or dose of a compound depends on the age, sex and weight of the patient, the current medical condition of the patient and the progression of the disease or disorder in the patient being treated. The skilled artisan is able to determine appropriate dosages depending on these and other factors.

A suitable dose of a compound of the present invention may be in the range of from about 0.01 mg to about 5,000 mg per day, such as from about 0.1 mg to about 1,000 mg, for example, from about 1 mg to about 500 mg, such as about 5 mg to about 250 mg per day. The dose may be administered in a single dosage or in multiple dosages, for example from 1 to 4 or more times per day. When multiple dosages are used, the amount of each dosage may be the same or different. For example, a dose of 1 mg per day may be administered as two 0.5 mg doses, with about a 12-hour interval between doses.

It is understood that the amount of compound dosed per day may be administered, in non-limiting examples, every day, every other day, every 2 days, every 3 days, every 4 days, or every 5 days. For example, with every other day administration, a 5 mg per day dose may be initiated on Monday with a first subsequent 5 mg per day dose administered on Wednesday, a second subsequent 5 mg per day dose administered on Friday, and so on.

The compounds for use in the method of the present invention may be formulated in unit dosage form. The term "unit dosage form" refers to physically discrete units suitable as unitary dosage for patients undergoing treatment, with each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, optionally in association with a suitable pharmaceutical carrier. The unit dosage form may be for a single daily dose or one of multiple daily doses (e.g., about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form may be the same or different for each dose.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures, embodiments, claims, and examples described herein. Such equivalents were considered to be within the scope of this invention and covered by the claims appended hereto. For example, it should be understood, that modifications in reaction conditions, including but not limited to reaction times, reaction size/volume, and experimental reagents, such as solvents, catalysts, pressures, atmospheric conditions, e.g., nitrogen atmosphere, and reducing/oxidizing agents, with art- recognized alternatives and using no more than routine experimentation, are within the scope of the present application.

It is to be understood that wherever values and ranges are provided herein, all values and ranges encompassed by these values and ranges, are meant to be encompassed within the scope of the present invention. Moreover, all values that fall within these ranges, as well as the upper or lower limits of a range of values, are also contemplated by the present application.

The following examples further illustrate aspects of the present invention. However, they are in no way a limitation of the teachings or disclosure of the present invention as set forth herein.

EXAMPLES

The invention is now described with reference to the following Examples. These Examples are provided for the purpose of illustration only, and the invention is not limited to these Examples, but rather encompasses all variations that are evident as a result of the teachings provided herein.

Materials and Methods Antibodies, Plasmids and Reagents.

Antibodies were obtained from the following: mouse C-terminus APP clone c 1/6.1 (from P. Mathews, Nathan Kline Institute, NY, USA), mouse APP N-terminus 4A 22C11 (Millipore), rabbit APP C-terminus A8717, mouse anti-Myc clone 9E10, rabbit LC3IIB and mouse β-actin (Sigma), mouse cleaved Notchl clone D3B8 and rabbit PSEN1 CTF clone D39D1 (Cell Signaling), mouse LAMP1, TGN38 and mouse EEA1 (BD

Biosciences), rat LAMP1 clone 1D4B and mouse MP6R clone 22d4 (DSHB), rabbit TGN46 (AbD Serotech) and rabbit cathepsin D (Dr. Stefan Honing, University of Cologne, Institute of Biochemistry, Germany).

Fluorescent secondary antibodies (Alexa Fluor 488, 594) were obtained from Jackson Immunoresearch Laboratories and IR-conjugated secondary antibodies (IRDye680, IRDye800) were obtained from Li-Cor Biosciences. Peroxidase-conjugated secondary antibodies were obtained from Cell Signaling.

Cyclopamine (0.5-10 μΜ) was purchased from LC Laboratories; L-685,458 (2 μΜ), cycloheximide (50 μg/ml) and DMSO were purchased from Sigma. pCS2-Myc-AENotch was used for overexpression studies. pMst-APP-Gal4 was originally developed by Cao & Sudhof, 2001, Science 293: 115-120. Human Chmp2a- GFP construct was from Dr. Elias Spiliotis (Drexel University, Department of Biology). Cell Culture and Transfection.

HeLa cells were maintained at 37°C, 5% C0 ₂ in complete DMEM (Corning) supplemented with 10% FBS (Altanta Biologicals), 100 units/ml penicillin and 100 mg/ml streptomycin (Corning), 2 mM L-glutamine (Corning). Cells were grown to 80% confluence and serum starved (0.5%FBS DMEM) for 24 hours prior to pharmacological or genetic manipulation. For pharmacological manipulation, drugs were diluted in 0.5%FBS DMEM. For genetic overexpression experiments, cells were grown to 80% confluence then transfected using with TurboFect Transfection Reagent (Thermo Scientific) according to manufacturer's protocol. Culture media was removed 24 hours post transfection and cells were collected or further treated with pharmacological agents (0.5% FBS DMEM) for an additional 24 hours.

Primary Neuron Culture.

Primary cortical neuron cultures were isolated from postnatal day 1 (PI)

Sprague-Dawley rat pups. Briefly, cortices were dissected out, minced, treated with papain (100 Units; Worthington Biochemicals) for 15 minutes at 37°C. Following, tissue was treated with Type II-O trypsin inhibitor from chicken egg white (Sigma) for 15 minutes at 37°C.

Tissue was washed with fresh Neurobasal medium (Invitrogen) supplemented with B-27

(Invitrogen), 2 mM L-glutamine, 100 Units/ml penicillin, and 100 mg/ml streptomycin.

Tissue was triturated, centrifuged at 1000 rpm for 10 minutes then resuspended in the fresh, complete Neurobasal medium. 2 x 10 ⁶ Cells per 35mm well were plated onto poly-DL-lysine

(50 μg/ml; Sigma) coated tissue culture plates. Cortical neurons were treated with pharmacological agents on DIV6 (days in vitro) for 24 hours and lysates collected for further biochemical analysis.

Drosophila stocks and genetics.

Drosophila husbandry was performed as described in Chakraborty, et al. 2011, PLoS One. 6:e20799. For experiments utilizing the γ-secretase reporter GMR-APP- Gal4; UAS-Grim/Cyo model (Guo, et al., 2003, Hum.Mol.Genet. 12:2669-2678), flies were crossed on standard cornmeal agar food supplemented with cyclopamine (100 nM) or DMSO vehicle control (0.1%) and collected 24 hours post eclosion. Their compound eye was inspected. Assessment of penetrance and severity of the rough-eye phenotype was accomplished by photographing the compound eye using a Canon PowerShot S70 digital camera mounted to a Leica Mz 125 stereomicroscope.

Severity of rough-eye phenotype was scored + (mild) to +++ (severe). One

"+" refers to where less than ½ of the eye was apoptotic and therefore appears "rough". A score of "++" (moderate) defined increased penetrance, where apoptosis affected

approximately ½ of the eye. Severe "+++" rough-eye phenotype described when more than ½ of the eye appeared "rough" and eye size significantly reduced. For objective

quantification, five blinded lab personnel analyzed all experiments.

Immunoblotting.

Lysates were collected in complete RIPA buffer (50 mM Tris-HCL, pH 8.0, 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS, 1% NP-40) supplemented with Halt Protease and Phosphatase Inhibitor and EDTA (ThermoFisher). Lysates were briefly cleared at 20,000 x g at 4°C, and stored at -20°C. Protein concentrations were determined using the BCA assay kit according to manufacturer's protocol (Pierce). 40 μg of lysate were supplemented with NuPAGE LDS Sample Buffer (Invitrogen) and heated to 75°C for 10 minutes. Protein was separated on 4-12% NuPAGE BisTris gels (Invitrogen) using MES running buffer (Invitrogen) then transferred onto Immobilon PVDF membrane (Millipore). Odyssey blocking buffer (Li-Cor Biosciences) was used for blocking and resuspending primary and secondary antibodies. Membranes were scanned using Li-Cor Odyssey infrared scanning instrument. Αβ ELISA.

HeLa cells and primary rat cortical neurons were treated with pharmacological agents for 24 hours and conditioned supernatants collected and cleared at 20,000 x g for 20 minutes at 4°C. Fresh cleared supernatants were used for Αβ40 ELISA kit (Wako, Japan) according to the manufacturer's protocol. Briefly, samples were diluted 1: 1 using kit diluent, incubated overnight at 4°C, and compared to the ELISA kit positive control and negative control (diluent alone). Samples were incubated and analyzed using a luminescence plate reader.

In vitro y-secretase assay. A cell-free γ-secretase activity assay that utilizes a fluorogenic peptide substrate corresponding to the APP γ-secretase cleavage site was used (Farmery, et ah, 2003, J. Biol. Chem. 278:24277-24284; Sarajarvi, et al, 2011, Mol. Cell. Biol. 31:2326-2340). HeLa cells grown to 100% confluence in 150 mm culture dishes were collected in ice cold PBS and pelleted at 5,000 rpm for 5 minutes. The pellet was homogenized in 500 μΐ Buffer B (20 mM HEPES pH 7.5, 150 mM KC1, 2 mM EGTA, protease & phosphatase inhibitors) using a 27-gauge needle. The resulting homogenate was cleared at 45,000 rpm at 4°C for 1 hour. Supernatant was stored at -80°C while pellet was washed with 500 μΐ Buffer B and passed through 27-gauge needle on ice. The suspension was cleared again at 45,000 rpm for 1 hour at 4°C. Supernatant was discarded and pellet resuspended in 75 μΐ Buffer B + 1%

CHAPSO and passed through 27-gauge needle on ice. The resulting membrane samples were solubilized on a rotator at 4°C for 2 hours. Solubilized samples were cleared at 45,000 rpm for 1 hour at 4°C, supernatant (total cell membrane) was collected and pellet discarded. Total protein was determined using BCA assay(Pierce) and 200 μg of protein were used for in vitro γ-secretase activity assay. Briefly, membranes were resuspended in γ-secretase assay buffer (100 mM Tris-HCl pH 6.8, 4 mM EDTA, 0.5% CHAPSO), and pre-treated with vehicle control, L-685,458 or cyclopamine. Since the membrane prep enriches total γ-secretase in sample, amount of pharmacological agent was increased accordingly. Thus, 20 μΜ of drug in total vehicle volume of 1 ml per were used. 150 μΐ total volume per well of 96- well plate was used. Membranes were pre-treated for 3 hours at 37 °C, 5% C0 ₂ then fluorogenic γ- secretase substrate (Calbiochem, EMD Millipore) was added to membranes and further incubated at 37 °C, 5% C0 ₂ for the indicated time points at which time membranes were removed and fluorescence was measured using plate reader (Promega). BSA was used as negative control in place of membranes .

Subcellular Fractionation.

HeLa cells grown to 80% confluence in 100 mm culture dishes were treated with 5 μΜ cyclopamine or DMSO for 24 hours, rinsed and collected in PBS, and cleared at 1000 rpms for 7 minutes. The cell pellet was resuspended in homogenization buffer (250 mM sucrose, 150 mM NaCl, 25 mM Tris, 1 mM EDTA, protease & phosphatase inhibitor cocktail) and homogenized (ball-bearing 12 mm-clearance cell buster). Homogenates were cleared at 1000 x g for 15 minutes at 4°C and post- nuclear supernatant was loaded into discontinuous density gradient (50%, 30%, 10%) medium (OptiPrep, Sigma) in Opti-Seal centrifuge tubes (Beckman). Homogenates were spun at 30,000 rpms for 19 hours at 4°C and 300 μΐ fractions collected.

Immunofluorescence.

Cells were fixed using 4% PFA, 0.1% Triton-X-100, blocked in 2% BSA for 30 minutes and incubated with primary antibodies overnight at 4°C. Cells were rinsed with PBS and stained with secondary antibodies at room temperature for 1 hour, washed with PBS and mounted. Cells were imaged using Olympus Fluoview 1000 inverted confocal microscope. Quantification of 3D confocal image stacks was accomplished using Slidebook 5.0 or Volocity Image analysis software (PerkinElmer).

Surface Biotinylation.

HeLa cells were treated with 5 μΜ cyclopamine or DMSO for 24 hours. Cells were placed on ice to halt membrane dynamics, rinsed with ice cold PBS and incubated with Sulfo-NHS-SS-biotin (1 mg/ml in PBS; Thermo Scientific) for 40 minutes on ice with gentle rocking. Biotin was quenched with 100 mM glycine in PBS for 15 minutes. Cells were collected in PBS and pelleted at 500 x g for 5 minutes at 4°C. The pellet was lysed in 200 μΐ standard RIPA lysis buffer containing protease and phosphatase inhibitors. Lysate was sheared using a 27-gauge needle on ice and solubilized for 2 hours at 4°C on rotator. Lysate was cleared by centrifugation at 10,000 x g for 5 minutes, and 50 μΐ from each sample were used for "total" protein analysis. The rest of the supernatant was loaded into a capped spin column (Pierce; 69725) with NeutrAvidin-coated agarose resin (Thermo Scientific) at a 1: 1 ratio and incubated overnight at 4°C on rotator. Columns were centrifuged at 10,000 rpm for 1 minute, and flow-through ("unbound" control) was collected and saved. Resin was washed several times with complete RIPA. Then 50 μΐ of NuPAGE LDS Sample Buffer (Invitrogen) with 5% β-mercapto ethanol was loaded into each column and incubated for 30 minutes at room temperature on shaker. To collect the surface biotinylated protein, columns were centrifuged at maximum speed for 2 minutes. Biotinylated protein was separated on 4-12% NuPAGE BisTris gels (Invitrogen) then transferred and membrane probed for surface APP. Non-biotinylated lysates were collected as control samples. Biotinylated "surface" samples were compared to "total" lysate samples.

Statistical Analysis.

All graphs and diagrams represent mean values + standard error of all triplicates from at least three independent experiments. Either two-tailed or one-tailed Student's ί-test was used to compare two treatment groups and calculate significance from at least three independent experiments (* /?<0.05, **/?<0.01, *** /?<0.005).

For in vivo Drosophila melanogaster experiments, G-test (goodness of fit) was used to determine significance of phenotypic penetrance in experimental populations. Degree of significance and corresponding p value criteria for G-test were identical to previously mentioned Student' s i-test.

Example 1: Cyclopamine treatment results in APP C-terminal fragment accumulation.

To test whether cyclopamine modulates APP metabolism, primary rat cortical neurons were treated with cyclopamine. An appreciable change in the full-length APP

(FLAPP) holoprotein was not observed after 24 hours of 5 μΜ cyclopamine treatment (Figs 2A-2B). However, the 8-12 kDa APP products of a- and β-secretase (a- and β-CTFs;

collectively known as APP-CTFs) significantly increased when compared to vehicle control treated neurons (p=0.0190) (Figs. 2A & 2C). To determine if these effects can be observed in other models, HeLa cells were utilized because they are easily manipulated and have been previously utilized to study APP processing and trafficking.

Using naive HeLa cells, cyclopamine time and dose dependence experiments were performed. Cells were treated for 24 hours with increasing concentrations of cyclopamine from 0.5 to 10 μΜ (Fig. 2D). Compared to vehicle control (Fig. 2E, dotted line), a significant increase in APP-CTF levels was observed with as little as 0.5 μΜ cyclopamine (p=0.000615) (Fig. 2E). No change in FL-APP was observed in cells exposed to 0.5, 1, and 5 μΜ of drug (Fig. 2E). A small, yet significant, increase in FL-APP was observed upon 10 μΜ cyclopamine treatment.

To address time dependence of cyclopamine' s effects on APP-CTF

accumulation, a time course experiment was performed. Since robust increases in APP-CTFs were observed after 24 hours with as little as 0.5 μΜ of drug, in certain embodiments using 5 μΜ of cyclopamine may significantly increase APP-CTF levels within a shorter exposure time. Accumulation of APP-CTFs was evident by 3 hours of exposure (p=0.000488) and further accumulation continued for the remainder of the time course (by 24 hours p=9.50x10 ^{" 6} ) (Figs. 2F-2G). The lack of significant changes in FL-APP levels upon 5 μΜ cyclopamine exposure suggests APP gene transcription is not altered. In fact, qPCR was used to analyze APP mPvNA in naive HeLa cells upon cyclopamine treatment, and no changes in APP transcript levels were observed as compared to vehicle control.

APP proteolysis is initiated by a- or β-secretase. This cleavage liberates soluble N-terminal APP ectodomains (sAPP). Treatment of naive HeLa cells with cyclopamine did not alter sAPP levels (Figs. 2H-2I). Without wishing to be limited by any theory, this suggests that increase in APP-CTFs is not due to modulation of a- or β-secretase cleavage of APP by cyclopamine. The observed increase in APP-CTFs and the lack of change in FL-APP levels resembles the effects of γ-secretase inhibitors, but to a diminished degree (Figs. 2J-2L).

Example 2: Cyclopamine decreases γ-secretase mediated cleavage of APP in vitro and in vivo.

The fact that cyclopamine treatment increased levels of APP-CTFs, analogously to γ-secretase inhibitor treatment, suggests that in certain embodiments cyclopamine may decrease levels of cleavage products of APP-CTFs by γ-secretase, namely Αβ and the APP intracellular domain (AICD). To test this hypothesis, naive HeLa and primary rat cortical neuron cells were exposed to cyclopamine for 24 hours. Cyclopamine- treated cells secreted significantly less Αβ compared to vehicle control in primary cortical neurons and HeLa cells (p=0.00567, p=0.000914; respectively) (Fig. 3A).

The other product of γ-secretase cleavage, AICD, is difficult to detect. Thus, an APP-Gal4 construct was used in detection (Cao & Sudhof, 2001, Science 293: 115-120; Zhang, et ah , 2007, Mol.Neurodegener. 2: 15). HeLa cells transiently overexpressing APP- Gal4 were exposed to cyclopamine for 24 hours, and AICD-Gal4 levels were detected using Western blot analysis. The γ-secretase inhibitor L-685,458 served as a positive control, because it prevents AICD generation. In comparison to vehicle control, cyclopamine significantly decreased AICD-Gal4 levels (/?=0.000228) (Figs. 3B-3C). However, these effects were much more modest than those observed upon γ-secretase inhibition with L- 685,458.

To determine if the observed effects were specific to APP, γ-secretase cleavage of Notch was monitored in response to cyclopamine treatment. Similar to AICD, endogenous NICD is also difficult to detect. To overcome this difficulty, Myc-AENotch was transiently overexpressed in HeLa cells, which were then treated with cyclopamine for 24 hours. Cyclopamine significantly decreased NICD levels (p=8.87xl0 ^~5 ) to a similar extent as observed in Αβ and AICD levels (Figs. 3D-3E). These effects on AICD and NICD were much more modest than those observed upon γ-secretase inhibition. Similar to endogenous APP-CTFs, cyclopamine increased APP-CTF-Gal4 levels (Fig. 3F). No change in AENotch levels was observed suggesting that the effects could be specific (Fig. 3F). Given these results and the availability of an in vivo γ-secretase reporter, the ability of cyclopamine to modulate γ-secretase cleavage of APP in vivo was tested. The Drosophila melanogaster γ-secretase reporter has been described (Guo, et ah, 2003, Hum. Mol. Genet. 12:2669-2678). These transgenic flies express the APP γ-secretase substrate, APP-C99-Gal4, specifically in the fly eye ommatidia. These flies also carry a UAS element upstream of GRIM, a cell death activator. Upon γ-secretase cleavage of APP-C99-Gal4, the resulting AICD-Gal4 can bind to the UAS element and induce GRIM expression. GRIM expression leads to death of ommatidia and results in a rough-eye phenotype.

To test whether cyclopamine decreases γ-secretase mediated cleavage of APP- C99-Gal4, APP-C99-Gal4; UAS-GRIM flies were raised on normal, vehicle or cyclopamine supplemented food. Flies were collected one day post eclosion and their eyes were scored for rough-eye phenotype. Flies raised on cyclopamine displayed decreased severity of the rough- eye phenotype (p=2.40xl0 ^~34 ) (Figs. 3G-3K). 10% of the flies raised on cyclopamine displayed "severe" rougheye phenotype compared to the 47% raised on vehicle food. While only 10% of the vehicle treated flies displayed "mild" rough-eye phenotype, 53% of cyclopamine treated flies displayed this phenotype (Fig. 3K). These data demonstrate that cyclopamine treatment decreases γ-secretase mediated cleavage of APP-CTFs in vitro and in vivo. Example 3: Cyclopamine does not alter γ-secretase activity.

Since in vivo and in vitro cyclopamine treatment leads to decreased γ-secretase cleavage of APP-CTFs, the possibility that cyclopamine inhibits γ-secretase activity was investigated. One major step in γ-secretase complex maturation is the autoproteolysis of Presenilinl (PSENl) to form the active N- and C-terminal fragments. Thus, detection of the PSEN1-CTF is an indicator of an active γ-secretase complex.

Naive HeLa cells were exposed to cyclopamine for 24 hours and observed an increase in APP-CTFs levels. However, PSENl -CTF levels did not change in response to cyclopamine treatment (Figs. 4A-4B). To assess overall γ-secretase activity, an in vitro, fluorescence based activity assay was used (Sarajarvi, et ah, 2011, Mol. Cell. Biol. 31:2326- 2340). Total cellular membranes were isolated from naive HeLa cells and these membranes were treated with vehicle, L-685,458 or cyclopamine. Treatment with L-685,458 decreased cleavage of the fluorogenic γ-secretase peptide substrate resulting in decreased fluorescence intensity over time. Treatment with cyclopamine did not alter γ-secretase activity (Fig. 4C). These results suggest that cyclopamine decreases γ-secretase mediated cleavage of APP without directly affecting γ-secretase activity. Without wishing to be limited by any theory, one mechanism that could explain these results is that cyclopamine mediates a change in the subcellular localization of APP and/or γ-secretase. Example 4: Cyclopamine alters APP-CTF subcellular localization.

Proteolytic processing of APP is dependent on its subcellular localization. To investigate if cyclopamine alters APP subcellular localization, naive HeLa cells were exposed to cyclopamine for 0, 6, or 24 hours, and APP subcellular distribution was visualized using immunofluorescence. Analogous to the time course experiment in which increased APP- CTFs were observed by Western blot (Fig. 2G), here cyclopamine treatment induced significant accumulation of APP positive puncta detected with an antibody raised to the APP C-terminus (p=0.00120) (Figs. 5A-5C). Visualization of APP distribution using an antibody specific to the N-terminal portion of APP did not reveal similar cyclopamine-induced APP puncta. In fact, a lack of colocalization was observed between the N- and C-terminal APP antibodies in the cyclopamine-induced APP puncta (Figs. 5D-5E). This suggests that the cyclopamine-induced puncta are APP-CTFs, and not FL-APP nor sAPP. FL-APP is not a suitable substrate for γ-secretase cleavage. In certain embodiments, the increase in APP-CTF subcellular puncta and the lack of change in FL-APP and sAPP protein levels indicate that cyclopamine does not alter APP biosynthetic pathway. In other embodiments, cyclopamine induces alterations in APP-CTF endocytosis.

To further investigate this latter possibility, surface APP levels were measured using cell surface biotinylation and Western blot analysis. Naive HeLa cells were treated with cyclopamine for 24 hours, and significant decrease in surface FL-APP (p=0.00446) was observed (Figs. 5F-5G). Therefore, the observed accumulations of APP-CTF positive puncta coupled with decreased surface FL-APP suggests that cyclopamine alters internalization and possibly retrograde trafficking that is required for γ-secretase mediated cleavage of APP- CTFs.

Example 5: Cyclopamine alters retrograde trafficking and promotes APP-CTFs localization to lysosomes.

Upon endocytosis, FL-APP and APP-CTFs are localized to early endosomes, then sorted to either one of three possible trafficking pathways. One route is for FLAPP to be recycled back to the plasma membrane. For a- or β-secretase cleaved APP fragments, APP- CTFs, a second route is available, which allows these fragments to be retrogradely trafficked to the trans-Golgi network (TGN) for γ-secretase cleavage. Finally, APP-CTFs can be trafficked to the lysosome for degradation. To gain insight into these possibilities, the subcellular localization of APP-CTFs was investigated using immunofluorescence and subcellular fractionation.

To initially investigate where cyclopamine-induced APP-CTFs accumulate, co-immunofluorescence was used to identify the subcellular compartment(s) to which these APP-CTF puncta are localized (Figs. 6-7). Specifically, markers of early endosomes (EEAl), late endosomes (MP6R), trans-Golgi network (TGN46), autophagosomes (LC3), and lysosomes (LAMP1) were assessed for accumulation of APP-CTFs. Upon cyclopamine treatment the total intensity of EEAl, MP6R, LC3 and LAMP1 significantly increased respectively), while no change was observed in TGN46 total intensity (Figs. 8A-8E). These data suggest that cyclopamine alters subcellular trafficking.

To determine to which subcellular compartment APP-CTFs localize, colocalization of APP-CTFs was quantified with these markers. While the overall co- localization is low, upon cyclopamine treatment there was a significant increase in APP colocalization with EEAl, MP6R and LAMP1 positive puncta (/?= 1.17xl0 ^"6 , /?= 1.12xl0 ¹⁴ , and p=4.39x10 - ^" 33 ; respectively) (Figs. 8F-8H). There was a significant reduction in colocalization of APP-CTFs with TGN46 (/?=3.21xl0 ^~5 ) (Fig. 81). No change in

colocalization of APP-CTFs with LC3 was observed (Fig. 8J). In addition, APP-CTF positive puncta were detected in close association with the ESCRT multivesicular body (MVB) markers, TsglOl and Chmp2a (Figs. 9A-9B). In certain embodiments, these data indicate that cyclopamine decreases retrograde trafficking of APP-CTFs to the TGN, while increasing trafficking to lysosomes.

To independently verify that APP-CTF localization is altered upon cyclopamine treatment, subcellular fractionation of vehicle and cyclopamine treated HeLa cells (Figs. 10A-10B) was utilized. Very modest changes in the distribution of subcellular markers such as EEAl and LAMP1 were observed upon cyclopamine treatment. With respect to APP-CTF distribution, in vehicle treated cells 69% of APP-CTFs are found in fractions 5, 6 and 7, which partially overlap with the TGN marker (TGN46) (Figs. IOC &

10H). However, in cyclopamine treated cells, a shift in APP-CTF distribution was observed; APP-CTFs in fractions 5, 6, and 7 decreased to 37% while an increase was observed in fractions 9 and 10. These latter fractions are enriched for the lysosomal marker LAMP1 (Figs. IOC & 10G). In contrast to APP-CTFs, an observable change in FL-APP distribution was not observed upon cyclopamine treatment (Fig. 10D). Thus cyclopamine decreases trafficking of APP-CTFs to the TGN where γ-secretase mediated Αβ generation occurs, and increases APP-CTF transport to the lysosome. Since no change in the distribution of PSEN1- CTF and FL-APP was observed upon cyclopamine treatment, this suggests the effects are specific to APP-CTFs.

In certain embodiments, increased localization to lysosomes could lead to increased degradation of APP-CTFs. Surprisingly, cyclopamine treatment caused increased APP-CTF levels. Without wishing to be limited to any theory, cyclopamine may attenuate lysosomal degradation of APP-CTFs.

Example 6: Cyclopamine decreases APP-CTF lysosomal degradation.

To investigate if cyclopamine affects APP-CTF degradation, HeLa cells were pre-treated with cyclopamine for 24 hours, then cycloheximide was added to inhibit protein synthesis for an additional 0-4 hours. At the end of these additional 4 hours, APP-CTFs decreased by 76% in vehicle treated cells, but only by 38% in cyclopamine treated cells

(p=0.00218) (Figs. 11A-11B). Cyclopamine treatment nearly doubled APP-CTFs' half-life from 2 hours to 3.6 hours (Fig. 1 IB), and did not alter FL-APP rate of degradation

(/?=0.0645) (Fig. 11C).

To test if these APP-CTF degradation changes are due to decreased lysosomal maturation, cathepsin D levels, a reliable marker of lysosomal maturation, were monitored. A modest but significant (p=0.030) decrease in the ratio of mature (31 kDa) to immature (53 kDa) cathepsin D levels was observed (Figs. 1 lD-1 IE). Together, our results suggest that cyclopamine leads to increased preferential retrograde trafficking of APP-CTFs to lysosomes and decreased lysosomal degradation of these APP-CTFs.

Example 7: Accumulation of APP-CTFs is Smo & cyclopamine dependent

Stable Smo Knock-down HeLa cells were transiently transfected with human Myc-Smo to validate shRNA efficiency. As illustrated in Fig. 13, Smo knock-down led to accumulation of APP-CTFs. Cyclopamine treatment of Smo knock-down cells led to further accumulation of APP-CTFs. Treating naive HeLa cells with GANT61 or GDC0449 did not alter FL-APP nor APP-CTFs. These data suggest that Smo is a novel modulator of APP metabolism. Additionally, the APP metabolism effects observed were specific to

cyclopamine and not other SHh antagonists. Example 8: Cyclopamine rescues AD -like pathology in a Drosophila model of AD

The UAS/GAL4 system was used to express human APP and human BACE in the CNS of drosophila; driven by elav promoter. E/av/APP/BACE flies were raised on food supplemented with 100 nM cyclopamine or vehicle control.

As illustrated in Fig. 14, cyclopamine treated flies had significantly more

APP-CTF in the brain than vehicle control. E/av/APP/BACE flies were raised on food containing cyclopamine or vehicle, and their negative geotaxis response was measured every two days. Cyclopamine significantly rescued motor neuron deficit seen in AD Drosophila flies raised on 100 nM cyclopamine as compared to vehicle control.

The results presented herein indicate that cyclopamine treatment alters APP metabolism, and establish Smoothened as a novel regulator of APP metabolism. Without wishing to be limited by theory, cyclopamine may have a biological target besides Smo. As demonstrated herein, γ-secretase activity was not altered by cyclopamine treatment, suggesting other cell factors may be involved. Cyclopamine was found to rescue motor neuron Alzheimer's pathology in AD model of Drosophila, and identification of Smo and cyclopamine as novel regulators of APP metabolism suggests novel therapeutic avenues for treatment of AD.

Example 9:

Cyclopamine and the compound LY2940680, which binds to the Smoothened

(Smo) receptor and potentially inhibits Hedgehog (Hh) signaling, were fed to

E/flv/APP/BACE flies. As illustrated in Fig. 15, both cyclopamine and LY2940680 suppressed external phenotypes observed in AD Drosophila flies.

The effects of cyclopamine on the memory deficit observed in AD Drosophila flies was investigated. AD Drosophila flies were treated with DMSO (in the absence of cyclopamine) or with cyclopamine. As illustrated in Fig. 16A, there was no difference in memory phase between AD trained flies treated with DMSO and AD sham flies, which indicates that DMSO did not rescue the memory deficit of the AD Drosophila flies. As illustrated in Fig. 16B, there was a significant difference between in memory phase between AD trained flies treated with cyclopamine and AD sham flies, which indicates that cyclopamine rescued the memory deficit of the AD Drosophila flies.

The mechanism by which cyclopamine causes APP-CTF accumulation was investigated. As demonstrated in Fig. 17A, HeLa cells were treated with a constant concentration of cyclopamine (5 mM) and increasing concentrations of the Hedgehog agonist SAG (or 3-chloro-N-((lR,4R)-4-(methylamino)cyclohexyl)-N-(3-(pyridin -4-yl)benzyl) benzo[b]thiophene-2-carboxamide). Treatment with SAG allowed for the rescue of canonical SHh signaling, but no effect was observed on APP-CTFs accumulation (Figs. 17A-17B). This suggests that SHh does not mediate cyclopamine' s effects on APP.

Treatment of HeLa cells with the Smo antagonist GDC-0449 (Fig. 18 A) did not cause significant changes in FL-APP or APP-CTFs (Figs. 18B-18C).

Smoothened knockdown in HeLa cells was then performed. For this experiment. Smo shRNA and nonspecific control shRNA were used. HeLa cells were transfected cells with either shRNA, and then with Myc-Smo (Fig. 19A). Using western blot, an approximately 50% decrease in Smo protein levels was detected in cells treated with Smo shRNA as compared to cells treated with control shRNA (Fig. 19B). Further, upon Smo knockdown accumulation of APP-CTFs was observed, similarly to the case of cyclopamine treated cells. These results suggest Smo has as role in modulating APP metabolism.

Without wishing to be limited by any theory, GDC-0449 is structurally unrelated to cyclopamine but also targets canonical Smo activity, and the results disclosed herein may indicate a novel and previously uncharacterized function of Smoothened.

As illustrated in Figs. 20A-20D, inhibition of SHh N-terminus domain (SHhN) with the Hedgehog pathway antagonist 5E1 altered APP metabolism, with accumulation of APP-CTFs (Figs. 20A-20B). On the other hand, treatment of the HeLa cells with GANT61 (which is an inhibitor for Glil/2-induced transcription) did not alter APP proteolysis (Figs. 20C-20D). This data suggests that SHhN, Ptch, and Smo modulate APP while the

downstream Gli's do not.

As demonstrate herein, cyclopamine decreased γ-secretase mediated cleavage of APP. In order to obtain further insight into this process, several SHh loss-of-function (lof) alleles were tested using this model, γ-secretase reported flies were crossed to w- control, Smo, Ptch and Hh lof alleles (Figs. 21A-21E). With smo ^lof flies, the γ-secretase mediated C- 99 proteolysis was ameliorated, similar to the in vitro results reported herein. On the other hand, Ptch ^lof flies exacerbated the severity of the rough eye phenotype. These results are consistent with the model wherein in Ptch ^lof flies there is no inhibition of Smo, leading to exacerbated SHh signaling. In the case of Hh ^lof flies, there was a rescue of rough eye phenotype. Thus, the result with Hh ^lof flies is analogous to the in vitro 5E1 treatment (Fig. 20B). These results are consistent with the in vitro data and suggest that cyclopamine' s effect takes place through a γ-secretase mediated pathway. The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety.

While the invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the present invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.