(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date 14 October 2010 (14.10.2010) WO 2010/117727 A2

(51) International Patent Classification: TECHNOLOGY TRANSFER, NATIONAL INSTI¬ A61K 31/407 (2006.01) A61K 31/7105 (2006.01) TUTES OF HEALTH [US/US]; 601 1 Executive Boule A61K 31/40 (2006.01) A61K 31/711 (2006.01) vard, Ste 325, MSC 7660, Bethesda, Maryland A61K 38/16 (2006.01) A61P 25/00 (2006.01) 20892-7660 (US). (21) International Application Number: (72) Inventors; and PCT/US2010/029056 (75) Inventors/Applicants (for US only): ROGERS, Jack [USAJS]; 63 Sunnyside Ave., Arlington, Massachusetts (22) International Filing Date: 02474 (US). TANZI, Rudolph E. [US/US]; 3 Oceanside 29 March 2010 (29.03.2010) Drive, Hull, Massachusetts 02045 (US). MOIR, Robert (25) Filing Language: English [US/US]; 6 1 Tilden Rd, Scituate, Massachusetts 02066 (US). GREIG, Nigel [US/US]; 11 Anne Brent Garth, (26) Publication Language: English Phoenix, Maryland 2 113 1 (US). FRIEDLICH, Avi L. (30) Priority Data: [US/US]; 8 Dwyer Circle, Medford, Massachusetts 02155 61/164,729 30 March 2009 (30.03.2009) US (US). (71) Applicants (for all designated States except US): THE (74) Agents: KUGLER DEYOUNG, Janice et al; Fish & GENERAL HOSPITAL CORPORATION [US/US]; Richardson P.C , P.O. Box 1022, Minneapolis, Minnesota 55 Fruit Street, Boston, Massachusetts 021 14 (US). NA¬ 55440-1022 (US). TIONAL INSTITUTE OF AGING OFFICE OF

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(54) Title: PHENSERINE AND POSIPHEN FOR TREATING NEUROP SYCHIATRIC AND NEURODEGENERATIVE CON DITIONS

Figure IA (57) Abstract: Described are methods for treating synucle- inopathy in a subject, by administering to the subject a ther 453 Abha-Syn apeutically effective dose of one or both of POSIPHEN and phenserine. 4OD

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Figure 1C (81) Designated States (unless otherwise indicated, for every GM, KE, LR, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, kind of national protection available): AE, AG, AL, AM, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, TM), European (AT, BE, BG, CH, CY, CZ, DE, DK, EE, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, MC, MK, MT, NL, NO, PL, PT, RO, SE, SI, SK, SM, HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, ML, MR, NE, SN, TD, TG). ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, Published: NO, NZ, OM, PE, PG, PH, PL, PT, RO, RS, RU, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, — without international search report and to be republished TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. upon receipt of that report (Rule 48.2(g)) (84) Designated States (unless otherwise indicated, for every kind of regional protection available): ARIPO (BW, GH, PHENSERINE AND PQSIPHEN FOR TREATING NEURQPSYCHIATRIC AND NEURODEGENERATIVE CONDITIONS

CLAIM OF PRIORITY This application claims the benefit of U.S. Provisional Patent Application Serial No. 61/164,729, filed on March 30, 2009, the entire contents of which are incorporated by reference herein.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with Government support under Grant Nos. NS10874 and NS059434 awarded by the National Institutes of Health. The Government has certain rights in the invention.

TECHNICAL FIELD This invention relates to novel uses for phenserine [(-)-phenserine tartrate] and POSIPHEN [(+)-phenserine tartrate], particularly for the treatment of neuropsychiatric conditions associated with production of alpha-synuclein protein.

BACKGROUND Phenserine, and its positive stereoisomer POSIPHEN, are presently in clinical assessment for Alzheimer's disease (AD) as blockers of amyloid precursor protein translation (anti-amyloid) (see, e.g., ClinicalTrials.gov Identifier: NCT01072812). The drugs have been well-tolerated in Phase I trials.

SUMMARY An inter-relationship exists between the Parkinson's disease causative protein, alpha-synuclein, and the Alzheimer's Aβ-amyloid plaque protein. Phenserine reached clinical assessment for AD as an anticholinesterase and blocker of amyloid precursor protein (APP) translation. As demonstrated herein, this well-tolerated compound also reduced neural alpha-synuclein expression over a similar dose range as APP. POSIPHEN, a (+) stereoisomer of phenserine that has no anticholinesterase activity, has also shown a capacity to lower alpha-synuclein expression. Thus in one aspect, the invention provides methods for treating a synucleinopathy in a subject. The methods include identifying a subject having a synucleinopathy; selecting the subject on the basis that they have a synucleinopathy; and administering to the subject a therapeutically effective dose of one or both of POSIPHEN and phenserine. In some embodiments, the synucleinopathy is selected from the group consisting of spectrum neurodegenerative diseases associated with aberrant production of alpha-synuclein (e.g., Parkinson's disease, with Lewy bodies, Lewy body variant of Alzheimer's disease, multiple system atrophy, Parkinsonism dementia of Guam, and with brain iron accumulation type I) and spectrum neuropsychiatric disorders associated with aberrant production of alpha- synuclein (e.g., REM sleep behavioral disorders, alcohol and cocaine dependence, and anxiety disorders). In some embodiments, the methods include administering to the subject a therapeutically effective dose of POSIPHEN. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS FIG. IA is a pair of line graphs illustrating the effect of treatment with phenserine or POSIPHEN™ on levels of α-synuclein (lower lines) or amyloid precursor protein (APP), relative to an actin standard. FIG. IB is the structure of (-)-phenserine. FIG. 1C is the structure of (+)-POSIPHEN. FIG. 2 is a schematic of a luciferase reporter construct (top) and a bar graph showing the effect of APP Blocker #9, Phenserine, and POSIPHEN on expression of the reporter construct. FIG. 3 is a pair of schematics showing a luciferase reporter constructs with (left) and without (right) an alpha-synuclein 5'UTR sequence, and two bar graphs showing the effects of APP Blocker #9, Phenserine, and POSIPHEN on expression of the two constructs in H2A cells (left) and H4-pGL3 cells (right).

DETAILED DESCRIPTION Described herein are methods of treating subjects suffering from disorders associated with aberrant production of alpha-synuclein, e.g., neuropsychiatric and neurodegenerative disorders. The methods include identifying a subject in need of such treatment, e.g., on the basis that they are suffering from a disorder associated with production of alpha-synuclein, and administering a therapeutically effective amount of the (-) isomer phenserine or the (+) isomer POSIPHEN™. The methods can further include monitoring the subject for an improvement in one or more parameters of their condition, e.g., a measure of cognitive impairment or function.

a-Synuclein

α-synuclein (α-syn) is an approximately 15 kd protein expressed from the SNCA gene on Chromosome 4 (at 4q21). The official name is synuclein, alpha (non A4 component of amyloid precursor). See, e.g., GenelD: 6622. There are two isoforms in humans, including alpha-synuclein isoform NACP 140 (mRNA: nCBI Ace. No. NM_000345.2, protein: NP_000336.1) and alpha-synuclein isoform NACP112 (mRNA: NM_007308.1 protein: NP_009292.1). The NACP140 variant is the longer transcript and encodes a longer isoform than the NACPl 12 variant, which lacks an alternate in-frame segment, compared to variant NACP 140, resulting in a shorter protein that has a distinct C-terminus, compared to isoform NACP 140. The genomic sequence is available atNC_000004.10 (nt 90977156-90865728, complement). Information regarding α-synuclein in other species is available in the Gene database, e.g., at GenelD Nos. 20617 (Mus musculus); 29219 (Rattus norvegicus); 641350 (Sus scrofa); 706985 (Macaca mulata); 395393 (Gallus gallus); and 393397 (Danio rerio). As previously described (Friedlich et al., MoI Psychiatry. 12(3):222-3 (2007); USPTO 20080003570), alpha synuclein is regulated at the post-transcriptional level through a translation enhancer element in its mRNA 5' untranslated region. This translational enhancer may be targeted for drug discovery to modulate expression of alpha synuclein protein levels. As one theory, not meant to be limiting, this translational enhancer may be successfully targeted by phenserine and POSIPHEN.

Neuropsychiatry Synucleinopathies Overproduction of the alpha-synuclein is associated with neuronal dysfunction and cell death. The α-synuclein protein has been linked to at least some forms of Parkinson's disease and has also been implicated in pathogenesis of spectrum neurodegenerative diseases (Trojanowski and Lee, (2003) Ann N Y Acad Sci 991: 107- 110; Ueda et al., (1993) Proc Natl Acad Sci U S A 90:11282-11286; Xia et al., (2001) JAlzheimers Dis 3:485-494), which may include Parkinson's disease, dementia with Lewy bodies, Lewy body variant of Alzheimer's disease, multiple system atrophy, Parkinsonism dementia of Guam, and neurodegeneration with brain iron accumulation type I (Jellinger, (2004) J Neural Transm 111:1219-1235; Dickson et al., (1999) Am J Pathol 155:1241-1251; Galvin et al., (2000) Am J Pathol 157:361-368), in addition to neuropsychiatric conditions such as the mood disorders, psychotic disorders, anxiety disorders, and substance use disorders. In each of these synucleinopathies, α- synuclein is postulated to undergo conformational change and oligomerization, resulting in a toxic gain of function and subsequent neuronal dysfunction or neurodegeneration, coinciding with deposition of α-synuclein aggregates, most commonly in Lewy bodies, but also in dystrophic neuritis, axonal spheroids, and glial cytoplasmic inclusions. (Lee et al., (2004) Trends Neurosci 27:129-134). In addition to its established role in neurodegenerative diseases, α-synuclein has been linked now more broadly to spectrum neuropsychiatric disorders, which may include REM sleep behavioral disorders (Boeve et al., (2007) Brain 130:2770-2788), alcohol and cocaine dependence, and anxiety disorders α-synuclein has been shown to regulate the norepinephrine and serotonin transporters and to importantly influence synaptic dopamine neurotransmission (Yu et al., (2005) MoI Neurobiol 31:243-254; Chua and Tang, (2006) J Cell MoI Med 10:837-846), and there is now solid evidence that genetic polymorphisms in α-synuclein influence risk for developing alcohol dependence. In a family based study, eight single nucleotide polymorphisms provided evidence of association with the phenotype of alcohol craving multiplex alcoholic families of European American descent (Foroud et al, (2007) Alcohol Clin Exp Res 31:537-545). With a case control approach, two polymorphic repeats within the α- synuclein gene were significantly common in alcohol-dependent patients compared with healthy controls (see, e.g., Bonsch et al., Hum MoI Genet 2005;14(7):967-71) and the disease associated repeats significantly correlated with levels of expressed α- synuclein message ribonucleic acid (Bonsch et al., (2005) Hum MoI Genet 14:967- 971). In patients with alcohol dependence, the α-synuclein promoter has been found to be hypermethylated (Bonsch et al. (2005) Neuroreport 16:167-170). Furthermore, elevated α-synuclein message ribonucleic acid levels are found in patients with obsessive alcohol craving (Bonsch et al., (2005c) Alcohol Clin Exp Res 29:763-765). In an animal model of alcohol dependence α-synuclein maps to a quantitative trait locus for alcohol preference and is differentially expressed in alcohol preferring and alcohol non-preferring rats (Liang et al., (2003) Proc Natl Acad Sci U S A 100:4690- 4695). Interestingly, methamphetamine associated psychosis may also be linked to α- synuclein (Kobayashi et al., (2004) Ann N Y Acad Sci 1025:325-334) with significant association between three single nucleotide polymorphism in α-synuclein intron 1 and methamphetamine associated psychosis in female subjects, but not in males. In cocaine using patients, serum concentrations of α-synuclein have been found to be significantly higher as compared with age-matched drug-free controls (Mash et al., (2007) Drug Alcohol Depend). Postmortem brain from cocaine users and age-matched drug-free control subjects demonstrate that α-synuclein levels in the dopamine cell groups of the substantia nigra/ventral tegmental complex are elevated threefold in chronic cocaine users compared with normal age-matched subjects, accompanied by changes in the expression of α-synuclein message ribonucleic acid in the substantia nigra and ventral tegmental area. (Mash et al., (2003) J Neurosci

23:2564-257 1) In another post-mortem study immunoblot analysis in the ventral putamen showed that α-synuclein protein was increased in striatal synaptosomes from cocaine users compared with age-matched drug-free controls (Qin et al., (2005) Neuroreport 16:1489-1493). In animal models of cocaine abuse α-synuclein is also implicated. Cocaine administration to rats leads to overexpression of α-synuclein protein and message ribonucleic acid in the hippocampus, the and the tegmentum (Brenz Verca et al., (2003) Eur J Neurosci 18: 1923-1938). In summary, a growing body of evidence linking α-synuclein to mental health and suggests that next generation therapeutics for alcohol and cocaine dependence may lower α-synuclein. A causative rather than an epiphenomenal role for α-synuclein in disease is established in Parkinson's disease by the presence of α-synuclein mutations that segregate in rare families with autosomal dominant Parkinson's disease (Polymeropoulos et al., (1997) Science 276:2045-2047; Gwinn-Hardy, (2002) Mov Disord 17:645-656). Most patients with Parkinson's disease, however, do not carry α- synuclein mutations, and the neurodegenerative cascade is thought to result from increased concentrations of wild-type α-synuclein. Indeed, overproduction of wild type α-synuclein is known to cause disease, because in some families with autosomal dominant Parkinson's disease, the disease segregates with α-synuclein gene multiplication (Singleton et al., (2003) Science 302:841). Also, overexpression of wild type α-synuclein is sufficient to cause a degenerative Lewy body pathology in mice (Giasson et al., (2002) Neuron 34:521-533; Hashimoto et al., (2003) Ann N Y Acad Sci 991:171-188). Thus, factors that upregulate steady state levels of soluble α- synuclein are potentially relevant to pathogenesis.

Currently 1.5 million individuals in the US suffer from Parkinson's disease. Four million individuals in the US suffer from Alzheimer's disease. Over 30 million individuals in the US suffer from alcohol dependence at some point in their lives and over 60 million individuals in the US suffer from an anxiety disorder at some point in their lives. Administering phenserine or POSIPHEN to suppress alpha-synuclein is useful in the treatment or prevention of at least some forms of all the said disorders, i.e., those forms associated with aberrant production of α-synuclein. The methods described herein can be used to treat any disorder associated with aberrant production of α-synuclein, e.g., spectrum neurodegenerative diseases (e.g., Parkinson's disease, dementia with Lewy bodies, Lewy body variant of Alzheimer's disease, multiple system atrophy, Parkinsonism dementia of Guam, and neurodegeneration with brain iron accumulation type I); and spectrum neuropsychiatric disorders (e.g., REM sleep behavioral disorders, alcohol and cocaine dependence, and anxiety disorders). The methods include identifying a subject having one of these disorders, and selecting the subject on the basis of that identification (e.g., selecting a patient on the basis of a diagnosis with one of the disorders). A diagnosis can be made based on standard diagnostic criteria, using methods known in the art, e.g., using clinical criteria with or without biomarkers or functional metrics, e.g., as described in the Diagnostic and Statistical Manual of Mental Disorders, 4th. Edition (DSM-IV). In general the subjects treated by the present methods will not have Alzheimer's disease (AD).

Phenserine/POSIPHEN™ Phenserine, a phenylcarbamate of (-)-, is a new potent and highly selective (AChE) inhibitor that was developed for the treatment of Alzheimer's disease (AD) (Klein, 2007; Lahiri et al., 2007). Phenserine is presently being developed for potential use in preventing neurotoxicity following exposure to chemical organophosphorus nerve agents, including the chemical warfare agents , , and VX. Phenserine and methods for making phenserine are described in U.S. Pat. Nos. 4,831,155; 5,171,750; 5,306,825; 5,409,948; 5,734,062; 6,495,700 and WO/2003/059909; see also Greig et al., Med. Res. Rev. 15(1):3-31 (1995); and Iijima et al., Psychopharmacology (Berl). 1993;112(4):415-20 (1993). POSIPHEN™ is the positive isomer of phenserine and possesses no anti cholinesterase activity. See, e.g., Lahiri et al,. J Pharmacol Exp Ther. 320(l):386-96 (2007) and Klein, Expert Opin Investig Drugs. 16(7): 1087-97 (2007). POSIPHEN recently underwent a dose escalating Phase 1 clinical assessment in humans and was found also to be well tolerated. Both drugs have been characterized as inhibitors of APP translation through the APP 5' UTR mRNA translational regulatory mechanism and have been investigated for therapeutic efficacy in Alzheimer's disease. In some embodiments, for use in the methods described herein, Phenserine and/or POSIPHEN can be incorporated into pharmaceutical compositions. Such compositions typically include the compound (i.e., Phenserine and/or POSIPHEN as an active agent) and a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carriers" includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Pharmaceutical compositions are typically formulated to be compatible with its intended . Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. Oral compositions generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring. Systemic administration of a therapeutic compound as described herein can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art. For administration by inhalation, the compounds are typically delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer. Such methods include those described in U.S. Patent No. 6,468,798. The therapeutic compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery. Therapeutic compounds comprising nucleic acids can be administered by any method suitable for administration of nucleic acid agents, such as a DNA vaccine. These methods include gene guns, bio injectors, and skin patches as well as needle- free methods such as the micro-particle DNA vaccine technology disclosed in U.S. Patent No. 6,194,389, and the mammalian transdermal needle-free vaccination with powder-form vaccine as disclosed in U.S. Patent No. 6,168,587. Additionally, intranasal delivery is possible, as described in, inter alia, Hamajima et al., Clin. Immunol. Immunopathol, 88(2), 205-10 (1998). Liposomes (e.g., as described in U.S. Patent No. 6,472,375) and microencapsulation can also be used. Biodegradable targetable microparticle delivery systems can also be used (e.g., as described in U.S. Patent No. 6,471,996). In one embodiment, the therapeutic compounds are prepared with carriers that will protect the therapeutic compounds against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Such formulations can be prepared using standard techniques, or obtained commercially, e.g., from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to selected cells with monoclonal antibodies to cellular antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,8 11. Dosage, toxicity and therapeutic efficacy of the therapeutic compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography. A therapeutically effective amount of a therapeutic compound (i.e., an effective dosage) depends on the therapeutic compounds selected. The compositions can be administered one from one or more times per day to one or more times per week; including once every other day. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of the therapeutic compounds described herein can include a single treatment or a series of treatments. In some embodiments, the dosage of phenserine is about 0.1 mg to 100 mg, e.g., about 20 to 60 mg, e.g., about 20 mg, 30 mg, 40 mg, 50 mg, or 60 mg daily. In rodents, phenserine can be administered in effective doses of 0.25 to 10.0 mg/kg (larger doses induce disabling centrally mediated (due to overdrive), and the toxic dose is in the ballpark of 20 to 25 mg/kg). In humans, phenserine can generally be administered in doses no higher than 20 mg (BID); 15 mg (BID) was routinely given and effective in AD patients (see Kadir et al., Ann Neurol.

63(5):62 1-3 1 (2008)). POSIPHEN can be administered in far higher doses than phenserine (in rodents - up to 150 mg/kg i.p. or p.o.; in humans: 120 mg has no adverse actions, 160 mg has some adverse actions (nausea)). In some embodiments, the dosage of POSIPHEN is about 1 mg to 500 mg/kg, daily, e.g., about 100-200 mg, e.g., about 10 mg, 20 mg, 40 mg, 80 mg, 100 mg, 120 mg, 140 mg, or 160 mg. The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration. EXAMPLES The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

Example 1 - POSIPHEN and Phenserine Modulate Expression of Alpha- Synuclein Because alpha-synuclein is regulated at the post-transcriptional level in a manner similar to that of APP (USPTO 20080003570; Friedlich et al, MoI Psychiatry. 12(3):222-3 (2007)), the present inventors sought to determine if POSIPHEN and phenserine also modulate expression of alpha synuclein (α-syn). The effect of POSIPHEN and phenserine on (α-syn) expression was determined by immunoblotting 20 µg of cell lysates with antibodies specific for α-syn (72-10), APP (22Cl 1), and beta-actin for standardization. No cell toxicity was observed for either phenserine of POSIPHEN (MTT and LDH assays). The dose responsive capacity of POSIPHEN and phenserine to reduce APP and α-syn was then graphed (Quanty-1 program using a phosphoimager (Biorad)). The results are shown in Figure IA. The average of 3 separate experiments revealed the half-inhibition of APP expression by phenserine was 5 uM, with an IC-50 of 7 uM for α-syn inhibition. By comparison, POSIPHEN dose responsively suppressed expression of α-syn and APP, each with an IC-50 of 5 uM. These results show that the two drugs, with known safety profiles in humans, in addition to their known effects in suppressing APP, are able to suppress α-syn and may therefore be useful in treating diseases whose pathogenesis or pathophysiology involves alpha-synculein production.

Example 2 - Generation of Constructs The firefly (Photinus pyralis) luciferase reporter gene will be translationally regulated either by the 46 residue α-synuclein mRNA 5'UTR or the 100 nucleotide prion protein mRNA 5' UTR. Within each construct, a EGFP (green fluorescent protein) gene is translated from a single bicistronic mRNA to control for rate of transcription. To generate pIRES (α-synuclein 5'UTR), we synthesized the entire 46 base sequence of the α-synuclein 5' UTR with a hindIII site at the 5' end and a Nco I site at the 3' end (the sequence is CCCCAAGCTTTCGGAGTGGCCATTCGACGACAGTGT- GGTGTAAAGGAATTCATTAGCCATGGTTTT (SEQ ID NO: 1); Integrated DNA Technologies, Coralville IA) and then ligated the insert into a pGL-3 vector (Promega, Madison WI) immediately in front of the luciferase gene, under the control of a SV40 promoter. The plasmid construct was amplified in bacteria in medium containing ampicillin. The colonies were isolated, and the generated cassette was confirmed from band size migration after treatment with EcoRl and separately with HindIII and Xbal . BamH I restriction enzyme pGL3 containing the α-synuclein 5' UTR insert upstream of the luciferfase promoter was excised from the pGL-3 construct by Hind III and Xba I digestion. The excised fragment will be blunt-ended by klenow DNA polymerase I treatment. This fragment will be then ligated to Smal digested linearized pIRES-EGFP vector (Clontech, Mountain View, CA). The plasmid construct was amplified in bacteria in medium containing kanamycin. The colonies were isolated, and the PCR (QIAGEN, Valencia, CA) generated cassette was confirmed from band size after treatment with BamH I restriction enzyme. B i directional DNA sequencing confirmed the identity of the clones using GL2 and RV3 primers, the sequencing primers for pGL-3 (Payton et al, (2003) J MoI Neurosci 20:267-275). The construct obtained permits both the gene of interest, i.e., α- synuclein mRNA 5'UTR driven by luciferase, and the EGFP gene to be translated from a single bicistronic mRNA To generate pIRES(PrP mRNA 5'UTR, two complementary oligonucleotides

(Sigma) were annealed to each other (30µg of each in a total volume of 1 ml of 10 mM Tris pH 7.5, 50 mM NaCl, ImM EDTA) at 95°C for lmin and then allowed to cool slowly to room temperature to form the double stranded 5'UTR of PrP variant 2 (Liao et al., (1986) Science 233:364-367; Hsiao et al., (1989) Nature 338:342-345). The oligonucleotide sequences are: AGCTTCCCCCTCGGCCCCGCGCGTCGCCTGTCCTCCGAGCCAGTCGCTGAC AGCCGCGGCGCCGCGAGCTTCTCCTCCCTCACGACCGAGAGCAGTCATTA C (sense; SEQ ID NO:2) and CATGGTAATGACTGCTCTGGTCGTGAGGAGAGGAGAAGCTCG- CGGCGCCGCGGCTGTCAGCGACTGGCTCGGAGGACAGGCGACGCGCGGG- GCCGAGGGGGA (antisense; SEQ ID NO:3). The annealed PrP 5'UTR oligo nucleotides were first cloned into pGL-3. This clone was then digested with Hind III and Xba I, blunt ended and cloned into Sma I digested pIRES-EGFP vector as described before. This was named pIRES(PrP mRNA 5'UTR) construct. To generate the dicistronic construct, the pIRES vector (Clontech), which contained an internal ribosome entry site element (IRES) followed by enhanced green fluorescent protein gene, is utilized. A cDNA cassette encoding 46 nt a-syn mRNA 5- UTR was cloned into the multiple cloning site of pIRES2 between unique XhoI/EcoRI sites. A luciferase reporter gene (Luc; Promega, Madison, WI) or the red fluorescent protein reporter gene (dsREDN-1; Clontech) was subsequently ligated downstream of the EcoRI/BamHI sites.

Example 3 - Transient transfection 8 x 105 neuroblastoma SH-SY5Y cells were seeded for 24 hours in 60 mm2 dishes with 5 ml Dulbenco's modified medium containing 10% fetal bovine serum. For transfection, cells were first rinsed 3x in PBS and then treated with 5 µg of purified DNA combined with PoIyFect transfection reagent and growth medium, according to the manufacturer's specifications (Qiagen). After a 24 hours incubation, cells were washed in PBS 3x, trypsonized and plated to 95% confluence in 96 well plates and allowed to seed for 12 hours in Dulbenco's modified medium containing 10% fetal bovine serum and ampicillin. Cells were exposed to desferoxamine (0- 100 µM, Calbiochem, La Jolla, CA) or iron citrate (0-10 µg/µl, NIST, Gaithersburg,MD), for the specific times indicated. Cytoplasmic protein lysates were prepared by homogenizing the cells in midRIPA buffer (25mM Tris pH 7.4, 1% NP40, 0.5% sodium deoxycholate, 15mM NaCl, protease inhibitors, RNase inhibitor, 1OmM DTT). After 48 hours of treatment with iron citrate or DFO, cell viability was confirmed by microscopic examination of each well.

Example 4 - Luciferase Assays Cells were lysed with Ix BRIGHT-GLO™ luciferase assay buffer and the lysates combined with BRIGHT-GLO luciferase assay substrate warmed to 37°, according to the specifications of the manufacturer (Promega, Madison, WI). All experiments were performed in triplicate. Automated luciferase assays were performed with a Wallac 1420 multi-label counter. Green fluorescence protein expression was quantified at 489/509 nm. Expression of green fluorescent protein within the pGL3 vector, controlled by a SV40 late poly(A) signal was used to control for transfection efficiency. For experiments utilizing the pIRES dicistronic construct the red fluorescence is measured at wavelength of 558/583-nm.

Example 5 - Stable transfections The SH-SY5Y neuroblastoma cell line will be transfected with AMAXA cell line Nucleofactor kit V (AMAXA biosystems, Gaithersburg, MD) following manufacturer's instructions, utilizing the constructs pIRES (α-synuclein mRNA 5'UTR) and pIRES(PrP mRNA 5'UTR). Once transfected cells are be obtained, they are then split and a stock maintained. Transfection efficiency is determined by counting GFP positive cells.

Example 5 - Western blotting Protein assays (BCA; Pierce, Rockford, IL) are performed from supernatants, and 25 µg protein will electrophoresed on a 0.1% sodium dodecyl sulfate/10% polyacrylamide gel. The proteins are then electrophoretically transferred to Hybond- C (Amersham). The nitrocellulose is soaked in 5% nonfat powdered milk for 1 hour and then exposed to primary antibody to a 1:1000 dilution for polyclonal rabbit anti- IRPl (α Diagnostics International, San Antonio, TX) or 1:000 dilution for Anti-IRP2 (kind gift from Dr. T. Rouault and S. Cooperman) or1:500 dilution of rabbit polyclonal antibody 72-10 against hα-syn (gift of Eliezer Masliah). After 3 washes in buffer, the nitrocellulose is incubated with an alkaline phosphatase-conjugated secondary antibody (1:5000). After 3 additional washes in buffer, the signal is visualized with an enhanced chemiluminescent reagent (CDP Star, Tropix). Following exposure, films are scanned as jpeg files and bands will be quantified with NIH Image software.

Example 6 - POSIPHEN and phenserine treatment in cultured cells SY5Y cells were allowed to grow in complete media (10% fetal calf serum, 2 mM glutamine in DMEM) for 2 days to reach 70% confluence. Then, the media was removed and replaced with fresh media (2 ml of DMEM) containing 0 to 10 µM

POSIPHEN or phenserine, and the cells were incubated at 37°C, 5% CO2 for 48 hours. Cell viability was assessed by measurement of lactate dehydrogenase levels. From cell lysates, α-syn protein and mRNA levels and rate of α-syn translation are determined as described above. Cells stably transfected with pIRES (α-synuclein mRNA 5'UTR)/pIRES(PrP mRNA 5'UTR ware also utilized, and after treatment these cells were lysed for luciferase assays as described above. The results, shown in Fig. IA, show that POSIPHEN and phenserine decreased levels of α-synuclein in a dose-dependent manner in cultured neural cells (SH-SY5Y), with POSIPHEN showing more efficacy than phenserine.

Example 7 - Posiphen inhibits alpha synuclein 5'UTR directed translation: The effects of POSIPHEN and phenserine on α-synuclein 5'UTR-driven luciferase expression was evaluated in H2A neural cells, stably transfected with an α- syn 5'UTR- luciferase construct. The results, shown in Fig. 2, showed that POSIPHEN was a highly selective inhibitor of α-synuclein 5'UTR activity since this stereoisomer of phenserine inhibited α-synuclein 5'UTR driven luciferase expression. By contrast, phenserine, and the known APP translation #9 blocker, did not suppress alpha-synuclein 5'UTR conferred translation in these cells. Interestingly, phenserine and APP blocker #9 increased α-syn 5'UTR conferred translation. As one theory, not meant to be limiting, the mechanism of action of POSIPHEN is as a highly selective blocker of alpha synuclein 5'UTR activity, whereas phenserine (which has the same chemical structure as, but is a stereoisomer of, posiphen) was previously shown to selectively inhibit translation driven by the APP 5'UTR. These results indicate that POSIPHEN potently and stereoselectively decreased α-synuclein 5'UTR directed translation of a downstream luciferase reporter gene in cultured neural cells. This activity correlated with the capacity of POSIPHEN to inhibit alpha-synclein expression in SH-SY5Y cells (Fig. IA). Phenserine appears to suppress alpha-synuclein at lower potency than POSIPHEN (Fig. IA), and may act via other sequences in the alpha synuclein gene. In additional experiments, the ability of POSIPHEN to selectively inhibit alpha-synuclein 5'UTR-conferred luciferase expression in H2A neural cells (α-syn 5'UTR positive stable transfectants) was evaluated in pGL3-H4 cells (**pGL3-H4 serves as an experimental control since these cells were stably transfected with pGL3, which is the same as the H2A construct but lacks the α-synuclein 5'UTR). Confirming selectivity, as shown in Fig. 3, POSIPHEN also increased luciferase expression in these cells. (N=3/ expt, 3 separate transfections)). Example 8 - Animal Model The efficacy of POSIPHEN as a protective agent against PD lesioning will be evaluated in vivo in an animal model of PD created by l-methyl-4-phenyl-l,2,3,6- tetrahydropyridine (MPTP) treatment and in alpha-synuclein PD transgenic models. MPTP is a neurotoxin that causes degeneration of nigrostriatal neurons by inhibiting oxidative phosphorylation. MPTP causes motor impairment that resembles PD in humans. The alpha-synuclein transgenic mice develop Lewy body pathology, nigrostriatal degeneration, and motor impairment, all of which usually occur in human patients with Parkinson's disease and related disorders.

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OTHER EMBODIMENTS It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. WHAT IS CLAIMED IS:

1. POSIPHEN for use in the treatment of a synucleinopathy.

2. Phenserine for use in the treatment of a synucleinopathy.

3. The use of claim 1 or 2, wherein the synucleinopathy is selected from the group consisting of spectrum neurodegenerative diseases associated with aberrant production of alpha-synuclein; and spectrum neuropsychiatric disorders associated with aberrant production of alpha-synuclein.

4. The use of claim 3, wherein the spectrum neurodegenerative diseases associated with aberrant production of alpha-synuclein is selected from the group consisting of Parkinson's disease, dementia with Lewy bodies, Lewy body variant of Alzheimer's disease, multiple system atrophy, Parkinsonism dementia of Guam, and neurodegeneration with brain iron accumulation type I.

5. The use of claim 3, wherein the spectrum neuropsychiatric disorders associated with aberrant production of alpha-synuclein is selected from the group consisting of REM sleep behavioral disorders, alcohol and cocaine dependence, and anxiety disorders.

6. A method of treating a synucleinopathy in a subject, the method comprising: identifying a subject having a synucleinopathy; selecting the subject on the basis that they have a synucleinopathy; and administering to the subject a therapeutically effective dose of one or both of POSIPHEN and phenserine.

7. The method of claim 6, comprising administering to the subject a therapeutically effective dose of POSIPHEN.