IDENTIFICATION OF COMPOUNDS THAT PROTECT AGAINST AMYLOID DISEASES

FIELD OF THE INVENTION

The present invention relates to methods of inhibiting aggregation-mediated proteotoxicity, methods of inhibiting the onset of symptoms of an amyloid disease and methods of identifying compounds that inhibit aggregation-mediated proteotoxicity.

BACKGROUND OF THE INVENTION

It has been known for decades that more than a dozen unrelated proteins can undergo aberrant assembly in vivo to form filaments or fibrils, which are generally referred to as amyloid (for reviews, see Sacchettini and Kelly, Nature Rev. 1 :267-75, 2002; Stefani and Dobson, J. MoI. Med. 81 :678-99, 2003; Lansbury and Lashuel, Nature 443:774-79, 2006). The presence of extracellular or intracellular aggregates of a specific polypeptide molecule is a hallmark of the recognized amyloid diseases; such aggregates promoting aggregation-mediated proteotoxicity. The polypeptides involved include full-length proteins {e.g., lysozyme and immunoglobulin light chains), peptides {e.g., amylin and atrial natriuretic factor) and fragments of larger proteins produced as a result of normal or abnormal endoproteo lytic processing {e.g., Alzheimer's β-amyloid peptide). Abnormal processing is often the result of misfolding (Golde et ah, Science 255:728-30, 1992; Shoji et ah, Science 258:126-29, 1992; Kimberly et al., J. Biol. Chem. 275:3173-78, 2000). Nearly all full-length amyloidogenic proteins are secreted, suggesting that these proteins misfold after export (Kelly, Structure 5:595-600, 1997; Moyer and Balch, Emerging Therap. Targets 5:165-76, 2001). In some cases the proteins involved have wild-type sequences, as in sporadic forms of the diseases, but in other cases these are variants resulting from genetic mutations associated with familial forms of the diseases.

The presence of misfolded or aggregated proteins triggers a complex biological response, leading to the expression, among others, of the genes for heat shock proteins (Hsp, or molecular chaperone proteins) and proteins involved in the

ubiquitin-proteasome pathway (Sherman and Goldberg, Neuron 29: 15-32, 2001). The evolution of such complex biochemical machinery makes clear the fact that it is necessary for cells to isolate and rapidly and efficiently clear any unfolded or misfolded protein as soon as it appears. Until recently, the main amyloid diseases were thought to be restricted to

Alzheimer's disease (AD), Parkinson's disease (PD), reactive amyloidosis and the systemic amyloidoses {e.g. , immunoglobulin-light-chain-, transthyretin- and gelsolin- based diseases) (Westermark et al, Proc. Natl. Acad. ScL USA 87:2843-45, 1990; Hurle et al., Proc. Natl. Acad. ScL USA 91 :5446-50, 1994; Selkoe, Science 275:630- 31, 1997; Kiuru, Amyloid 5:55-66, 1998). However, amyloid-like disorders are likely far more widespread than previously thought, and could include many common neurodegenerative and neuromuscular pathologies, as well as prion disease. The most intensely studied amyloid disease is AD, where characteristic brain plaques contain β- amyloid. The primary biochemical component of β-amyloid is a 39- to 43-amino acid peptide derived from the β-amyloid precursor protein (APP) through endoproteolysis (Glenner and Wong, Biochem. Biophys. Res. Commun. 83:885-90, 1984).

The deposition of proteins in the form of amyloid fibrils and plaques is the characteristic feature of more than twenty degenerative conditions affecting either the central nervous system or a variety of peripheral tissues. As discussed herein, these conditions include AD, Parkinson's disease and several forms of fatal systemic amyloidoses, and are of enormous importance in the context of present-day human health and welfare. Therefore, there remains a need for the development of effective therapies for the treatment and prevention of aggregation-mediated proteotoxicity and the identification of compounds that inhibit aggregation-mediated proteotoxicity.

BRIEF SUMMARY OF THE INVENTION

Compositions and methods for inhibiting protein aggregation and aggregation- mediated proteotoxicity in a subject are provided. Compositions include at least one of a flavone, a biguanide, a furanofiavonoid, 5-Aminoimidazole-4-carboxamide 1-β- D-ribofuranoside, trimethadione, morin, and combinations thereof. The compositions can be used in conjunction with other compounds known to inhibit protein aggregation to enhance their activities. The compositions are provided in therapeutically effective amounts to a subject in need thereof to inhibit protein aggregation. The compositions find use in treating and preventing amyloid diseases,

such as neurodegenerative diseases, associated with protein aggregation and aggregation-mediated proteotoxicity.

Also provided are methods for identifying compounds that inhibit protein aggregation. The methods include the expression of a β-amyloid peptide and determining the effect of a test compound on paralysis and/or protein aggregation. In one embodiment, an assay utilizing a C. elegans modified to express a β-amyloid peptide is utilized.

DETAILED DESCRIPTION OF THE INVENTION

Introduction

Compositions and methods for inhibiting protein aggregation and aggregation- mediated proteotoxicity in a subject are provided. The compositions can be used in conjunction with other compounds known to inhibit protein aggregation to enhance their activities. The compositions are provided in therapeutically effective amounts to a subject in need thereof to inhibit protein aggregation. The compositions find use in treating and preventing neurodegenerative diseases associated with protein aggregation and aggregation-mediated proteotoxicity.

An amyloid disease (or amyloidosis) is any of a group of disparate conditions of diverse etiologies characterized by the accumulation of amyloid in various organs and tissues of the body, with accompanying impairment of vital function. Amyloid is the generic term for a group of diverse but specific protein deposits which are seen in a number of different diseases. Amyloid deposits are metabolically inert but interfere physically with organ structure and function. Though diverse in their occurrence, all amyloid deposits have common morphologic properties, stain with specific dyes (e.g., Congo red) and have a characteristic red-green birefringent appearance in polarized light after staining. They also share common ultrastructural features and common X- ray diffraction and infrared spectra. The associated disease states may be inflammatory, hereditary or neoplastic, and the deposition can be local, generalized or systemic. There are three major systemic forms of amyloidosis: primary, secondary and familial. In addition, there are two major localized forms, β-amyloid peptide (Aβ) and islet amyloid polypeptide (IAPP, which occurs in the pancreas of type-2 diabetes subjects), as well as several miscellaneous forms (e.g., Aβ ₂ -microglobulin associated with chronic hemodialysis).

Primary amyloidosis (AL) is a monoclonal plasma cell disorder in which the abnormal protein is an immunoglobulin, usually a light chain fragment (i.e., Bence Jones protein) but occasionally a heavy chain fragment (AH amyloidosis). These chains either have an aberrant structure or are processed abnormally, resulting in the formation of insoluble deposits. Common sites for deposition include the skin, nerves, heart, gastrointestinal tract (including tongue), kidney, liver, spleen, and blood vessels. Mild plasmacytosis occurs in the bone marrow, suggestive of multiple myeloma, but most AL subjects do not have true multiple myeloma (however, about 10-20% of subjects with multiple myeloma also develop AL amyloidosis). Secondary amyloidosis (AA) can occur secondary to several infectious, inflammatory and malignant (e.g., myeloma) conditions and is caused by the degradation of the acute -phase reactant serum amyloid A (SAA) protein. Common causative infections include tuberculosis (TB), bronchiectasis, osteomyelitis, and leprosy. Inflammatory conditions include rheumatoid arthritis (RA), juvenile RA, Crohn's disease, and familial Mediterranean fever. Inflammatory cytokines (e.g., IL- 1 , tumor necrosis factor and IL-6) that are produced in these disorders cause increased hepatic production of the precursor protein SAA, which circulates in the serum. AA amyloidosis shows a predilection for the spleen, liver, kidneys, adrenals, and lymph nodes, but involvement of the heart and peripheral or autonomic nerves is also known. Familial amyloidosis results from accumulation of a normal or mutated version of a plasma protein (e.g., transthyretin (TTR) in familial amyloid polyneuropathy I or senile systemic amyloidosis). Nearly all of the abnormal protein is produced by the liver, and over 80 mutations of the gene for TTR have been identified, all inherited in an autosomal dominant pattern. Other rare hereditary amyloidoses result from mutations of other physiologic proteins, including apolipoprotein A-I (familial amyloid polyneuropathy III), lysozyme (familial non- neuropathic amyloidosis), fibrinogen (hereditary renal amyloidosis), gelsolin (Finnish hereditary systemic amyloidosis), superoxide dismutase (familial amyotrophic lateral sclerosis), and cystatin C (hereditary cerebral amyloid angiopathy); these amyloidoses have various systemic and localized effects.

Localized amyloidoses are those that tend to involve a single organ system and include Alzheimer's disease (Aβ peptides), spongiform encephalopathies (prion), Parkinson's disease (α-synuclein), Huntington's disease (Huntingtin), type-2 diabetes

(IAPP), medullary carcinoma of the thyroid (calcitonin fragment), and atrial amyloidosis (atrial natriuretic factor).

Inhibition of Aggregation-Mediated Proteotoxicity The invention provides methods of inhibiting aggregation-mediated proteotoxicity in a subject or inhibiting the onset of symptoms of an amyloid disease in a subject, including administering to the subject a composition comprising a compound described herein for practicing the invention. "Aggregation-mediated proteotoxicity" refers to the pathological outcomes associated with the intracellular and/or extracellular accumulation of insoluble fibrillar proteins (amyloid) in affected tissues and organs. By "inhibiting", with respect to inhibiting aggregation-mediated proteotoxicity or inhibiting the onset of symptoms of an amyloid disease, is intended (i) preventing or treating the pathological outcomes associated with the intracellular and/or extracellular accumulation of amyloid, for example, causing clinical symptoms of proteotoxicity or the amyloid disease not to develop in a subject that may be predisposed to the same but does not yet experience or display symptoms; (ii) restraining proteotoxicity or the amyloid disease, for example, arresting the development of the disease or its clinical symptoms; (iii) ameliorating proteotoxicity or the amyloid disease, for example, delaying onset of clinical symptoms in a susceptible subject or a reduction in severity of some or all clinical symptoms; or (iv) relieving proteotoxicity or the amyloid disease, for example, causing regression of some or all clinical symptoms.

As used herein, "subject" includes organisms in which aggregation-mediated proteotoxicity or amyloidosis can occur, or which are susceptible to amyloid diseases, for example, the amyloid diseases described herein. Thus, "subject" includes both human, laboratory and veterinary organisms, for example, humans, non-human primates, dogs, cats, mice, horses, cows, and the like. In one embodiment, the compounds described herein for practicing the invention prevent or inhibit amyloid protein assembly into insoluble fibrils which, in vivo, are deposited in various organs, or they facilitate clearance of pre-formed deposits or slow deposition in subjects already having amyloid deposits. In another embodiment, the compounds may also prevent amyloid protein, in its soluble, oligomeric form or in its fibrillar form, from binding or adhering to a cell surface and causing cell damage or toxicity. In yet another embodiment, the compounds may block amyloid-induced cellular toxicity. In

still another embodiment, the compounds may block amyloid-induced neurotoxicity. In a further embodiment, the compounds may enhance clearance from a specific organ (e.g. , the brain) or decrease concentration of amyloid protein in such a way that amyloid fibril formation is prevented in the target organ.

Compounds for Practicing the Invention

The compounds described herein for practicing the invention include a flavone, a biguanide, a furanoflavonoid, 5-Aminoimidazole-4-carboxamide 1-β-D- ribofuranoside (AICAR), trimethadione (TMD; 3,5,5-trimethyloxazolidine-2,4- dione), morin (2-(2,4-dihydroxyphenyl)-3,5,7-trihydroxy-chromen-4-one), and combinations thereof. In one embodiment, a method of inhibiting aggregation- mediated proteotoxicity in a subject includes administering to the subject a composition comprising a flavone, a biguanide, a furanoflavonoid, 5- Aminoimidazole-4-carboxamide 1 -β-D-ribofuranoside, trimethadione, morin, and combinations thereof. In another embodiment, a method of inhibiting the onset of symptoms of an amyloid disease in a subject includes administering to the subject a composition comprising a flavone, a biguanide, a furanoflavonoid, 5- Aminoimidazole-4-carboxamide 1 -β-D-ribofuranoside, trimethadione, morin, and combinations thereof. By "flavone" is intended a class of compounds based on the backbone of 2-

Phenyl-4H-l-benzopyran-4-one (2-Phenylchromone), which has the structure illustrated in Formula (1).

(1)

Naturally occurring fiavones are plant pigments and serve as the basis for flavonoids. Exemplary fiavones include, but are not limited to, 3',4',-dihydroxy-B-napthoflavone; 3,5,7-trihydroxy-3',4',5'-trimethoxyflavone; 3,6-dimethoxyflavone; 3,7- dihydroxyflavone; 3,7-dimethoxyflavone; 3-hydroxyflavone; 4'-hydroxy-alpha-

naphthoflavone; 4'-methoxy-alpha-naphthoflavone; 5,7,4'-trimethoxyflavone; 5,7- dimethoxyflavone; 6,7-dihydroxyflavone; 6-hydroxy-7-methoxyflavone; 7,8- Benzoflavone; 7,8-dimethoxyflavone; 7-hydroxy-3-methylflavone; 7-hydroxyflavone; 4',5,7-trihydroxyflavone (apigenin); 5,6,7-trihydroxyflavone (baicalein); and 7,4'- dihydroxy-6-methoxyisoflavone (glycitein). In a particular embodiment, a flavone is 3,7-dihydroxyflavone; 7-hydroxy-3-methylflavone; 7-hydroxyflavone; or 7,4'- dihydroxy-6-methoxyiso flavone, which have the structures illustrated in Formula (2), Formula (3), Formula (4), and Formula (5), respectively.

As used herein, a "biguanide" includes any of a group of substituted derivatives of 2-carbamimidoylguanidme, which has the structure illustrated in Formula (6).

The biguanides are commonly used as oral antihyperglycemic agents; they increase insulin action in peripheral tissues, and by inhibiting gluconeo genesis decrease hepatic glucose production. Exemplary biguanides include, without limitation, metformin (2-(N,N-dimethylcarbamimidoyl)guanidine) and phenformin (2-(N'- phenethylcarbamimidoyl)guanidine), which have the structures illustrated in Formula (7) and Formula (8), respectively.

By a "furanoflavonoid" is intended a flavonoid containing a furan ring structure, for example, karanjin (3-methoxyfurano-2',3',7,8-flavone), which has the structure illustrated in Formula (9).

Additional compounds useful according to the invention include AICAR, trimethadione and morin, which have the structures illustrated in Formula (10), Formula (11) and Formula (12), respectively.

H ₃ C ry _x H ₃ C' >°

O CH ₃

(H)

Still further compounds useful according to the invention include coumarins, such as, for example, 6-ethoxy-3(4'-hydroxyphenyl)-4-methylcoumarin and 7- diethylamino-3(4'-methoxyphenyl)coumarin. In addition to the compounds specifically noted above, the assays described according to the present invention are also useful for identifying further compounds having activity for use in treatment of various disorders, such as neurodegenerative diseases, such activity arising from modulation of one or more aspects of aggregation- mediated proteotoxicity. As noted in relation to the above compounds, the activity of the compounds of the invention can include prevention or inhibition of amyloid protein assembly into insoluble fibrils, clearance of pre-formed deposits or slowing deposition in subjects already having amyloid deposits. The activity of the compounds may also include prevention of amyloid protein, in its soluble, oligomeric form or in its fibrillar form, from binding or adhering to a cell surface and causing cell damage or toxicity, or enhancing amyloid clearance from a specific organ {e.g., the brain).

The compounds provided above according to Formula (1) through Formula (12) are not intended to limit the scope of active compounds provided according to the

present invention. On the contrary, the invention encompasses the additional compounds recited herein, multiple variants of the recited compounds, as well as further compounds identifiable according to the methods and assays described herein. In particular, the present invention also encompasses pharmaceutically acceptable esters, amides, salts, or solvates of the various compounds described herein.

Biologically active variants of the compounds described herein for practicing the invention are particularly also encompassed by the methods of the present invention. Such variants should retain the biological activity of the compounds described herein (i.e., the ability to inhibit aggregation-mediated proteotoxicity). According to one embodiment of the invention, suitable biologically active variants comprise analogues and derivatives of the compounds described herein for practicing the invention. As used herein, an "analogue" refers to a compound in which one or more individual atoms or functional groups have been replaced, either with a different atom or a different functional, generally giving rise to a compound with similar properties. Indeed, a single compound, such as those described herein, may give rise to an entire family of analogues having similar activity and, therefore, usefulness according to the present invention. Likewise, a single compound, such as those described herein, may represent a single family member of a greater class of compounds useful according to the present invention. Accordingly, the present invention fully encompasses not only the compounds described herein, but analogues of such compounds, particularly those identifiable by methods commonly known in the art and recognizable to the skilled artisan.

A "derivative", as used herein, comprises a compound that is formed from a similar, beginning compound by attaching another molecule or atom to the beginning compound. Further, derivatives, according to the invention, encompass one or more compounds formed from a precursor compound through addition of one or more atoms or molecules or through combining two or more precursor compounds.

The present invention also includes stereoisomers of the compounds described herein for practicing the invention, where applicable, either individually or admixed in any proportions. Stereoisomers may include, but are not limited to, enantiomers, diastereomers, racemic mixtures and combinations thereof. Such stereoisomers can be prepared and separated using conventional techniques, either by reacting enantiomeric starting materials, or by separating isomers of compounds of the present invention. Isomers may include geometric isomers. Examples of geometric isomers

include, but are not limited to, cis isomers or trans isomers across a double bond. Other isomers are contemplated among the compounds of the present invention. The isomers may be used either in pure form or in admixture with other isomers of the compounds described herein. The present invention further includes prodrugs and active metabolites of the compounds described herein for practicing the invention. A prodrug includes any compound which, when administered to a subject, is converted in whole or in part to a compound as described herein. An active metabolite is a physiologically active compound which results from the metabolism of a compound described herein for practicing the invention, or a prodrug thereof, when such compound or prodrug is administered to a subject.

Pharmaceutical Compositions

In some embodiments, a therapeutically effective amount of a composition comprising a flavone, a biguanide, a furanoflavonoid, 5-Aminoimidazole-4- carboxamide 1-β-D-ribofuranoside, trimethadione, morin, and combinations thereof is administered to a subject to inhibit aggregation-mediated proteotoxicity or the onset of symptoms of an amyloid disease. A "therapeutically effective amount" of a composition comprising a flavone, a biguanide, a furanoflavonoid, 5- Aminoimidazole-4-carboxamide 1 -β-D-ribofuranoside, trimethadione, morin, and combinations thereof is an amount which, when administered to a subject, is sufficient to achieve a desired effect in a subject being treated with that composition. For example, this may be the amount of a composition useful in preventing, ameliorating and/or treating disease associated with amyloid fibril formation, aggregation or deposition in a subject. Ideally, a therapeutically effective amount of a composition comprising a flavone, a biguanide, a furanoflavonoid, 5-Aminoimidazole-4- carboxamide 1 -β-D-ribofuranoside, trimethadione, morin, and combinations thereof is an amount sufficient to prevent, ameliorate and/or treat disease associated with amyloid fibril formation, aggregation or deposition in a subject without causing a substantial cytotoxic effect in the subject.

The effective amount of a composition comprising a flavone, a biguanide, a furanoflavonoid, 5-Aminoimidazole-4-carboxamide 1 -β-D-ribofuranoside, trimethadione, morin, and combinations thereof useful for preventing, ameliorating and/or treating disease associated with amyloid fibril formation, aggregation or

deposition in a subject will depend on the subject being treated, the specific type and severity of the affliction and the manner of administration of the composition. Responses to a therapeutically effective amount of a composition comprising a flavone, a biguanide, a furanoflavonoid, 5-Aminoimidazole-4-carboxamide 1-β-D- ribofuranoside, trimethadione, morin, and combinations thereof can include, for instance, inhibition of amyloid protein assembly into insoluble fibrils, clearance of pre-formed deposits or a decrease in the rate of deposition of amyloid deposits. As described herein, the present invention contemplates pharmaceutical compositions including one or more of a flavone, a biguanide, a furanoflavonoid, 5- Aminoimidazole-4-carboxamide 1 -β-D-ribofuranoside, trimethadione, and morin. Delivery systems and treatment regimens useful for such compounds are known and can be used to administer these compounds as therapeutics. In addition, representative embodiments are described herein.

The compounds described herein for practicing the invention can be administered in the form of an ester, amide, salt, solvate, prodrug, metabolite, derivative, or the like, provided it maintains pharmacological activity according to the present invention. Esters, amides, salts, solvates, prodrugs, and other derivatives of the compounds of the present invention may be prepared according to methods generally known in the art, such as, for example, those methods described by J. March, Advanced Organic Chemistry: Reactions, Mechanisms and Structure, 4 ^th Ed. (New York: Wiley-Interscience, 1992).

Examples of pharmaceutically acceptable salts of the compounds described herein for practicing the invention include acid addition salts. Salts of non- pharmaceutically acceptable acids, however, may be useful, for example, in the preparation and purification of the compounds. Suitable acid addition salts according to the present invention include organic and inorganic acids. Preferred salts include those formed from hydrochloric, hydrobromic, sulfuric, phosphoric, citric, tartaric, lactic, pyruvic, acetic, succinic, fumaric, maleic, oxaloacetic, methanesulfonic, ethanesulfonic, p-toluenesulfonic, benzesulfonic, and isethionic acids. Other useful acid addition salts include propionic acid, glycolic acid, oxalic acid, malic acid, malonic acid, benzoic acid, cinnamic acid, mandelic acid, salicylic acid, and the like. Particular example of pharmaceutically acceptable salts include, but are not limited to, sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, phosphates, monohydrogenphosphates, dihydrogenphosphates, metaphosphates, pyrophosphates,

chlorides, bromides, iodides, acetates, propionates, decanoates, caprylates, acrylates, formates, isobutyrates, caproates, heptanoates, propiolates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, butyne-l,4-dioates, hexyne-1,6- dioates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxyenzoates, phthalates, sulfonates, xylenesulfonates, phenylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, γ- hydroxybutyrates, glycolates, tartrates, methanesulfonates, propanesulfonates, naphthalene- 1 -sulfonates, naphthalene-2-sulfonates, and mandelates.

An acid addition salt may be reconverted to the free base by treatment with a suitable base. Preparation of basic salts of acid moieties which may be present on a compound described herein for practicing the invention may be prepared in a similar manner using a pharmaceutically acceptable base, such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, triethylamine, or the like. Esters of the compounds described herein for practicing the invention may be prepared through functionalization of hydroxyl and/or carboxyl groups that may be present within the molecular structure of the compound. Amides and prodrugs may also be prepared using techniques known to those skilled in the art. For example, amides may be prepared from esters, using suitable amine reactants, or they may be prepared from anhydride or an acid chloride by reaction with ammonia or a lower alkyl amine. Moreover, esters and amides of compounds of the invention can be made by reaction with a carbonylating agent (e.g. , ethyl formate, acetic anhydride, methoxyacetyl chloride, benzoyl chloride, methyl isocyanate, ethyl chloroformate, methanesulfonyl chloride) and a suitable base (e.g., 4-dimethylaminopyridine, pyridine, triethylamine, potassium carbonate) in a suitable organic solvent (e.g., tetrahydrofuran, acetone, methanol, pyridine, N,N-dimethylformamide) at a temperature of 0 ⁰ C to 60 ⁰ C. Prodrugs are typically prepared by covalent attachment of a moiety, which results in a compound that is therapeutically inactive until modified by an individual's metabolic system. Examples of pharmaceutically acceptable solvates include, but are not limited to, compounds according to the invention in combination with water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, or ethanolamine.

Methods of administering a composition comprising a compound described herein for practicing the invention include, but are not limited to, intrathecal,

intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, intradural, intracranial, intraventricular, and oral routes. A composition comprising a compound described herein for practicing the invention may be administered by any convenient route, including, for example, infusion or bolus injection, topical, absorption through epithelial or mucocutaneous linings (for example, oral mucosa, rectal and intestinal mucosa, and the like) ophthalmic, nasal, and transdermal, and may be administered together with other biologically active agents. Administration can be systemic or local. In some instances, injection may be facilitated by a catheter, for example, attached to a reservoir. Pulmonary administration can also be employed (for example, by an inhaler or nebulizer), for instance using a formulation containing an aerosolizing agent.

In a specific embodiment, it may be desirable to administer a pharmaceutical composition locally to the area in need of treatment. This may be achieved by, for example, and not by way of limitation, local or regional infusion or perfusion during or following surgery, topical application (for example, wound dressing), injection, catheter, suppository, or implant (for example, implants formed from porous, non-porous, or gelatinous materials, including membranes, such as sialastic membranes or fibers), and the like. In one embodiment, a pump may be used (see, e.g., Langer, Science 249: 1527-33, 1990; Sefton, Crit. Rev. Biomed. Eng. 14:201-40, 1987; Buchwald et ah, Surgery 88:507-16, 1980; Saudek et ah, N. Engl. J. Med. 321 :574-79, 1989). In one specific example, administration is achieved by intravenous, intradural, intracranial, intrathecal, or epidural infusion of a pharmaceutical composition using a transplanted minipump. Such minipump may be transplanted in any location that permits effective delivery of the therapeutic agent to the target site. In another embodiment, administration can be by direct injection at the site (or former site) of a tissue that is to be treated. In another embodiment, a pharmaceutical composition is delivered in a vesicle, in particular liposomes (see, e.g., Langer, Science 249: 1527-33, 1990; Treat et al, in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, N.Y., pp. 353-365, 1989).

In yet another embodiment, a pharmaceutical composition can be delivered in a controlled release system. In another embodiment, polymeric materials can be used (see, e.g., Ranger et al., Macromol. Sci. Rev. Macromol. Chem. 23:61-67, 1983; Levy et al, Science 228: 190-02, 1985; During et al, Ann. Neurol. 25:351-56, 1989;

Howard et al., J. Neurosurg. 71 :105-12, 1989). Other controlled release systems, such as those discussed in the review by Langer (Science 249: 1527-33, 1990), can also be used.

The vehicle in which a compound described herein for practicing the invention is delivered can include pharmaceutically acceptable compositions known to those of skill in the art. For instance, in some embodiments, compounds described herein for practicing the invention are contained in a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" means approved by a regulatory agency of the federal or a state government or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeia for use in animals, and, more particularly, in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the composition is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions, blood plasma medium, aqueous dextrose, and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. The medium may also contain conventional pharmaceutical adjunct materials such as for example, pharmaceutically acceptable salts to adjust the osmotic pressure, lipid carriers such as cyclodextrins, proteins such as serum albumin, hydrophilic agents such as methyl cellulose, detergents, buffers, preservatives and the like.

Examples of pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like. The pharmaceutical composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The pharmaceutical composition can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations, and the like. The pharmaceutical composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. A more complete explanation of parenteral pharmaceutical carriers can be found in Remington: The Science and Practice of Pharmacy (19 Edition, 1995) in chapter 95.

Compositions comprising a compound described herein for practicing the invention can be formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection.

The ingredients in various embodiments are supplied either separately or mixed together in unit dosage form, for example, in solid, semi-solid and liquid dosage forms such as tablets, pills, powders, liquid solutions, or suspensions, or as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water or saline can be provided so that the ingredients may be mixed prior to administration.

The amount of a compound described herein for practicing the invention that will be effective depends on the nature of the amyloid disorder or condition to be treated, as well as the stage of the disorder or condition. Effective amounts can be determined by standard clinical techniques. The precise dose to be employed in the formulation will also depend on the route of administration, and should be decided according to the judgment of the health care practitioner and each subject's circumstances. An example of such a dosage range is from about 1 mg/day to about 1000 mg/day, including about 10 mg/day, 20 mg/day, 30 mg/day, 40 mg/day, 50 mg/day, 60 mg/day, 70 mg/day, 80 mg/day, 90 mg/day 100 mg/day, 125 mg/day, 150 mg/day, 175 mg/day, 200 mg/day, 225 mg/day, 250 mg/day, 275 mg/day, 300 mg/day, 350 mg/day, 400 mg/day, 450 mg/day, 500 mg/day, 550 mg/day, 600 mg/day, 650 mg/day, 700 mg/day, 750 mg/day, 800 mg/day, 850 mg/day, 900 mg/day, 950 mg/day, 1000 mg/day, and other such values between about 1 mg/day to about 1000 mg/day, for a patient having approximately 70 kg body weight, in single or divided doses. In some particular embodiments, a target concentration of a flavone, a biguanide, a furanoflavonoid, 5-Aminoimidazole-4-carboxamide 1-β-D- ribofuranoside, trimethadione, or morin in a target cell or tissue is between 0.1 and 10 mg/kg body weight in single or divided doses.

The specific dose level and frequency of dosage for any particular subject may be varied and will depend upon a variety of factors, including the activity of the specific compound, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, and severity of the amyloid condition of the host undergoing therapy.

The compositions comprising a compound described herein for practicing the invention can be administered at about the same dose throughout a treatment period, in an escalating dose regimen, or in a loading-dose regime (for example, in which the loading dose is about two to five times the maintenance dose). In some embodiments, the dose is varied during the course of a treatment based on the condition of the subject being treated, the severity of the amyloid disease or condition, the apparent response to the therapy, and/or other factors as judged by one of ordinary skill in the art. In some embodiments long-term treatment with a disclosed compound described herein for practicing the invention is contemplated.

In another embodiment of the invention, the pharmaceutical composition comprising the therapeutically effective dose of a compound described herein for practicing the invention is administered intermittently. By "intermittent administration" is intended administration of a therapeutically effective dose of a compound described herein for practicing the invention, followed by a time period of discontinuance, which is then followed by another administration of a therapeutically effective dose, and so forth. Administration of the therapeutically effective dose may be achieved in a continuous manner, as for example with a sustained-release formulation, or it may be achieved according to a desired daily dosage regimen, as for example with one, two, three, or more administrations per day. By "time period of discontinuance" is intended a discontinuing of the continuous sustained-released or daily administration of the compound described herein for practicing the invention. The time period of discontinuance may be longer or shorter than the period of continuous sustained-release or daily administration. During the time period of discontinuance, the level of the compound in the relevant tissue is substantially below the maximum level obtained during the treatment. The preferred length of the discontinuance period depends on the concentration of the effective dose and the form of compound used. The discontinuance period can be at least two days, at least four days or at least one week. In other embodiments, the period of discontinuance is at

least one month, two months, three months, four months or greater. When a sustained-release formulation is used, the discontinuance period can be extended to account for the greater residence time of the compound. Alternatively, the frequency of administration of the effective dose of the sustained-release formulation can be decreased accordingly. An intermittent schedule of administration of a compound described herein for practicing the invention can continue until the desired therapeutic effect, and ultimately treatment of the disease or disorder, is achieved.

Amyloid Diseases As described herein, methods of the invention include a method of inhibiting the onset of symptoms of an amyloid disease in a subject, comprising administering to the subject a therapeutically effective amount of a composition comprising a compound described herein for practicing the invention. In one embodiment, the amyloid disease is a neurodegenerative disease. As used herein, "neurodegenerative disease" includes conditions that affect brain function, including conditions affecting movement, conditions affecting memory and conditions related to dementia. The area of the brain affected in a neurodegenerative disease may be the stroma including the vasculature, or the parenchyma including functional or anatomical regions, or neurons themselves. Exemplary neurodegenerative diseases include, but are not limited to, Alzheimer's disease, Parkinson's disease, Huntington's disease, and spongiform encephalopathies .

In one embodiment of the invention, the subject is a human, for example, a human at risk for Alzheimer's disease. Alzheimer's disease is a neurodegenerative disease characterized by progressive cognitive deterioration and is the most common type of dementia. The pathological process consists principally of neuronal loss or atrophy, together with an inflammatory response to the deposition of amyloid plaques and neurofibrillary tangles. The most frequent type of amyloid in the brains of subjects with Alzheimer's disease is composed primarily of Aβ peptide fibrils, β- amyloid peptide is a 39-43 amino acid peptide derived by proteolysis from a larger protein known as β- Amyloid Precursor Protein (APP). Mutations in APP result in familial forms of Alzheimer's disease, in addition to cerebral amyloid angiopathy, and senile dementia, characterized by cerebral deposition of plaques composed of Aβ fibrils and other components. The familial form of Alzheimer's disease represents only approximately 10% of the subject population. Most occurrences of Alzheimer's

disease are sporadic cases where APP and Aβ do not possess any mutation. The structure and sequence of Aβ peptides of various lengths are well known in the art. Such peptides can be made according to methods known in the art, or extracted from the brain according to known methods (see, e.g., Glenner and Wong, Biochem. Biophys. Res. Comm. 122:1131-35, 1984; Glenner and Wong, Biochem. Biophys. Res. Comm. 129:885-90, 1984). In addition, various forms of the peptides are commercially available.

Alzheimer's disease predisposing factors identified or proposed in the scientific literature include, inter alia, age {e.g., being over 40 years old, such as over 50 years old, over 60 years old, over 70 years old, over 80 years old, over 85 years old, over 90 years old, or over 95 years old), certain genotypes (e.g., presenilin-1, presenilin-2 and APP missense mutations associated with familial AD; α-2- macroglobulin and LRP-I genotypes associated with late -onset AD), environmental factors (e.g., exposure to aluminum), past history of infection by specific viral and bacterial agents (e.g., herpes simplex virus and Chlamydia pneumoniae), and certain vascular factors (e.g., hypertension and diabetes). In a further embodiment, a human subject is shown to be at risk for Alzheimer's disease by a cognitive test such as Clinical Dementia Rating (CDR), Alzheimer's Disease Assessment Scale-Cognition (ADAS-Cog), Disability Assessment for Dementia (DAD), or Mini-Mental State Examination (MMSE). The subject may exhibit a below average score on a cognitive test, as compared to a historical control of similar age and educational background. The subject may also exhibit a reduction in score as compared to previous scores of the subject on the same or similar cognition tests.

In determining the CDR, a subject is typically assessed and rated in each of six cognitive and behavioral categories: (1) memory, (2) orientation, (3) judgment and problem solving, (4) community affairs, (5) home and hobbies, and (6) personal care. The assessment may include historical information provided by the subject, or preferably, a corroborator who knows the subject well. The subject is assessed and rated in each of these areas and the overall rating, (0, 0.5, 1, 2, or 3) determined. A rating of 0 is considered normal. A subject with a CDR of 0.5 is characterized by mild consistent forgetfulness, partial recollection of events and "benign" forgetfulness, while ratings of 1, 2 and 3 correspond to mild, moderate and severe dementia, respectively. See, for example, Morris, Neurology 43:2412-14, 1993.

Another means to evaluate cognition, particularly in a subject suspected of having Alzheimer's disease, is the ADAS-Cog, or a variation termed the Standardized Alzheimer's Disease Assessment Scale (SADAS). Both tests are commonly used as an efficacy measure in clinical drug trials of Alzheimer's disease and related disorders characterized by cognitive decline. While ADAS-Cog and SADAS were not designed to diagnose Alzheimer's disease per se, they are useful in characterizing symptoms of dementia and are relatively sensitive indicators of dementia progression. See, for example, Doraiswamy, Neurology 48: 1511-17, 1997. The ADAS-cog is designed to measure, with the use of questionnaires, the progression and the severity of cognitive decline as seen in Alzheimer's disease on a 70-point scale. The ADAS-cog scale quantifies the number of wrong answers; consequently, a high score on the scale indicates a more severe case of cognitive decline. Annual deterioration in untreated Alzheimer's disease patients is approximately 8 points per year. See, for example, Raskind, J. Clin. Psychiatry 2:134-38, 2000. The DAD scale has been developed to measure a subject's ability to perform the activities of daily living, and may be assessed according to self care (i.e., dressing and personal hygiene) and instrumental activities (e.g. , housework, cooking and using household devices). The objectives of the DAD scale include quantitatively measuring functional abilities in activities of daily living in subjects with cognitive impairments and to help delineate areas of cognitive deficits that may impair performance in activities of daily living. The DAD is administered through an interview with the caregiver. It measures actual performance in activities of daily living of the subject as observed over a two week period prior to the interview. The scale assesses the following domains of activities: hygiene, dressing, telephoning, continence, eating, meal preparation, outing activities, finance and correspondence, medication use, leisure and housework. A total score is obtained by adding the rating for each question; higher scores represent less disability, while lower scores indicate more dysfunction. See, for example, Gelinas et al., Am. J. Occ. Ther. 53:471-81, 1999. The MMSE is a means to evaluate the onset of dementia and the presence of global intellectual deterioration, as seen in Alzheimer's disease and multi-infarct dementia. The MMSE is scored from 1 to 30. The MMSE does not evaluate basic cognitive potential, as, for example, does the so-called "IQ test," but rather tests intellectual skills. A person of "normal" intellectual capabilities will score a 30 on the

MMSE objective test. Scores progressively lower than 30 correspond to very mild, mild, moderate, and severe dementia, respectively. See, for example, Folstein, J. Psychiatr. Res. 12:189-98, 1975.

In another embodiment of the invention, the subject is a human, for example, a human at risk for Parkinson's disease. Parkinson's disease is a neurodegenerative disease characterized by muscle rigidity, tremor, a slowing of physical movement (bradykinesia), and in extreme cases, a loss of physical movement (akinesia). The primary symptoms are the results of decreased stimulation of the motor cortex by the basal ganglia, normally caused by the loss of pigmented dopamine-secreting (dopaminergic) cells. The mechanism by which the brain cells in Parkinson's disease are lost includes an abnormal accumulation of the protein α-synuclein bound to ubiquitin in the damaged cells. Without being bound by theory, it is believed that the α-synuclein-ubiquitin complex cannot be directed to the proteosome, due to a defect in the machinery that transports proteins between the endoplasmic reticulum and the Golgi apparatus. This protein accumulation forms proteinaceous cytoplasmic inclusions called Lewy bodies.

The Unified Parkinson's Disease Rating Scale (UPDRS) is the primary clinical tool used to assist in diagnosis and determine severity of PD, and includes the following sections: (1) Mentation, behavior, and mood; (2) Activities of daily living; (3) Motor examination; (4) Complications of therapy; (5) Modified Hoehn and Yahr Staging; and (6) Schwab and England Activities of Daily Living Scale. See, for example, Fahn S., Marsden CD., Calne D. B., and Goldstein M., eds. Recent Developments in Parkinson 's Disease, VoI 2. Macmillan Health Care Information, Florham Park, NJ, 1987, pp 153-63; 293-304.

Identification of Compounds That Inhibit Proteotoxicity

Methods of identifying compounds that inhibit aggregation-mediated proteotoxicity in a subject are also disclosed herein. Any compound that has the ability (whether or not ultimately realized) to: (1) prevent or inhibit amyloid protein assembly into insoluble fibrils; (2) facilitate clearance of pre-formed amyloid deposits; (3) slow amyloid deposition in subjects already having amyloid deposits; (4) prevent amyloid protein (soluble and/or fibrillar) from binding or adhering to a cell surface; or (5) block amyloid-induced cellular toxicity is contemplated for use in the methods of the invention.

Libraries, such as combinatorial chemical libraries, useful in the disclosed methods include, but are not limited to, analogous organic syntheses of small compound libraries (Chen et al, J. Am. Chem. Soc, 116:2661-62, 1994), oligocarbamates (Cho et al, Science, 261 :1303-05, 1993), small organic molecule libraries (see, e.g., benzodiazepines, Baum, C&EN, Jan 18, page 33, 1993; isoprenoids, U.S. Pat. No. 5,569,588; thiazolidionones and methathiazones, U.S. Pat. No. 5,549,974; pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholmo compounds, U.S. Pat. No. 5,506,337; benzodiazepines, U.S. Pat. No. 5,288,514), and the like. Libraries may include a varying number of compounds (members), such as up to about 100 members, such as up to about 1000 members, such as up to about 5000 members, such as up to about 10,000 members, such as up to about 100,000 members, such as up to about 500,000 members, or even more than 500,000 members.

A. Identification of Compounds Using C. elegans

In one embodiment, a method of identifying a compound that inhibits aggregation-mediated proteotoxicity in a subject, includes: (1) providing a first C. elegans expressing a β-amyloid peptide; (2) determining the rate of paralysis in the first C. elegans; (3) exposing a second C. elegans expressing the β-amyloid peptide to a test compound; (4) determining the rate of paralysis in the second C. elegans; and (5) comparing the rates of paralysis of the first and second C. elegans, where the test compound is identified as a compound that inhibits aggregation-mediated proteotoxicity in a subject when the rate of paralysis in the second C. elegans is less than the rate of paralysis in the first C. elegans. In a non-limiting example, the β- amyloid peptide is expressed in body wall muscles.

Transgenic C. elegans strains have been engineered to express human proteins associated with neurodegenerative diseases. These model systems include transgenic worms expressing Aβ (Alzheimer's disease; see, e.g., Link, Proc. Natl. Acad. Sd. USA 92:9368-72, 1995; Link et al, Neurobiol. Aging 24:397-413, 2003), polyglutamine repeat proteins (Huntington's disease; see, e.g., Steffan and Thompson, Expert Opin. Ther. Targets 7:201-13, 2003), and α-synuclein (Parkinson's disease; see, e.g., Vartiainen et ah, Exp. Gerontol. 41 :871-76, 2006). In these invertebrate models, many aspects of the human diseases are reproduced. For example, transgenic C. elegans strain CL2006 constitutively expresses (under the direction of the unc-54

promoter) human Aβi_ ₄₂ within body wall muscles (i.e. , those muscles below the neck), resulting in paralysis (Link, Proc. Natl. Acad. Sd. USA 92:9368-72, 1995). A similar phenotype is seen in C. elegans strain CL4176, where the expression of Aβi_ ₄₂ depends on a temperature up-shift from 16 to 23 ⁰ C (Link et al., Neurobiol. Aging 24:397-413, 2003).

As used herein, "β-amyloid peptide" includes any amyloidogenic peptide produced from APP. Native human APP is encoded by a single 400-kb gene comprised of 18 exons on chromosome 21. Alternative mRNA splicing gives rise to three APP isoforms. Two forms, APP751 and APP770 contain a Kunitz-protease inhibitor (KPI) region; the third, APP-695, lacks the KPI segment. Sequences of particular interest are those which are disease-linked. Examples of disease-linked mutations include: a mutation at codon 693 (of APP770), linked to Dutch congophilic angiopathy (Levy et al., Science 248:1124-26, 1990); a valine -isoleucine mutation at codon 717 (of APP770), linked to familial AD (Goate et al, Nature 349:704-06, 1991 ), enhanced Aβi_42 production is reported for APP with this mutation (Cai et al. , Science 259:514-16, 1993; Suzuki et al, Science 264:1335-40, 1994); a mutation wherein the valine at codon 717 is replaced by phenylalanine or glycine (Chartier- Harlin et al, Nature 353:844-46, 1991; Murrell et al, Science 254:97-99, 1991); a mutation wherein alanine is replaced by glycine at codon 692, causing both congophilic angiopathy and AD (Hendriks et al, Nature Gen. 1 :218-21, 1992); a double mutation at codons 670 and 671, resulting in a substitution of the normal lysine-methionine dipeptide by asparagine-leucine (Mullan et al, Nature Gen. 1 :345- 47, 1992), APP with this double mutation is associated with increased Aβi^o secretion (Citron et al, Nature 360:672-74, 1992; Cai et al, Science 259:514-16, 1993). In some embodiments, β-amyloid peptide is a 39-43 amino acid peptide derived by proteolysis from APP, such as, for example, Aβi_ ₄₂ .

"Determining the rate of paralysis" in C. elegans involves an assessment or measurement of muscle function in the worms as a function of time. For example, assessment or measurement of muscle function can be evaluated on a minute-by- minute basis, such as every minute, every five minutes, every ten minutes or every thirty minutes, or on an hourly basis, such as every hour, every two hours, every six hours, or every twelve hours. Assessment or measurement of muscle function can be by visual inspection or by automated means. In one embodiment, determining the rate of paralysis includes measurement of muscle function by visual inspection. In

another embodiment, determining the rate of paralysis includes measurement of muscle function by an automated system.

Measurement of muscle function in C. elegans by visual inspection is a well known procedure to those of ordinary skill in the art, and includes probing (i. e. , physically contacting) the worms, for example, with a pick, and looking for muscle contractions below the neck. In the absence of muscle contractions, the probing is generally repeated several more times (e.g., at the same and/or different locations on the worm) to ensure that there are no muscle contractions. The absence of muscle contractions indicates that the worm is paralyzed. Measurement of muscle function in C. elegans can also be automated. By "automated" is intended an assessment or measurement of muscle function by any means that reduces human operator involvement in the measurement and/or analysis of muscle function, and generally includes mechanical and/or electronic components. In one embodiment, automated measurement of muscle function in C. elegans includes measurement of Ca ²⁺ flux within muscle cells. In another embodiment, automated measurement of muscle function in C. elegans includes measurement of the rate of ATP hydrolysis within muscle cells. In yet another embodiment, automated measurement of muscle function in C. elegans includes measurement of one or more reporter proteins.

Measurement of Ca ²⁺ flux within muscle cells can be determined by methods well known to those of ordinary skill in the art, including, for example, Ca ²⁺ measurements with fluorescent indicators, such as fluorescent Ca ²⁺ indicators excited with UV light and fluorescent Ca ²⁺ indicators excited with visible light, and Ca ²⁺ measurements with bioluminescent indicators. Fluorescent probes that show a spectral response upon binding Ca ²⁺ enable investigations into changes in intracellular free Ca ²⁺ concentrations using fluorescence microscopy, flow cytometry and fluorescence spectroscopy. Exemplary UV light-excitable, ratiometric Ca ⁺ indicators include fura-2, indo-1, fluo-3, fluo-4, Calcium Green, quin-2, fura-4F, fura-5F, fura- 6F, and indo-5F (Invitrogen, Carlsbad, CA; see also, Haugland and Johnson, Intracellular Ion Indicators in Fluorescent and Luminescent Probes for Biological Activity, 2nd Ed., Mason, Ed., pp. 40-50, 1999). Exemplary visible light-excitable Ca ²⁺ indicators include fluo-3, fluo-4, rhod-2, Calcium Green, Calcium Yellow, Calcium Orange, Calcium Crimson, and the Oregon Green 488 BAPTA indicators (Invitrogen, Carlsbad, CA). Aequorin, a photoprotein originally isolated from

luminescent jellyfish and other marine organisms, is an exemplary bioluminescent Ca ⁺ indicator (Invitrogen, Carlsbad, CA).

Measurement of the rate of ATP hydrolysis within muscle cells is also determined by methods well known to those of ordinary skill in the art. For example, the rate of ATP hydrolysis can be measured with an enzymatic-coupled β-NADH fluorometric technique in which the regeneration of ATP from ADP and phospho(e«o/)pyruvate is catalyzed by pyruvate kinase (see, e.g. , Kenney et al. , Can. J. Physiol. Pharmacol. 72:1361-67, 1994). This reaction is coupled to the oxidation of NADH to NAD ⁺ and to the reduction of pyruvate to lactate; these reactions are catalyzed by lactate dehydrogenase. For each mole of ADP produced, 1 mole of NADH, a fluorescent compound, is oxidized to NAD ⁺ , a nonfluorescent compound. Thus the rate of decrease in NADH fluorescence is proportional to the rate of ATP hydrolysis by the muscle cells.

Reporter proteins can also be used for automated measurement of muscle function in C. elegans. DNA microarray analysis of worms that express Aβi_42 has revealed increased expression of several genes in response to Aβi_ ₄₂ expression. Under conditions of high Aβi_ ₄₂ proteotoxicity (i.e., paralysis), these genes are turned on, while they are turned off under conditions of low Aβi_ ₄₂ proteotoxicity (i.e., no paralysis). Reporter constructs containing the promoters of these genes operably- linked to a reporter of interest (e.g., luciferase or a fluorescent protein, such as green fluorescent protein (GFP)) can be used to monitor muscle function in an automated system. Such a system will include a means for analyzing reporter activity (as a surrogate for muscle function), such as a spectrophotometer or a fluorescence- activated cell sorter. In some embodiments, the C. elegans are deficient in the expression of one or more genes, such as, for example, one or more genes that regulate lifespan and/or youthfulness. In both mammals and worms, insulin/insulin growth factor (IGF)-I -like signaling (IIS) plays a prominent role in lifespan and youthfulness (Kenyon, Cell 120:449-60, 2005). In C. elegans, the sole insulin/IGF- 1 receptor, DAF-2, initiates the transduction of a signal that causes the phosphorylation of the FOXO transcription factor, DAF- 16, preventing its translocation to the nucleus (Lin et al., Nat. Genet. 28:139-45, 2001). This negative regulation of DAF-16 compromises expression of its target genes and shortens the worms' lifespans. Thus, knockdown ofdaf-2 expression creates long-lived, youthful worms (Kenyon et al., Nature 366:461-64, 1993). In

worms, life-span extension due to reduced daf-2 activity is also dependent upon Heat Shock Factor 1 (HSF-I), and increased expression oϊhsf-1 extends worm lifespan in a daf-16 dependent manner (Hsu et al, Science 300:1142-45, 2003).

Without being bound by theory, it is believed that two opposing mechanisms, regulated by the IIS pathway, protect worms from aggregation-mediated proteotoxicity {e.g., Aβi_ ₄₂ mediated toxicity): the HSF-I transcriptome regulates the primary proteotoxicity protection pathway, a disaggregation process coupled to proteolysis of monomorized amyloid protein {e.g., whereas the DAF-16 transcriptome regulates an active aggregation process that converts the more toxic oligomeric amyloid aggregates {e.g., Aβi_ ₄₂ aggregates) into high molecular weight aggregates of lower toxicity (Cohen et al, Science 313:1604-10, 2006).

By "deficient" in expression, is intended any statistically significant lower level of gene expression than that seen in a control, such as 10% lower, 20% lower, 30% lower, 40% lower, 50% lower, 60% lower, 70% lower, 75% lower, 80% lower, 85% lower, 90% lower, 95% lower, or 100% lower. An assessment of gene expression can be achieved by any number of methods well known in the art, including, for example, measurement of RNA levels {e.g., using Northern blotting or reverse transcription PCR), measurement of protein levels {e.g., using Western blotting, or immunohistochemistry) and measurement of protein activity {e.g., measuring the disaggregation activity of HSF-I and/or the aggregation activity of DAF-16). Methods for determining disaggregation/aggregation activities are described elsewhere herein.

Knockdown of gene expression in C. elegans can be achieved by one of ordinary skill in the art using well known methods. For example, the level and/or activity of a polypeptide may be modulated by employing a polynucleotide that is not capable of directing, in a worm, the expression of a protein or an RNA. For example, polynucleotide constructs may be designed that can be employed in methods for altering or mutating a genomic nucleotide sequence in an organism. Such polynucleotide constructs include, but are not limited to, RNA:DNA vectors, RNA:DNA mutational vectors, RNA:DNA repair vectors, mixed-duplex oligonucleotides, self-complementary RNA:DNA oligonucleotides, and recombino genie oligonucleobases. Such nucleotide constructs and methods of use are known in the art. See, e.g., U.S. Patent Nos. 5,565,350; 5,731,181; 5,756,325;

5,760,012; 5,795,972; and 5,871,984. See also, WO 98/49350, WO 99/07865, WO 99/25821, and Beetham et al, Proc. Natl. Acad. ScL USA 96:8774-78, 1999.

Many techniques for gene knockdown or silencing are well known to one of skill in the art, including, but not limited to, antisense technology (see, e.g., Sheehy et al., Proc. Natl. Acad. ScL USA 85:8805-09, 1988; and U.S. Patent Nos. 5,107,065; 5,453,566; and 5,759,829); cosuppression (see, e.g., Jorgensen, Trends Biotech. 8:340-44, 1990; Flavell, Proc. Natl. Acad. ScL USA 91 :3490-96, 1994; Finnegan et al, Bio/Technology 12:883-88, 1994; and Neuhuber et al, MoI. Gen. Genet. 244:230- 41, 1994); RNA interference (see, e.g., Napoli et al, Plant Cell 2:279-89, 1990; Sharp, Genes Dev. 13:139-41, 1999; Zamore et al, Cell 101 :25-33, 2000;

Montgomery et al, Proc. Natl. Acad. ScL USA 95: 15502-507, 1998; and U.S. Patent No. 5,034,323); virus-induced gene silencing (see, e.g., Burton et al, Plant Cell 12:691-705, 2000; and Baulcombe, Curr. Op. Plant Bio. 2: 109-13, 1999); target- RNA-specific ribozymes (see, e.g., Haseloff et al, Nature 334: 585-91, 1988); hairpin structures (see, e.g., Smith et al, Nature 407:319-20, 2000; Chuang and Meyerowitz, Proc. Natl. Acad. ScL USA 97:4985-90, 2000; Stoutjesdijk et al, Plant Physiol. 129:1723-31, 2002; Waterhouse and Helliwell, Nat. Rev. Genet. 4:29-38, 2003; Panstruga et al, MoI. Biol. Rep. 30: 135-40, 2003; Wesley et al, Plant J. 27:581-90, 2001; Wang and Waterhouse, Curr. Opin. Plant Biol 5:146-50, 2001; WO 98/53083; WO 99/53050; and WO 02/00904); ribozymes (see, e.g. , Steinecke et al, EMBO J. 11 :1525-30, 1992; and Perriman et al, Antisense Res. Dev. 3:253-63, 1993); oligonucleotide-mediated targeted modification (see, e.g., WO 03/076574 and WO 99/25853); Zn-fmger targeted molecules (see, e.g., WO 01/52620; WO 03/048345; and WO 00/42219); and other methods or combinations of the above methods known to those of skill in the art.

In a non-limiting example, RNA interference (RNAi) is used to decrease the activity of a C elegans gene, such as daf-2, hsf-1 and/or daf-16. Briefly, feeding worms bacteria expressing double-stranded RNA (dsRNA) for a gene of interest (e.g. , daf-2, hsf-1 and/or daf-16 dsRNA) decreases the activity of the gene of interest. In a further embodiment, a method of identifying a compound that inhibits aggregation-mediated proteotoxicity in a subject, includes: (1) providing a first C elegans expressing a β-amyloid peptide; (2) preparing an extract from the first C. elegans; (3) determining a disaggregation activity of the extract from the first C. elegans; (4) exposing a second C elegans expressing the β-amyloid peptide to a test

compound; (5) preparing an extract from the second C. elegans; (6) determining a disaggregation activity of the extract from the second C. elegans; and (7) comparing disaggregation activities of the extracts of the first and second C. elegans, where the test compound is identified as a compound that inhibits aggregation-mediated proteotoxicity in a subject when the disaggregation activity of the extract from the second C. elegans exceeds the disaggregation activity of the extract from the first C. elegans. In a non-limiting example, the β-amyloid peptide is expressed in body wall muscles.

By "determining a disaggregation activity" of an extract from a C. elegans is intended assessing the ability of an extract prepared from one or more worms to disaggregate amyloid fibrils, for example, β-amyloid fibrils such as Aβi_ ₄₂ fibrils. The worms can be wild-type C. elegans, worms treated with one or more test compounds and/or worms deficient in the expression of one or more genes. Methods for measuring disaggregation of amyloid fibrils are well known in the art. In one non- limiting example, post-debris supernatant (PDS) from homogenized worms is used in an in-vitro assay to measure disaggregation of amyloid fibrils (see, Cohen et ah, Science 313:1604-10, 2006).

Briefly, time dependent disaggregation of pre-aggregated amyloid fibrils in- vitro, in the presence and absence of worm PDS is quantified. The pre-aggregated amyloid fibrils {e.g., Aβi_ ₄₂ fibrils) can be labeled, for example, with a fluorescent dye, such as Thioflavin-T. Thioflavin-T is used in the fluorometric determination of amyloid fibrils in-vitro. In the absence of amyloid fibrils, the dye fluoresces faintly at the excitation and emission maxima of 350 and 438 nm, respectively. In the presence of amyloid fibrils, there is a bright fluorescence with this dye at the excitation and emission maxima of 450 and 482 nm, respectively; fluorescence change is linear from 0 to 2.0 μg/ml of amyloid fibrils. One or more protease inhibitors can be included to exclude the possibility of monitoring proteasomal and/or proteolytic degradation, rather than disaggregation. Pre- and post-disaggregation samples can also be assayed by Western blot analysis, using an amyloid-specific antibody {e.g., an amyloid-specific monoclonal antibody) to visualize fibrillar and monomelic amyloid protein.

In yet a further embodiment, a method of identifying a compound that inhibits aggregation-mediated proteotoxicity in a subject, includes: (1) providing a C. elegans expressing a β-amyloid peptide; (2) preparing a plurality of equivalent extracts from

the C. elegans; (3) determining a disaggregation activity of a first extract from the plurality of equivalent extracts; (4) exposing a second extract from the plurality of equivalent extracts to a test compound; (5) determining a disaggregation activity of the second extract; and (6) comparing disaggregation activities of the first and second extracts, where the test compound is identified as a compound that inhibits aggregation-mediated proteotoxicity in a subject when the disaggregation activity of the second extract exceeds the disaggregation activity of the first extract. In a non- limiting example, the β-amyloid peptide is expressed in body wall muscles.

In another embodiment, high throughput screening methods involve providing a combinatorial chemical library containing a large number of potential therapeutic compounds (/. e. , compounds with the potential to inhibit aggregation-mediated proteotoxicity in a subject). Such combinatorial libraries are then screened in one or more assays as described herein to identify those library members (particularly chemical species or subclasses) that display a desired characteristic activity (such as inhibiting amyloid protein assembly into insoluble fibrils or facilitating clearance of pre-formed amyloid deposits). The compounds thus identified can serve as conventional "lead compounds" or can themselves be used in potential or actual pharmaceutical compositions. In some instances, pools of candidate compounds can be identify and further screened to determine which individual or subpools of compound(s) in the collective have the desired activity.

B. Identification of Compounds Using a Non-Human Mammal

In another embodiment, a method of identifying a compound that inhibits aggregation-mediated proteotoxicity in a subject, includes: (1) providing a first mammal expressing a β-amyloid peptide; (2) preparing an extract from the first mammal; (3) determining a disaggregation activity of the extract from the first mammal; (4) exposing a second mammal expressing the β-amyloid peptide to a test compound; (5) preparing an extract from the second mammal; (6) determining a disaggregation activity of the extract from the second mammal; and (7) comparing disaggregation activities of the extracts of the first and second mammals, where the test compound is identified as a compound that inhibits aggregation-mediated proteotoxicity in a subject when the disaggregation activity of the extract from the second mammal exceeds the disaggregation activity of the extract from the first

mammal. In a non-limiting example, the β-amyloid peptide is expressed within the brain.

Transgenic non-human mammals, generally rodents, such as a mouse, have been engineered to over or under express numerous proteins, including many proteins associated with neurodegenerative diseases and animal lifespan. These models include, for example, transgenic mice expressing Familial Alzheimer's Disease (FAD)-linked presenilin 1 (PSl) and presenilin 2 (PS2) variants (Borchelt et al., Neuron 17:1005-13, 1996) and APP/APP variants (Borchelt et al. , Neuron 19:939-45, 1997), or both (Arendash et al, Brain Res. 891 :42-53, 2001), as well as IGF-I receptor knockout mice (Holzenberger et al. , Nature 421 : 182-87, 2003). A promoter from a gene expressed in a tissue of interest (e.g., the brain) in the host animal is employed for varying the phenotype of the transgenic animal.

A variety of promoter sequences can be used to control expression of coding sequences, such as APP coding sequences, in transgenic animals. These include, without limitation, the metallothionine promoter, from which expression can be regulated through modulation of zinc and glucocorticoid hormone levels (Palmiter et al., Nature 300:611-15, 1982); the rat neuron specific enolase gene promoter (Forss- Petter et al., Neuron 5:187-97, 1990); the human β-actin gene promoter (Ray et al., Genes Dev. 5:2265-73, 1991); the human platelet derived growth factor β chain gene promoter (Sasahara et al., Cell 64:217-27, 1991); the rat sodium channel gene promoter (Maue et al, Neuron 4:223-31, 1990); the human copper-zinc superoxide dismutase gene promoter (Ceballos-Picot et al, Brain Res. 552: 198-14, 1991); and promoters for members of the mammalian POU-domain regulatory gene family (Xi et al, Nature 340:35-42, 1989). The POU-domain is the region of similarity between the transcription factors Pit- 1 , Oct-1, Oct-2, and UNC-86, and represents a portion of the DNA-binding domain. These promoters provide for expression specifically within the neurons of transgenic animals.

Transgenic animals are prepared in a number of ways well known to the skilled artisan. A transgenic animal is one that has an extra or exogenous fragment of DNA in its genome. In order to achieve stable inheritance of the extra or exogenous DNA fragment, the integration event must occur in a cell type that can give rise to functional germ cells, either sperm or oocytes. Two animal cell types that can form germ cells and into which DNA can be introduced readily are fertilized egg cells and embryonic stem (ES) cells.

A exemplary method for making transgenic animals is by zygote injection, as described, for example, in U.S. Pat. No. 4,736,866. The method involves injecting DNA into a fertilized egg, or zygote, and then allowing the egg to develop in a pseudo-pregnant mother. The zygote can be obtained using male and female animals of the same strain or from male and female animals of different strains. The transgenic founder is bred to produce more animals with the same DNA insertion. In this method of making transgenic animals, the new DNA typically randomly integrates into the genome by a non-homologous recombination event. One to many thousands of copies of the DNA may integrate at one site in the genome. Another method is to use ES cells, which can be returned from in vitro culture to a "host" embryo where they become incorporated into the developing animal and can give rise to transgenic cells in all tissues, including germ cells. The ES cells are transfected in culture and then the mutation is transmitted into the germline by injecting the cells into an embryo. The animals carrying mutated germ cells are then bred to produce transgenic offspring.

In a similar fashion, knockout animals (both heterozygous knockouts and null mutants) can be produced. A exemplary method for making knockout animals uses a gene targeting vector that flanks an exon of the gene to be knocked out with a selectable cassette, such as a drug-resistance cassette {e.g. , neomycin-resistance), and a pair of loxP sites. The drug -resistance cassette provides a useful means for screening and selection of positively targeted zygotes, and both it and the exon of the gene to be knocked out can be deleted by Cre-lox recombination, producing a knockout allele (see, e.g., Holzenberger et al, Endocrinology 141 :2257-66, 2000; Holzenberger et al, Endocrinology 142:4469-78, 2001). Transgenic founder animals can be used to produce stable lines of transgenic animals that over or under express a protein of interest {e.g. , native or a mutant forms APP), or have one or more genes knocked out. Behavioral studies can be used to evaluate one or more effects mediated by the transgene, such as one or more effects mediated by native or mutant forms of APP. Exemplary behavioral studies include, but are not limited to, memory tests, such as the submerged platform and fear conditioning tests, and motor skills tests, such as the rota-rod, string agility and beam balance tests.

The submerged platform (or water maze) test is used to measure acquisition and memory retention. For purposes of analysis, a circular pool floor is divided into

four quadrants and an indiscernible platform is positioned in one of the quadrants below the water surface. Testing involves multiple trials per day over several days. In the course of daily testing, an animal is admitted successively into each of the quadrants and allowed to swim for a maximum time period (e.g., 60 seconds). Upon locating the platform (or after the maximum time period) the animal is permitted to remain on the platform for a time period (e.g., 30 seconds) prior to the next trial. Latency to find the platform for each of the trials and the average of the trials is recorded for each animal. On the day following the last day of acquisition testing, memory retention is determined in a single probe trial (e.g., 60 seconds). The submerged platform is removed from the water maze and the animal is released into the quadrant opposite that into which the submerged platform has been placed for the acquisition trials. Trials are recorded for subsequent analysis of swim path and swim speed. The percent of time spent in each quadrant is determined and statistically analyzed. See, for example, King et ah, Behav. Brain Res. 103:145-62, 1999. The fear conditioning test is also used to measure acquisition and memory retention. Briefly, the acquisition of cue fear consists of two phases, fear acquisition and testing. Conditional fear acquisition is induced by presenting audible cues (e.g. , a tone or white noise) that coterminates with mild foot shocks. Acquisition involves multiple repeats of the same cue. The time of freezing after the same cue (a consequence of fear) is recorded during the next several days; fear depends upon memory of the electric shocks. See, for example, King et ah, Behav. Brain Res. 103:145-62, 1999 and Cain et al, J. Neurosci. 22:9113-21, 2002.

The rota-rod test, for evaluation of motor skills, is performed essentially as described by Lynch et al. (Brain Res. 83:249-59, 1975). Briefly, an animal is required to run on a treadmill (e.g., Rota-Rod, Ugo Basile, Comerio, Italy) for a set period of time (e.g., 3-5 minutes) to maintain its position on top of a rod revolving at a set or accelerating speed. The protocol ends when the animal falls, or when the time has expired (in which case the animal is put back on the rod each time it falls). Total time on the rod or total number of falls are recorded and statistically analyzed. The string agility test is also a well known method of evaluating motor skills.

In order to measure forepaw grip capacity and agility, string (e.g., cotton string) is tautly suspended above a padded surface. Initially, an animal is permitted to grasp the string only by the forepaws, and then is released. In the course of a trial period (e.g., 60 seconds), a rating system is used to assess each animal, 0: animal unable to remain

on string following release; 1 : animal hangs by two forepaws for approximately 60 seconds; 2: animal attempts to climb string; 3: animal places two forepaws and one or both hindpaws around string; 4: animal places four paws and tail around string with lateral movement; and 5: animal escapes. See, for example, King et ah, Behav. Brain Res. 103:145-62, 1999.

An additional well known test for evaluating motor skills is the beam balance test. A narrow dowel beam {e.g. , about 1 cm wide) is fixed between two support columns above a padded surface. At either end of the beam is attached a platform. Each animal is administered several trials {e.g., three) during a single day of testing. The animal is placed in a perpendicular orientation at the center of the beam and released for a trial interval period {e.g., 60 seconds). The time required for the animal to fall from the beam is noted for each of the several trials. Alternatively, if the animal remains on the beam for the duration of the trial period, or escapes to either platform, the maximum interval period is recorded. The score for each trial, the average of the several trials and the number of escapes are recorded for each animal. See, for example, King et al, Behav. Brain Res. 103: 145-62, 1999.

Transgenic animals are also observed clinically to determine, inter alia, the age of onset of one or more effects mediated by the transgene, the duration of the one or more effects, the penetrance of the phenotype, and systemic or regional organ dysfunction {e.g., brain dysfunction). As discussed herein, an assessment of gene expression can be achieved by any number of methods well known in the art, including, for example, measurement of RNA levels {e.g., using Northern blotting or reverse transcription PCR), measurement of protein levels {e.g., using Western blotting, or immunohistochemistry) and measurement of protein activity. Various changes in phenotype are of interest. For example, in transgenic animals with modified APP expression, these changes may include progressive neurologic disease in the cortico -limbic areas of the brain expressed within a short period of time from birth, increased levels of expression of an APP gene above endogenous expression levels (generally accompanied by the development of a neurologic illness and premature death), gliosis and intracellular APP/Aβ accretions present in the hippocampus and cerebral cortex, diminished 2-deoxyglucose uptake/utilization and hypertrophic gliosis in the cortico-limbic regions of the brain, progressive neurologic disease characterized by diminished exploratory/locomotor behavior, and impaired performance on memory and learning tests.

Regions known to be affected by the amyloid disease of interest (e.g., the brain) are particularly reviewed for changes. When the amyloid disease of interest is AD, the regions reviewed include the cortico -limbic region, including APP/Aβ excretions, gliosis, changes in glucose uptake and utilization, and Aβ plaque formation. However, in strains of animals which are not long-lived, either naturally or when expressing high levels of APP, not all behavioral and/or pathological changes associated with a particular amyloid disease may be observed. For example, transgenic FVB/N mice expressing high levels of APP tend not to develop detectable Aβ plaques, whereas longer lived C57B6/SJL Fl mice expressing identical transgenes do develop amyloid plaques which are readily detected with Thioflavin-T and Congo red. Immunologic studies of various brain regions also are used to detect transgene product.

As used herein, the singular terms "a," "an" and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description. The following examples are offered by way of illustration and not by way of limitation.

EXPERIMENTAL

Example 1 : Identifying Compounds That Inhibit Aggregation-Mediated Proteotoxicity

Identification of compounds that inhibit aggregation-mediated proteotoxicity was carried out using transgenic C. elegans strain CL2006, which constitutively expresses human Aβi_ ₄₂ within body wall muscles under the direction of the unc-54 promoter, resulting in paralysis (Link, Proc. Natl. Acad. ScL USA 92:9368-72, 1995).

Paralysis Screening Bleaching and Plating

C. elegans (transgenic strain CL2006) were decontaminated and disrupted using bleach. After the final rinse of M-9 solution (42 mM Na ₂ HPO ₄ , 22 mM

KH ₂ PO ₄ , 86 mM NaCl, 1 mM MgSO ₄ ) the unhatched eggs were left in about 10 ml of M-9 solution. If an unusually high concentration of eggs were present, then they were split into two conical tubes, each having 10 ml total M-9 solution. The tube(s) was placed on a rocker in a 15 ⁰ C fridge. Twenty- four hours after placing on the rocker, the conical tubes were removed from the fridge, and inspected under a microscope for hatched worms. A centrifuge was used to pellet the worms, and all but 3 ml of the M-9 solution was removed; the worms were resuspended by shaking the tube a couple of times.

Worms were spotted onto primary plates (previously spotted with E. coli OP50) in 50 μl aliquots, taking care that the plates remained flat and that the entire worm aliquot landed within the spot of OP50 already on the plates. Once the worms reached the Ll stage, the plates were placed into the 15 ⁰ C fridge, again taking care to keep the plates flat.

Fileting

Thirty-seven hours (35 hours if the plates contained DMSO) after placing the Ll -stage worms into the fridge, the plates were removed from for fileting. Ten worms each were fϊleted onto ten plates (100 worms total) of the corresponding compound to be screened, or a corresponding control. Only worms that were moving normally were fϊleted. After all lifespans had been fϊleted, the plates were placed in a 25 ⁰ C fridge simultaneously.

Compounds screened included: 3',4',-dihydroxy-B-napthoflavone; 3,5,7- trihydroxy-3',4',5'-trimethoxyflavone; 3,6-dimethoxyflavone; 3,7-dihydroxyflavone; 3,7-dimethoxyflavone; 3-hydroxyflavone; 4'-hydroxy-alpha-naphthoflavone; 4'- methoxy-alpha-naphthoflavone; 5,7,4'-trimethoxyflavone; 5,7-dimethoxyflavone; 6,7- dihydroxyflavone; 6-ethoxy-3(4'-hydroxyphenyl)-4-methylcoumarin; 6-hydroxy-7- methoxyflavone; 7,8-Benzoflavone; 7,8-dimethoxyflavone; 7-diethylamino-3(4'- methoxyphenyl)coumarin; 7-hydroxy-3-methylflavone; 7-hydroxyflavone; AICAR; apigenin; baicalein; baicalein-5,6,7-trimethylether; carnitine; coenzyme QlO; dapson;

deprenyl; dehydroepiandrosterone (DHEA); dienestrol; flavone; genistein; glycetin; ibuprofen; karanjin; liquid nicotine; luteolin; metformin; morin; myricetin; naproxen; naringenin; nicotinic acid; alpha-phenyl-N-tert-butylnitrone (PBN); phenformin; phloretin; quercetin dihydrate; rutin trihydrate; sulfuretin; syringetin; tacrine; taxifolin; TMD; and DL-α-tocopherol.

Counting

Twenty hours (22 hours if the plates contained DMSO) after being placed in the 25 ⁰ C fridge, the plates were removed for observation and scoring of the worms. When paralysis of a worm was suspected, a pick was used for probing. When no muscle contractions behind the neck were observed, the worm was removed from the spot of OP50 and placed onto an area of clean agar for additional probing. If there was still no muscle contraction behind the neck, the worm was scored as "paralyzed," removed from the plate and flamed. Any worms that exhibited muscle contractions were scored as "alive." Plates were monitored for a total of eight hours, or until all worms were paralyzed. Selected results are shown in Table I.

"C" - control

All publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.