Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0085—Brain, e.g. brain implants; Spinal cord
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7088—Compounds having three or more nucleosides or nucleotides
  • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7088—Compounds having three or more nucleosides or nucleotides
  • A61K31/713—Double-stranded nucleic acids or oligonucleotides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

• Alzheimer's disease is characterized by. two pathological hallmarks, namely extracellular accumulation of plaques, which are aggregates of amyloid beta ( ⁇ ) peptides derived from proteolytic cleavages of amyloid precursor protein (APP) , and intracellular accumulation of hyperphosphorylated tau (Hardy & Selkoe (2002) Science 297:353-356).
• APP amyloid beta
• APP amyloid precursor protein
• hyperphosphorylated tau Hardy & Selkoe (2002) Science 297:353-356
• APP can be cleaved via two competing pathways, the alpha and the beta secretase pathways, which are distinguished by different subcellular sites of proteolysis and cleavage points within APP (Thinakaran & Koo (2008) J. Biol. Chem. 283:29615-29619).
• proteases are capable of producing the alpha-cleavage, after which the gamma- secretase complex that includes presenilin 1 as a catalytic subunit, further cleaves the APP fragment to produce small, non-amyloidogenic fragments .
• the beta-secretase pathway involves sequential cleavages by beta-secretase and gamma- secretase complexes, and generates ⁇ .
• APP and secretases are all membrane bound proteins/enzymes. Studies have shown that cholesterol content in cells can affect the production of ⁇ , in part by the ability of cholesterol to modulate the enzyme activities of various secretases in cell membranes
• HMGR HMG-CoA reductase
• LXRs liver X receptors
• Acyl-CoA Cholesterol Acyltransferase (ACAT) converts free cholesterol to cholesterol ester, and is one of the key enzymes in cellular cholesterol metabolism.
• ACAT Cholesterol Acyltransferase
• ACAT1 and ACAT2 also known as S0AT1 and SOAT2 . While both ACAT1 and ACAT2 are present in the liver and intestine, the cells containing either enzyme within these tissues are distinct, suggesting that ACATl and ACAT2 have separate functions. Both enzymes are potential drug targets for treating dyslipidemia and atherosclerosis.
• soluble ⁇ (42) was reduced by 34% in brain homogenates. Spatial learning was slightly improved and correlated with decreased ⁇ levels. In non-transgenic littermates, CP- 113,818 also reduced ectodomain shedding of endogenous APP in the brain.
• a 50% decrease in ACATl expression has also been shown to reduce cholesteryl ester levels by 22%, reduce proteolytic processing of APP, and decrease ⁇ secretion by 40% (Huttunen, et al . (2007) FEBS Lett. 581 ( 8 ): 1688-92 ) in an in vitro neuronal cell line.
• ACAT inhibition could serve as a strategy to DC0412WO.2 -4- PATENT treat Alzheimer's disease (Huttunen & Kovacs (2008) Neurodegener. Dis. 5 (3-4 ) : 212-4 ) .
• the present invention features methods for decreasing the size and density of amyloid plaques, decreasing cognitive decline associated with amyloid pathology, and treating Alzheimer's Disease by administering to a subject in need of treatment an agent that selectively inhibits the expression or activity of Acyl-CoA: Cholesterol
• the agent has an IC 5 o value for ACATl which is at no more than one half the corresponding IC50 value for ACAT2. In another embodiment, the agent does not inhibit the expression of ACAT2. In an alternative embodiment, the agent is a siRNA or microRNA molecule. In a further embodiment, the agent has an IC 50 value in the range of 1 nM to 100 ⁇ . In a particular embodiment of the invention, the agent is selectively delivered to the brain of the subject. In a specific embodiment, the agent is administered via a liposome or nanoparticle .
• Figure 1 shows amyloid beta 42 ( ⁇ 1-42) levels in the hemibrains of wild-type (WT) Alzheimer's Disease (AD) mice or AD mice lacking Acatl (Acatl "/ ⁇ ) after injection of PBS, or injection with adeno-associated virus (AAV) vectors harboring a negative control miRNA or with siRNA targeting ACATl (ACATl KD) .
• Mice were injected at 10 months of age and analyzed at 12 months of age.
• FIG. 2 depicts results of analysis of human full- length amyloid precursor protein (hAPP) levels and ⁇ 1-42 in the hemibrains of WT mice, AD mice and AD/Acat ⁇ _ mice.
• Results for ELISA assay levels of ⁇ 1-42 are shown in panel 2A.
• Sample pools of mouse brain homogenates in sucrose buffer were, subjected to formic acid extraction.
• the neutralized extracts were assayed for ⁇ 1-42 by ELISA.
• Formic acid extraction followed by ELISA was performed three separate times on different sample pools; each time the ELISA assay was run in duplicate or triplicate for each sample. Error bars represent standard error of the mean (SEM) .
• WT wild-type mice
• AD Alzheimer's disease mice
• AAV adeno-associated virus
• ACAT acyl-CoA: cholesterol acyltransferase
• AD Alzheimer' s disease
• hAPP human amyloid precursor protein
• PBS phosphate-buffered saline.
• Amyloid beta-peptide (Abeta or ⁇ ) accumulation in specific brain regions is a pathological hallmark of Alzheimer's disease (AD) . It has now been found that ACATl, but not ACAT2, plays a significant role in amyloid pathology of AD in vivo. Specifically, ACATl modulates the sizes and densities of amyloid plaques and cognitive decline manifested in a mouse model for the AD in vivo. Contrary to previous reports (Huttunen, et al. (2007) FEBS Lett.
• the present invention features compositions and methods for decreasing the size and density of amyloid plaques, decreasing cognitive decline associated with amyloid pathology, and treating AD.
• a subject having, suspected of having or predisposed to have AD is administered an effective amount of an agent that selectively inhibits the activity of ACATl so that the size and density of amyloid plaques in the subject are decreased, cognitive decline associated with amyloid pathology is decreased, and/or the DC0412WO.2 -7- PATENT progression of the AD is slowed or prevented, thereby treating AD.
• a "selective inhibitor of ACATl” or “ACATl-selective inhibitor” is any molecular species that is an inhibitor of the ACATl enzyme but which fails to inhibit, or inhibits to a substantially lesser degree ACAT2.
• Methods for assessing the selectively of ACATl inhibitors are known in the art and can be based upon any conventional assay including, but not limited to the determination of the half maximal (50%) inhibitory concentration (IC) of a substance (i.e., 50% IC, or IC 50 ) , the binding affinity of the inhibitor (i.e., Ki) , and/or the half maximal effective concentration (EC 50 ) of the inhibitor for ACATl as compared to ACAT2.
• a selective ACAT inhibitor is one wherein the IC 50 value for inhibition of ACATl is lower than the IC 50 value for inhibition of ACAT2.
• ACATl and ACAT2 proteins that can be employed in such assays are well-known in the art and set forth, e.g., in GENBANK Accession Nos . NP_000010 (human ACATl) and NP_005882 (human ACAT2 ) . See also U.S. Patent No. 5, 834, 283.
• a selective inhibitor of ACATl is an agent which has an IC 50 value for ACATl that is at least twice or, more desirably, at least three, four, five, or six times lower than the corresponding IC 50 value for ACAT2.
• a selective inhibitor of ACATl has an IC 5 o value for ACATl which is at least one order of magnitude or at least two orders of magnitude lower than the IC 50 value for ACAT2.
• K-604 has an IC 50 value of 0.45 ⁇ /L for human ACATl and 102.85 ⁇ /L for human ACAT2. As such K-604 is 229-fold more selective for ACATl than ACAT2.
• diethyl pyrocarbonate has been shown to inhibit ACATl with 4-fold greater activity
• Lada, et al. ((2004) J. Lipid Res. 45:378-386) teach a compound (designated therein as Compound 1A) , and derivatives thereof (designated Compounds IB, 1C, and ID) , which inhibit ACATl more efficiently than ACAT2 with IC 50 values 66- to 187-fold lower for ACATl than for ACAT2.
• Lee, et al. ((2004) Bioorg. Med. Chem. Lett. 14:3109-3112) teach methanol extracts of Saururus chinensis root that contain saucerneol B and manassantin B for inhibiting ACAT activity.
• Saucerneol B inhibited human ACAT-1 (hACATl)and human ACAT-2 (hACAT2 ) with IC 50 values of 43.0 and 124.0 ⁇ , respectively, whereas manassantin B inhibited hACAT-1 with an IC 50 value of 82.0 ⁇ , only exhibiting 32% inhibition of hACAT2 at a very high concentration of 1 mM.
• ACATl-selective inhibitors of the present invention have an IC50 value in the range of 1 nM to 100 ⁇ . More desirably, ACATl-selective inhibitors of the invention have an IC 50 value less than or equal to 100 ⁇ . Most DC0412WO.2 -9- PATENT desirably, ACATl-selective inhibitors of the invention have an IC 50 value in the nM range (e.g., 1 to 999 nM) .
• any conventional drug screening assay can be employed for identifying or selecting additional or more selective ACATl inhibitors or derivatives or analogs of known ACATl inhibitors. See, e.g., Lada, et al. (2004) J. Lipid Res. 45:378-386.
• Such agents can be identified and obtained from libraries of compounds containing pure agents or collections of agent mixtures. Examples of pure agents include, but are not limited to, proteins, peptides, nucleic acids, oligonucleotides, carbohydrates, lipids, synthetic or semi-synthetic chemicals, and purified natural products.
• agent mixtures include, but are not limited to, extracts of prokaryotic or eukaryotic cells and tissues, as well as fermentation broths and cell or tissue culture supernates.
• agent mixtures one may not only identify those crude mixtures that possess the desired activity, but also monitor purification of the active component from the mixture for characterization and development as a therapeutic drug.
• the mixture so identified may be sequentially fractionated by methods commonly known to those skilled in the art which may include, but are not limited to, precipitation, centrifugation, filtration, ultrafiltration, selective digestion, extraction, chromatography, electrophoresis or complex formation. Each resulting subtraction may be assayed for the desired activity using the original assay until a pure, biologically active agent is obtained.
• Library screening can be performed in any format that allows rapid preparation and processing of multiple reactions such as in, for example, multi-well plates of the DC0412WO.2 -10- PATENT
• Examples of conventional animal models of AD include but are not limited to models discussed in the scientific literature and cited herein. Many of these models are models where mice have been genetically altered to either express certain genes or to ablate expression of certain genes (transgenic mice) . Such transgenic mouse models have been well-accepted for use in screening drugs for potential therapeutic activity in humans and are commonly used in drug development.
• a "selective inhibitor of ACATl” specifically excludes molecules such as antisense molecules or ribozymes.
• the ACATl selective inhibitor is a molecule which selectively inhibits the expression of ACATl, without modulating the expression of ACAT2. While some RNAi molecules have been shown to induce significant neurotoxicity in brain tissue (McBride, et al . (2008) Proc. DC0412WO.2 -11- PATENT
• miRNA or microRNA refer to 19-25 nucleotide non-coding RNAs derived from endogenous genes that act as post-transcriptional regulators of gene expression. They are processed from longer (ca 70-80 nucleotide) hairpin-like precursors termed pre-miRNAs by the RNAse III enzyme Dicer. MicroRNAs assemble in ribonucleoprotein complexes termed miRNPs and recognize their target sites by antisense complementarity thereby mediating down-regulation of their target genes.
• target sequences for mouse ACATl microRNA molecules include, but are not limited to, those listed in Table 2 as SEQ ID NOs: 37-40.
• Artificial microRNAs against human ACATl gene e.g., GENBANK Accession No. NM_000019, incorporated by reference
• Exemplary microRNA sequences targeting human ACATl include, but are not limited, those listed in Table 4.
• microRNA against the ACATl gene in primates e.g., GENBANK Accession No. XM_508738, incorporated by reference
• SiRNA and/or microRNA molecules which selectively inhibit the expression of ACATl, can be administered as naked molecules or via vectors (e.g., a plasmid or viral vector such as an adenoviral, lentiviral, retroviral, adeno- associated viral vector or the like) harboring nucleic acids encoding the siRNA and/or microRNA.
• vectors e.g., a plasmid or viral vector such as an adenoviral, lentiviral, retroviral, adeno- associated viral vector or the like
• a vector used in accordance with the invention provides all the necessary control sequences to facilitate expression of the siRNA and/or microRNA.
• Such expression control sequences can DC0412WO.2 -12- PATENT include but are not limited to promoter sequences, enhancer sequences, etc.
• the siRNA and/or microRNA molecule is delivered by a non-viral delivery method, e.g., liposome, nanoparticle, or liposome- siRNA-peptide complex (Pulford et al. 2010. PloS One 5:ell085) .
• siRNA molecules of this invention may be modified by methods known in the art to increase stability, increase resistance to nuclease degradation or the like. These modifications are known in the art and include, but are not limited to modifying the backbone of the oligonucleotide, modifying the sugar moieties, or modifying the base.
• the invention features a siRNA molecule, wherein the siRNA molecule includes a sense region and an antisense region and wherein the antisense region has a nucleotide sequence that is complementary to a nucleotide sequence or a portion thereof of RNA encoded by the ACATl gene and the sense region has a nucleotide sequence that is complementary to the antisense region.
• the siRNA is composed of two nucleic acid molecules, e.g., a sense and antisense strand. In other embodiments, the siRNA is composed of one nucleic acid molecules, wherein the sense and antisense strand are connected by a linker.
• the purine nucleotides present in the antisense region include 2 ' -deoxy-purine nucleotides. In another embodiment, the purine nucleotides present in the antisense region include 2 ' -O-methyl purine nucleotides.
• the antisense region can include a phosphorothioate internucleotide linkage at the 3 1 end of the antisense region.
• the antisense region includes a glyceryl modification at the 3' DC0412WO.2 -13- PATENT end of the antisense region.
• any nucleotides present in a non-complementary region of the antisense strand are 2 ' -deoxy nucleotides. See, e.g., US 8,232,383; WO 00/44914; or WO 01/68836.
• selective inhibitors of ACATl find application in methods for decreasing the size and density of amyloid plaques, decreasing cognitive decline associated with amyloid pathology, and treating AD.
• such methods involve administering to a subject in need of treatment a selective inhibitor of ACATl in an amount that effectively reduces the activity of ACATl by at least 60% to as much as 100%, including levels of inhibition between 60% and 100%.
• Subjects benefiting from treatment with an agent of the invention include subjects confirmed as having AD, subjects suspected of having AD, or subjects predisposed to have AD (e.g., subjects with a family history of Down's syndrome or ones with a genetic predisposition to Alzheimer's disease).
• a subject can be any mammal including human, companion animals [e.g., dogs or cats), livestock ⁇ e.g., cows, sheep, pigs, or horses), or zoological animals (e.g., monkeys).
• the subject is a human.
• agents of the invention embrace in vivo applications, in vitro use of agents of the invention are also contemplated for examining the effects of ACATl inhibition on particular cells, tissues or regions of the brain. In addition to treatment, agents of the invention also find application in monitoring the phenotypic consequences (e.g., rate of plaque formation or rate of cognitive decline) of amyloid pathology in animal models of AD. DC0412WO.2 -14- PATENT
• an ACATl- selective inhibitor of the invention When used in therapeutic applications, will have the therapeutic benefit of decreasing the size and density of amyloid plaques in the subject, decreasing or slowing the cognitive decline associated with amyloid pathology in the subject, and/or treating AD in the subject as compared to subjects not receiving treatment with the ACATl-selective inhibitor.
• An ACATl-selective inhibitor of the invention is expected to decrease the size and density of amyloid plaques in a subject by any amount from 10% to 60% or more as compared to an untreated subject.
• an ACATl- selective inhibitor of the invention is expected to decrease or slow the cognitive decline associated by amyloid pathology by from any amount from 10% to 60% or more as compared to an untreated subject (e.g., as determined by commonly applied tests that would include but be limited to the Screen Information-Memory-Concentration Test, the Screen Orientation-Memory-Concentration Test, and the Short Test of Mental Status, or the Mini-Mental State Examination) .
• Cognitive assessment can include monitoring of learning and retaining new information (e.g., does the subject .
• monitoring handling of complex tasks e.g., can the subject follow a complex train of thought, perform tasks that require many steps such as balancing a checkbook or cooking a meal
• monitoring reasoning ability e.g., is the subject able to respond with a reasonable plan to problems at work or home, such as knowing what to do if the bathroom flooded
• monitoring subject's spatial ability and orientation e.g., can the subject drive, organize objects around the house, or find his or her way around familiar places
• monitoring language e.g., does the subject DC0412 O.2 -15- PATENT have difficulty finding words to express what he or she wants to say and with following conversations
• Successful clinical use of an ACATl-selective inhibitor can be determined by the skilled practitioner, such as a clinician or veterinarian, based upon routine clinical practice, e.g., by monitoring cognitive decline via methods disclose herein, monitoring or measuring levels of functional activities (e.g., the Functional Activities Questionnaire) , and monitoring or measuring levels of sensory impairment and physical disability according to methods known in the art.
• functional activities e.g., the Functional Activities Questionnaire
• ACATl-selective inhibitors can be formulated with a pharmaceutically acceptable carrier at an appropriate dose.
• Such pharmaceutical compositions can be prepared by methods and contain carriers which are well- known in the art. A generally recognized compendium of such methods and ingredients is Remington: The Science and Practice of Pharmacy, Alfonso R. Gennaro, editor, 20th ed. Lippincott Williams & Wilkins: Philadelphia, PA, 2000.
• a pharmaceutically acceptable carrier, composition or vehicle such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, is involved in carrying or transporting the agent in the subject 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 and must not be significantly injurious to the patient, although some level of toxicity can be expected as well.
• One of skill in the art would understand how to ensure that any agent DC0412WO.2 -16- PATENT used in a subject is one wherein the benefits to the subject outweigh the risks to the subject. Given the serious nature of AD, agents with some level of toxicity or risk to subject health could be tolerated and developed as a useful therapeutic agent to treat AD.
• Examples of materials which can 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; alginic acid; pyrogen-free water; isotonic saline; Ring
• wetting agents such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.
• compositions of the present invention can be administered parenterally (for example, by intravenous, intraperitoneal, subcutaneous or intramuscular injection) , topically, orally, intranasally, intravaginally, or rectally according to standard medical practices.
• parenterally for example, by intravenous, intraperitoneal, subcutaneous or intramuscular injection
• topically orally, intranasally, intravaginally, or rectally according to standard medical practices.
• the ACATl-selective inhibitor is selectively delivered to the brain.
• “selective delivery to the brain” or “selectively delivered to the brain” is intended to mean that the agent is administered directly to the brain of the subject ⁇ e.g., by a shunt or catheter; see, e.g., U.S. Patent Application No. 20080051691) , to the perispinal space of the subject without direct intrathecal injection (see, e.g., U.S. Patent No. 7,214,658), or in a form which facilitates delivery across the blood brain barrier thereby reducing potential side effects associated with ACATl inhibition in other organs or tissues.
• formulation of the agent into a nanoparticle made by polymerization of a monomer e.g., a. methylmethacrylate, polylactic acid, polylactic acid- polyglycolic acid-copolymer, or polyglutaraldehyde
• a stabilizer allows passage of the blood brain barrier without affecting other organs with the agent.
• a monomer e.g., a. methylmethacrylate, polylactic acid, polylactic acid- polyglycolic acid-copolymer, or polyglutaraldehyde
• AAV Adeno- Associated Virus
• the selected dosage level of an ACATl-selective inhibitor will depend upon a variety of factors including the activity of the particular agent of the present invention employed, the route of administration, the time of administration, the rate of excretion or metabolism of the particular agent being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular agent employed, the age, sex, weight, condition, general health and prior medical DC0412WO.2- -18- PATENT history of the patient being treated, and other factors well-known in the medical arts.
• a practitioner such as a physician or veterinarian, having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required based upon the administration of similar compounds or after routine experimental determination.
• the physician or veterinarian could start doses of an agent 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. This is considered to be within the skill of the artisan and one can review the existing literature on a specific agent or similar agents to determine optimal dosing .
• mice were fed ad libitum with standard chow diet, maintained in a pathogen-free environment in single- ventilated cages and kept on a 12 hour light/dark schedule.
• Acatl-/-Alz (Al-/Alz) and Acat2-/- /Alz (A2-/Alz) Mice.
• Acatl-/- and Acat2-/- mice (Meiner, et al. (1996) Proc. Natl. Acad. Sci. USA 93:14041-14; Buhman, et al. (2000) Nat. Med. 6:1341-1347) in C57BL/6 background are known in the art.
• the 3XTg-Alz mice (Alzheimer's disease mice) in hybrid 129/C57BL/6 background contain two mutant human transgenes, hAPP harboring Swedish mutation (hAPPswe) , and mutant htau (htau P 3oiL) under a neuron-specific promoter, and contain the knock-in mutant presenilin 1 (PSl M i 46 v) (Oddo, et al. (2003) Neuron 39:409-421). DC0412WO.2 -19- PATENT
• mice Tissue Isolation Animals were sacrificed by C0 2 asphyxiation. The brains, adrenals and livers were rapidly isolated. Mice brains were dissected into various regions on ice within 5 minutes and were either used fresh (for ACAT enzyme activity assay) or were rapidly frozen on dry ice for other usage.
• ACAT Activity Assay Immunoprecipitation (IP) and Immunoblot Analyses .
• Freshly isolated tissue samples were homogenized on ice in 50 mM Tris, 1 mM EDTA, pH 7.8 and solubilized in detergent using 2.5% CHAPS and 1 M KC1.
• the homogenates were centrxfuged at 100,000g for 45 minutes.
• the supernatants were used for ACAT activity assay in mixed micelles, and for IP and immunoblot analyses (Chang, et al. (1998) J. Biol. Chem. 273:35132-35141; Chang, et al. (2000) J. Biol. Chem. 275:28083-28092).
• RNA Isolation was isolated with TRIZOL reagent (Invitrogen) , stored at - 80°C, and used for RT-PCR experiments, using the protocol supplied by the manufacturer.
• Real-time PCR was performed using the DYNAMO HS SYBR Green qPCR kit (New England Biolabs) . Relative quantification was determined by using the delta CT method (Pfaffl, et al . (2002) Nucleic Acids Res. 30:e36) .
• Mouse ACAT1 and human APP primers were designed using Oligo 4.0 Primer Analysis Software.
• the PCR reaction conditions for amplification of ACAT1, ACAT2, GAPDH, NF120 and Human APP included an initial denaturation at 94 °C for 5 minutes. Subsequently, 40 cycles of amplification were performed which included: denaturation DC0412WO.2 -21- PATENT at 94°C for 10 seconds, annealing at 56°C for 20 seconds, and elongation at 72 °C for 29 seconds. Amplification conditions for the remaining primers listed in Table 1 were as previously described (Van Eck, et al. (2003) J. Biol. Chem. 278:23699-23705).
• Brain homogenates were prepared in sucrose buffer with protease inhibitors at 4°C according to a published protocol
• Contextual fear conditioning was performed according to a published protocol
• the auditory cue was from e2s (London, U.K.) .
• GoldWave software program was used to edit the auditory cue; Winamp software was used to play the cue sound using the speakers.
• the digital sound level meter (RadioShack) was used to adjust the cue sound level to 87 dB.
• Each mouse's behavior was recorded using a computer webcam (QuickCam from Logitech) and ANY-maze recording software. The videos were analyzed for freezing behavior, using time sampling at 5 second intervals.
• mice forebrains were homogenized and extracted using chloroform:methanol (2:1) (at 12 ml final vol. per mouse brain) , dried down under nitrogen, and re-dissolved in methanol. Ten percent of the sample was placed in a 2 ml GC/MS autosampler vial, dried down, and trimethyl-silyl derivatized overnight at room temperature with 0.5 ml TRI- SIL TBT (Pierce) . One microliter of derivatized sample (or 0.1 ⁇ for cholesterol measurements) was injected into a Shimadzu QP 2010 GC-Mass instrument.
• GC/MS analysis of sterols was performed according to known methods (Ebner, et DC0412 O.2 -23- PATENT al. (2006) Endocrinology 147:179-190) with modifications, using selected ion monitoring (cholesterol: 24 329, 353, 368, 458; desmosterol: 441, lanosterol: 393; 24S- hydroxycholesterol : 413) and standard curves for quantification .
• 3 H cholesterol at 10 Ci/mouse prepared in 3 ⁇ of 5 mM methyl beta-cylodextrin in PBS was injected into the right lateral ventricle with a glass syringe (in 2 minutes) . Mice were kept in cages for 3 hours, and then euthanized by C0 2 gas. The forebrains were removed; lipids were extracted and redissolved in methanol as described earlier. Ten percent of the redissolved sample was analyzed by TLC, using plates from Analtech, and solvent system hexanes: ethyl ether (anhydrous): acetic acid (60:40:1). The cholesterol and 3 H cholesterol ester (CE) bands were scraped off the TLC plate and counted. Percent cholesterol esterification was determined by dividing the CE count by the total 3 H- cholesterol count.
• Hippocampal neurons were isolated from Al+/Alz and Al-/Alz mice at postnatal day 5 according to standard protocols (Brewer (1997) J. Neurosci. Methods 71:143-155; Price & Brewer (2001) In Protocols for Neural Cell Culture. Fedoroff & Richardson, editors. Totowa, NJ: Humana Press, Inc. 255-264).
• Cells were seeded in 6-well DC0412WO.2 -24- PATENT dishes in triplicate at 300,000 cells/well, and grown in 3 ml/well Neurobasal A medium with lx B27, 0.5mM L-Gln and 5 ng/ml FGF for 14 days. Half of the medium was replaced with fresh media once every 7 days. Forty-eight hours after the second media replacement, 50 ⁇ of [ 3 H] sodium acetate (100 mCi/mmol) in phosphate-buffered saline (PBS) was added per well for 3 hours. Lipids in cells and in media were extracted, saponified, and analyzed by using the same TLC system described herein.
• PBS phosphate-buffered saline
• brain homogenates were prepared from wild-type, Acatl- DC0412WO.2 -25- PATENT
• mice /- (A1-) and Acat2-/- ⁇ A2- mice.
• Mouse ACATl is a 46-kDa protein (Meiner, et al. (1997) J. Lipid Res. 38:1928-1933).
• Immunoblot analysis showed that in homogenates prepared from mouse brain (but not from other mouse tissues) , a non-ACATl protein band appeared in the 46-kDa region; the presence of this non-specific band precluded the use of immunoblotting or histochemical staining to identify ACATl in the mouse brain.
• immunoprecipitation (IP) experiments were performed using detergent solubilized wild-type mouse brain extracts. The results of the IP experiment showed that ACAT activity could be efficiently immunodepleted by ACATl-specific antibodies, but not by control antibodies. Immunoblot analysis of the immunoprecipates was then performed.
• Example 3 ACATl-Deficient Alzheimer's Mice (Al-/Alz Mice)
• Example 4 Effect of Inactivating ACATl on ⁇ Deposition , hAPP Processing, and on hTau
• Results showed that the ⁇ 42 levels were significantly decreased (by -78 %) in Al-/Alz mice; the ⁇ 40 levels were also decreased, but the difference observed was not statistically significant.
• Control experiments showed that the brains of nontransgenic mice did not contain measurable ⁇ .
• hAPP level was also significantly reduced (by -62%) .
• hAPP protein levels there was no difference in hAPP mRNA levels between the Al+/Alz mice and the Al-/Alz mice.
• hAPP is synthesized in the endoplasmic reticulum in its immature form (with a molecular weight of -105-kDa) ; the immature form moves from the endoplasmic reticulum to the Golgi via a secretory pathway (Cai, et al. (2003) J. Biol. Chem.
• the Alz mice express both hAPP and endogenous (mouse) APP. It is possible that Al- may affect both the hAPP and the mAPP levels.
• a different antibody antigen 369 was used, which recognizes the C-terminal fragments of both hAPP and mAPP (Buxbaum, et al. (1990) Proc . Natl. Acad. Sci. USA 87:6003- 6006) .
• the results showed that there was no detectable difference in the total APP levels between the non-Tg, the DC0412WO.2 -29- PATENT
• Tau pathology is one of the hallmarks of AD. Accordingly, the effect of Al- on mutant human tau (htau) was analyzed in 3XTg-Alz mice. The results showed that at 4 months of age, Al- mice exhibited a significant decrease in htau (by ⁇ 57%) , but at 17 months of age, Al- mice had an increased level of hyperphosphorylated htau. No significant change was observed in the number of hippocampal neurofibrillary tangles between the Al+/Alz and the Al-/Alz mice. These results indicated that Al- does not attenuate tau pathology in Alz mice. DC0412 O.2 -30- PATENT
• Example 5 Effect of Inactivating ACATl on Cognitive
• 3 H-oleic acid Upon entering cells, 3 H-oleic acid is rapidly converted to 3 H-CE by ACA . Both the A1+ cells and the Al- cells synthesize CE; however, Al- cells synthesize 3 H-CE at a much reduced capacity compared to A1+ cells.
• the 3 H- sterols in the media of A1+ and Al- cells was also examined. The results showed that the 3 H-cholesterol content was not significantly different; in contrast, the 3 H-24SOH content DC0412WO.2 -34- PATENT in Al- cells was significantly ( ⁇ 56%) higher than that in A1+ cells. The percent of total 3 H-sterols secreted into the media was calculated and it was found that neurons secreted only about 2% of total 3 H-cholesterol, but secreted 13-15% of total 3 H-24SOH into the media.
• the current findings link cellular cholesterol trafficking with ACAT1 , CYP46A1, 24SOH synthesis, and HMGR at the endoplasmic reticulum.
• ACAT1 a resident enzyme located at the endoplasmic reticulum (Chang, et al. (2006) Annu. Rev. Cell Dev. Biol.
• agents that inhibit ACATl enzyme activity or decrease ACATl gene expression can ameliorate amyloid pathology, and have therapeutic value for treating AD in humans.
• agents that increase the concentration of 24SOH may help to combat AD by decreasing APP content in the DC0412WO.2 -36- PATENT brain.
• agent include, but are not limited to, 24SOH itself.
• the pre-microRNAs (including sense, antisense and loop regions) of microRNAs #54 and #55 were 5'-TGC TGT CCA GTA TCA GAA TGA ACC GGG TTT TGG CCA CTG ACT GAC CCG GTT CAC TGA TAC TGG A-3' (SEQ ID NO: 41) and 5'-TGC TGT ACA GTA GGA GTC CTT GGG TAG TTT TGG CCA CTG ACT GAC TAC CCA AGC TCC TAC TGT A-3' (SEQ ID NO: 42), respectively.
• NIH-3T3 mouse fibroblasts were transiently transfected with one of several rAAV vectors encoding EmGFP and microRNA (miR) #52, #53, #54 or #55. Forty-eight hours post-transfection, GFP-positive cells were harvested by FACS. GFP-positive cells were washed then lysed in 10% SDS and syringe homogenized. Twenty microgram of protein per sample was subjected to SDS-PAGE. After western blot analysis, bands were quantified with ImageJ. ACATl intensity was normalized to GAPDH as a loading control and expressed as relative intensity. The results of this analysis are presented in Table 3. DC0412WO.2 -37- PATENT
• a cohort of subjects fulfilling NINCDS-ADRDA criteria (McKhann, et al. (1984) Neurology 34:939-44) for probable or possible AD will be recruited. The median age of the sample group will be determined. Clinical diagnosis will be made independently by, e.g., a psychiatrist and neurologist based on a checklist for symptoms of the disease with strict adherence to NINCDS-ADRDA criteria. Cognitive assessment will be recorded by trained clinical research nurses using the MMSE (Mini Mental State Examination; Folstein et al.
• subjects will either receive regular doses of an ACATl-selective inhibitor or placebo.
• the rate of cognitive decline will be based on the average slope of MMSE points change per year. Differences in the average annual MMSE decline in the whole group by the presence or absence of the K variant of the ACATl-selective inhibitor will be assessed by the Mann-Whitney U test. The subjects will then be grouped into four categories depending on their baseline MMSE scores ⁇ e.g., >24; ⁇ 24 and >16; ⁇ 16 and >8; ⁇ 8 points). Differences in the average annual MMSE decline in the four categories by the presence or absence of the K variant of ACATl-selective inhibitor will be initially assessed by independent t-tests.
• CP113818 inhibits the processing of both human APP and mouse APP, whereas CI 1011 decreases the mature/immature ratio of hAPP.
• Al- only caused a decrease in the full-length human APP protein content and did not affect the mouse APP at any level or alter the mature/immature ratio of hAPP.
• Another important difference is that unlike the effect of A1-, CP113818 causes a reduction in the full-length hAPP content (Hutter-Paier, et al. (2004) supra).
• the differences in results indicate that the ACAT inhibitors used in the prior art are not selective for ACAT1, as evidenced by the differences in results seen with complete ablation of ACATl (ATI-) .
• ACAT is a member of the membrane bound O- acyltransferase (MBOAT) enzyme family (Hofmann (2000) Trends Biochem. Sci. 25:111-112), which includes sixteen enzymes with similar substrate specificity and similar catalytic mechanisms, but with diverse biological functions.
• MBOAT membrane bound O- acyltransferase
• ACAT inhibitors are hydrophobic, membrane active molecules (Homan & Hamelehle (2001) J. Pharm. Sci. 90:1859-1867). When administrated to cells, it is likely that they partition into membranes at high concentration, thereby perturbing membrane properties nonspecifically .
• CP113818 and CI 1011 are designated as ACAT inhibitors, they also may inhibit other enzymes in the MBOAT family, and/or interfere with other biological processes.
• the present data shows that inactivating the ACATl gene alone is sufficient to ameliorate amyloid pathology in the 3XTg-AD mouse model.
• Al- acts to reduce ⁇ load mainly by reducing the hAPP protein content .
• the action of Al- is similar to that of cerebrolysin, a peptide mixture with neurotrophic effects.
• APP contains three CRAC motifs, a consensus motif known to bind cholesterol (Epand (2008) Biochim. Biophys . Acta 1778:1576-1582). It is also possible that cholesterol and/or oxysterol may directly interact with the hAPP protein to accelerate its rate of degradation. Alternatively, 24SOH may act indirectly by reducing membrane cholesterol content.
• Cyp46al causes a near elimination in the 24SOH content, a decrease in cholesterol biosynthesis rate in the brain, and a decrease in cholesterol turnover in the brain; the total brain cholesterol content in the Cyp46al ' " mice remained unchanged; Cyp46al ⁇ / ⁇ did not affect the amyloid pathology in an AD mouse model (Lund, et al. (2003) J. Biol. Chem. 278:22980-22988; Kotti, et al. (2006) Proc. Natl. Acad. Sci. USA 103:3869-3874; Halford & Russell (2009) Proc. Natl. Acad. Sci.
• the present results show that in the Al-/Alz mice, a 30% increase in 24SOH in brain cholesterol content, a modest reduction in cholesterol biosynthesis rate, , and a significant reduction in amyloid pathology occurred.
• the Cyp46al gene knockout or Cyp46al overexpression in mice may have produced compensatory effects that did not occur in the Al- mice, and vice versa; thus a direct comparison of the results described above is difficult.
• the combined results suggest that 24SOH may play an auxiliary, but not an obligatory, role in affecting cholesterol metabolism and amyloid biology, and its effects may be cell- type dependent.
• ACATl a resident enzyme located at the ER (Sun, et al . (2003) J. Biol. Chem. 278:27688- 27694), removes a portion of ER cholesterol by converting it to CE. Al- leads to an increase in the ER cholesterol pool and raises the substrate level for CYP46A1, another ER resident enzyme. This leads to an increase in 24SOH
• cholesterol and/or 24SOH level in the neurons Barring the possible side effects caused by altering cholesterol
• Example 12 Effect of Recombinant Adeno-Associated Virus Expressing Acatl siRNA
• siRNA sequences (#52-#55; Table 2) targeting the mouse Acatl gene were inserted into an endogenous mouse microRNA (miR) scaffold using Invitrogen' s RNAi design tool.
• the artificial miRs were ligated into the mammalian expression vector pcDNA6.2-GW/EmGFP-miR.
• These AcatlmiR constructs were tested along with a negative control (NC) miR (5' -TACTGCGCGTGGAGACG-3 ' ; SEQ ID N0:9), which does not match the sequence of any known vertebrate gene, in NIH-3T3 mouse fibroblasts.
• NC negative control
• the miRs were delivered to the cells using a standard cDNA transfection protocol.
• This vector contained a strong and cell-type nonspecific promoter that expresses Acatl miRs in any cell type where the viral genome is expressed. For identification purpose, it also co-expresses the GFP with the miRs.
• These three constructs were used to produce three recombinant AAV viruses. To test the efficacy and specificity of these viruses, cultured primary hippocampal neurons isolated from the triple transgenic Alzheimer neurons from AD mice (AD/Acatl+/+ mice) were treated with the NC AAV, or with AAV that expressed miR containing siRNA Acatl #55.
• NC AAV or the Acatl AAV that includes both siRNA Acatl #54 and #55
• mice were also injected into the hippocampal region of the AD mice at 4 months of age. After a single bilateral injection, mice were allowed to recover. One month after injection, mice were sacrificed and the ACATl enzyme activities in the mouse brain homogenates were analyzed by using a standard ACAT enzyme activity assay in vitro. The result showed that when compared with the control values, the Acatl AAV reduced ACATl enzyme activity by 42%
• various inflammatory markers iba, GFAP, TNFalpha, and iNOS
• AD/ACATl '/' both mouse strains were at 10 months of age. After injections, mice were allowed to recover, and were sacrificed two months later (at 12 months of age) to determine ⁇ 1-42 content.
• the results showed that injecting AAV unexpectedly caused significant reduction of the ⁇ 1-42 levels in the AD mice. Additional results also showed that DC0412WO.2 -45- PATENT injecting the AAV that expresses the Acatl KD microRNA caused a clear reduction in the ⁇ 1-42 level (see Figure 1) .
• Acatl genetic ablation (Acatl- /-) caused a 60-80% reduction in the ⁇ 1-42 content; however, residual ⁇ 1-42 was still present in the brains of the AD/Acatl-/- mouse brain.
• Acatl AAV Figure 1
• NC AAV Figure 1
• siRNAs against ACATl can be employed to cause inhibition of ACATl enzyme activity and to cause significant ⁇ 1-42 reduction in the AD mouse brains in vivo, after cognitive deficit occurred in these mice.
• ⁇ 1-42 levels were significantly decreased in AD mice treated with AAV-Acatl compared with AD mice treated with AAV-NC or PBS. There was no difference in ⁇ 1-42 levels in AD mice treated with AAV-Acatl versus AD/Acatl - ⁇ mice, and also no significant difference in ⁇ 1-42 levels between the AD/ACAT "7" mice treated with PBS, AAV-NC or AAV-Acatl ( Figure 2A) . Amyloidpi-40 levels were also assayed by ELISA and levels in these mice were near the lower limit of detection in the assay and these were no significant differences detected among treatment groups. These data also provide in vivo evidence for the therapeutic efficacy of using siRNA molecules to reduce levels of amyloid proteins in an animal model of AD. These data support the use of such molecules to treat AD in humans.

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