BACKGROUND Somatostatin is a hormone with broad physiological activity including regulating other endocrine factors, cell growth, angiogenesis, and gut motility. Somatostatin exerts its action through 5 receptor subclasses (SSTR1-5). The SSTRs are members of the family of G-protein coupled receptors. Modulation of one or more subclasses of these receptors has therapeutic utility in a wide range of disease conditions.

One somatostatin analogue is the drug octreotide (Novartis), which is largely SSTR2 selective, with much less potency at SSTR5 and even less potency at SSTR3 in humans and rodents. Octreotide is FDA approved for acromegaly and palliation of neuroendocrine turnors.

SUMMARY Downregulation of the GH/IGF-1 axis can modulate the rate of aging. It is important for a wide range of disease settings. Although octreotide is effective for downregulating this axis from super-physiological levels, it is less effective for decreasing GH and IGF-1 levels from their baseline levels. In a wide range of disease conditions including diabetic retinopathy, diabetic nephropathy, and obesity, it is desirable to control this pathway over a broader dynamic range. A somatostatin agonist, e. g. , an agonist with selectivity for SSTR1 and/or SSTR2, can be used to downregulate IGF and GH levels and therefore has efficacy in treating diseases such as diabetic retinopathy, diabetic neuropathy, a neurodegenerative disorder such as Huntington's disease, obesity, and other disorders described herein.

Reducing GH/IGF-l/Insulin activity (i. e. , increasing forkhead and HSF-1 transcriptional activity) is beneficial in a broad spectrum of diseases. Experimental and clinical observations suggest that this can be achieved in mammals with somatostatin receptor (SSTR) agonists.

SSTR subtype-selective compounds can obtain the desired decreased activity through the GH/IGF-1/Insulin axis in rodents.

treating diseases such as diabetic retinopathy, diabetic neuropathy, a neurodegenerative disorder such as Huntington's disease, obesity, and other disorders described herein.

In one aspect, the invention features a method of treating or preventing a diabetic retinopathy with a modulator of SSTR, e. g. , an agonist of SSTR, e. g. , an agonist of SSTR1, 2,3, 4, or 5, e. g., SSTR1.

In one aspect, the invention features a method of treating or preventing a diabetic neuropathy with a modulator of SSTR, e. g. , an agonist of SSTR, e. g. , an agonist of SSTR1, 2,3, 4, or 5, e. g., SSTR1.

In one aspect, the invention features a method of treating or preventing obesity with a modulator of SSTR, e. g. , an agonist of SSTR, e. g. , an agonist of SSTR1, 2,3, 4, or 5, e. g., SSTR1.

In one embodiment, a disorder is hypothalamic obesity. For example, the compound can be administered to a subject identified as at risk for hypothalamic obesity or to a subject that has an abnormal (e. g. , extreme) insulin response to glucose.

In one aspect, the invention features a method of treating or preventing a neurodegenerative disorder (e. g. , a disorder caused at least in part by polyglutamine aggregation (such as Huntington's disease), Alzheimer's disease, Parkinson's diseases and so forth) with a modulator of SSTR, e. g. , an agonist of SSTR, e. g. , an agonist of SSTR1, 2,3, 4, or 5, e. g., SSTR1.

In another aspect, the invention features a method that includes evaluating a compound for its ability to modulate a disorder described herein, e. g. , by providing an animal or cellular model of the disorder, administering the compound to the animal or cell, and evaluating a parameter of the animal or cell, e. g. , a parameter associated with the disorder. Application U. S. S. N. 10/656,530 includes a description of some exemplary models.

In another aspect, the invention features a method that includes selecting a compound based on a somatostatin-like property (e. g. , ability to interact, agonize, or antagonize a SSTR, e. g., SSTR1, 2,3, 4, or 5), and evaluating a compound for its ability to modulate a disorder described herein, e. g. , by providing an animal or cellular model of the disorder, administering the compound to the animal or cell, and evaluating a parameter of the animal or cell, e. g. , a parameter associated with the disorder. U. S. S. N. 10/656,530 includes a description of some exemplary models.

In another aspect, the invention features a method that includes selecting a compound based on a somatostatin-like property (e. g. , ability to interact, agonize, or antagonize a SSTR,

e. g., SSTR1, 2,3, 4, or 5), and administering the compound to the animal or cell, and evaluating a parameter of the animal or cell, e. g. , a parameter associated with a disorder, e. g. ,. a disorder described herein.

In one embodiment, an agonist is used to treat a disorder described in U. S. S. N.

10/656,530 for other disease or disorder of aging.

In another aspect, the disclosure features a method of evaluating a compound for ability to modulate somatostatin receptor (SSTR) in a cell, the method including: providing a test compound; contacting the test compound to SSTR (e. g., SSTR 1, 2,5) ; evaluating interaction between the-test compound and SSTR ; contacting the test compound to a cell; and evaluating the activity and/or levels of GH/IGF-1 axis components in the cell.

In one embodiment, the GH/IGF-1 axis component is the forkhead or HSF-1 transcription factor. In one embodiment, evaluating interaction between the test compound and SSTR includes selecting an agonist of SSTR. In one embodiment, evaluating the activity of the GH/IGF-1 axis component includes evaluating productions of GH or IGF-1.

The method can include other features described herein.

In another aspect, the disclosure features a method for evaluating compounds for ability to modulate SSTR in a cell The method includes: providing a library of compounds; contacting each compound of the library to SSTR; evaluating interaction between each compound and SSTR; selecting a subset of compounds from the library based on the evaluated interactions; and for each compound of the subset, contacting the compound to a cell and evaluating the activity and/or levels of GH/IGF-1 axis components in the cell. In one embodiment, the GH/IGF-1 axis component is the forkhead or HSF-1 transcription factor.

The method can include other features described herein.

In another aspect, the disclosure features a method of evaluating a compound for ability to modulate SSTR in an organism, the method including: providing a test compound; contacting the test compound to SSTR; evaluating interaction between the test compound and SSTR; administering the test compound to a subject organism; and evaluating the subject organism for activity or levels of GH/IGF-1 axis components. In one embodiment, evaluating interaction between the test compound and SSTR includes selecting an agonist of SSTR, e. g. , an agonist of at least one, two, or more SSTR subtypes, or a particular combination of receptor subtypes (e. g. , 2 and 5, or 1 and 5). In one embodiment, evaluating the

subject organism includes evaluating IGF-1 levels in blood or sera. The method can further include evaluating activity of a pharmacodynamic marker.

In one embodiment, the GH/IGF-1 axis component is the forkhead or HSF-1 transcription factor.

The method can include other features described herein.

In another aspect, the disclosure features a method of evaluating a compound for ability to modulate SSTR in an organism. The method includes: providing a test compound; contacting the test compound to SSTR; evaluating interaction between the test compound and SSTR; administering the test compound to a subject organism; and evaluating the subject organism for pharmacodynamic markers, e. g. , one or more of GH, IGF-1, insulin, leptin, ghrelin, blood glucose, blood corticosterone, free fatty acids in the blood, blood cholesterol and HDL/LDL ratios, and blood triglycerides.

The method can include other features described herein.

In another aspect, the disclosure features a method for evaluating compounds for ability to modulate SSTR in an organism, the method including: providing a library of compounds; contacting each compound of the library to SSTR; evaluating interaction between each compound and SSTR; selecting a subset of compounds from the library based on the evaluated interactions; administering one or more compounds from the subset to a subject organism; and evaluating the subject organism for levels of GH/IGF-1 axis components.

For example, the GH/IGF-1 axis component is the forkhead or HSF-1 transcription factor. The method can include other features described herein.

In another aspect, the disclosure features a method for evaluating compounds for ability to modulate SSTR in an organism. The method includes: providing a library of compounds; contacting each compound of the library to SSTR (e. g. , an SSTR described herein); evaluating interaction between each compound and SSTR; selecting a subset of compounds from the library based on the evaluated interactions; administering compounds from the subset to a subject organism; and evaluating the subject organism for pharmacodynamic markers. Compounds can be selected that modulate at least two SSTRs, e. g. , 2 and 5 or 1 and 5.

Compounds can be administered individual or in pools to a test organism, e. g. , a rodent or other model organism described herein. Exemplary pharmacodynamic markers include one or more of : GH, IGF-1, insulin, leptin, ghrelin, blood glucose, blood corticosterone, free fatty acids in the blood, blood cholesterol and HDL/LDL ratios, and blood triglycerides.

In another aspect, the disclosure features a method of treating or preventing obesity in a subject, e. g. , a human, the method including administering an agonist of SSTR. For example, the obesity is hypothalamic obesity. Exemplary agonists include somatostatin, octreotide, or SOM 230. Other agonists can be used, alternatively or in combination. In one embodiment, the agonist modulates activity of at least two different SSTR subtypes. For example, the first and second SSTR subtypes are subtypes 2 and 5. In another example, the first and second SSTR subtypes are subtypes 1 and 5. The agonist (the first agonist) can modulate activity of a first SSTR subtype, and method further includes administering a second agonist that modulates activity at least a second SSTR subtype. For example, the first and second SSTR subtypes are subtypes 2 and 5. In another example, the first and second SSTR subtypes are subtypes 1 and 5.

The method can include other features described herein.

In another aspect, the disclosure features a method of treating or preventing an aggregative neurodegenerative disorder, the method including administering an agonist of SSTR.

Exemplary agonists include somatostatin, octreotide, or SOM 230. Other agonists can be used, alternatively or in combination. In one embodiment, the agonist modulates activity of at least two different SSTR subtypes. For example, the first and second SSTR subtypes are subtypes 2 and 5. In another example, the first and second SSTR subtypes are subtypes 1 and 5. The agonist (the first agonist) can modulate activity of a first SSTR subtype, and method further includes administering a second agonist that modulates activity at least a second SSTR subtype.

For example, the first and second SSTR subtypes are subtypes 2 and 5. In another example, the first and second SSTR subtypes are subtypes 1 and 5.

The method can include other features described herein.

In another aspect, the disclosure features a method of treating or preventing a diabetic retinopathy or diabetic nephropathy. The method including administering an agonist of SSTR.

Exemplary agonists include somatostatin, octreotide, or SOM 230. Other agonists can be used, alternatively or in combination. In one embodiment, the agonist modulates activity of at least two different SSTR subtypes. For example, the first and second SSTR subtypes are subtypes 2 and 5. In another example, the first and second SSTR subtypes are subtypes 1 and 5. The agonist (the first agonist) can modulate activity of a first SSTR subtype, and method further includes administering a second agonist that modulates activity at least a second SSTR subtype.

For example, the first and second SSTR subtypes are subtypes 2 and 5. In another example, the first and second SSTR subtypes are subtypes 1 and 5.

The method can include other features described herein.

In another aspect, the disclosure features a method of treating or preventing acromegaly, the method including administering an agonist of SSTR.

Exemplary agonists include somatostatin, octreotide, or SOM 230. Other agonists can be used, alternatively or in combination. In one embodiment, the agonist modulates activity of at least two different SSTR subtypes. For example, the first and second SSTR subtypes are subtypes 2 and 5. In another example, the first and second SSTR subtypes are subtypes 1 and 5.

The agonist (the first agonist) can modulate activity of a first SSTR subtype, and method further includes administering a second agonist that modulates activity at least a second SSTR subtype.

For example, the first and second SSTR subtypes are subtypes 2 and 5. In another example, the first and second SSTR subtypes are subtypes 1 and 5.

The GH/IGF-1 axis includes a series of extracellular and intracellular signalling components that have as a downstream target, the transcription factor Forkhead. The components can be divided into three categories: pre-IGF-1, IGF-1, and post-IGF-1 components."Pre-IGF-1 components"include GH, GHS, GHS-R, GHRH, GHRH-R, SST, and SSTR."Post-IGF-1 components"include IGF-1-R and intracellular signalling components including PI (3) kinase, PTEN phosphatase, PI (3,4) P2,14-3-3 protein, and PI (3,4, 5) P3 phosphatidyl inositol kinases, AKT serine/threonine kinase (e. g., AKT-1, AKT-2, or AKT-3), or a Forkhead transcription factor (such as FOXO-1, FOXO-3, or FOXO-4).

A"core component of the IGF-1 Receptor signalling pathway"refers to a component that is one of the following: (i) the IGF-1 receptor (IGF1R), (ii) a Forkhead transcription factor that responds to IGF1R signalling, or (iii) a protein that participates in signal transduction between IGF1R and the Forkhead transcription factor. Examples of proteins that participate in this signal transduction include PI (3) kinase, PTEN phosphatase, PI (3,4) P2, and PI (3,4, 5) P3 phosphatidyl inositol kinases, PDK-1 (3-phosphoinositide-dependent kinase-1), and AKT serine/threonine kinase (e. g., AKT-1, AKT-2, or AKT-3).

A"somatotroph axis signalling pathway component"refers to a protein that is one of the following: (i) a protein that is located in a somatotroph and that regulates GH release by the somatotroph, or (ii) a protein that directly binds to a protein in class (i). Exemplary somatotroph axis signalling pathway components of class (i) include cell surface receptors such as GHS-R, GHRH-R, and SSTR. Exemplary somatotroph axis signalling pathway components of class (ii) include GHRH, Ghrelin, and SST. As described herein, compounds that function analogously to SSTR agonists can be used instead of, or in conjunction with, a SSTR agonist. Such compounds

include other compounds that agonize the somatotroph axis signalling pathway. Somatostatin agonists include agonists of SSTR proteins.

The details of one or more embodiments of the invention are set forth in the accompa- nying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and the claims. This application incorporates U. S. S. N. 10/656, 530, filed September 5,2003, by reference in its entirety for all purposes. The contents of all references, pending patent applications and published patents, cited throughout this application are hereby expressly incorporated by reference.

DESCRIPTION OF DRAWINGS FIG. 1 is a schematic of an exemplary method for evaluating compounds.

DETAILED DESCRIPTION The GH/IGF-1/Insulin pathway regulates aging and is highly conserved through evolution. The longevity phenotype of the pathway was initially discovered in C. elegans, where certain loss of function mutations in Daf-2 (the worm orthologue of IGF-lReceptor/Insulin Receptor) increase life span by over two-fold. Long-lived Daf-2 mutant animals are resistant to hypoxia and toxicity from bacterial pathogens ; Daf-2 mutants are also resistant to protein aggregation and associated toxicities as a result of mutant huntingtin expression. Both the longevity and"disease"resistance phenotyp, es of Daf-2 mutants require two transcription factors: Daf-16, a member of the forkhead family, and heat shock factor-1 (HSF-1), which activates expression of genes encoding protection and repair functions, e. g. , anti-oxidants, chaperonins (small heat shock proteins), anti-microbial peptides. Mutations in genes encoding proteins within the GH/IGF-I/Insulin pathway in rodents also increase life span. In addition, the long- lived mutant rodents are resistant to a variety of diseases such as cancers, nephropathy and cognitive decline.

Useful somatostatin agonists include agents that are orally available and agents that have a somatostatin receptor subtype specificity that differs from the specificity of octreotide. For example, a compound that has specificity for SSTR1 can be useful since subtype 1 agonist activity may augment subtype 2 activity to lower IGF-1, inhibit angiogenesis and protect against diabetic end organ damage.

Somatostatin agonists can be used to modulate or treat metabolic disease, e. g. , diabetes and disorders characterized by multi-hormonal imbalances, hyperinsulinemia (primary focus to date), dysregulation of GH, IGF-1 system, and dysregulation of other important

autocrine/paracrine factors (e. g. VEGF, FGFs). Diabetes can cause, e. g. , progressive damage to eyes, kidneys, nerves, & arteries and presents major health problems.

Somatostatin agonists can be used to modulate or treat diabetic retinopathy. Diabetic retinopathy is a frequent end-organ complication of diabetes. It can cause of blindness among working age individuals. Despite aggressive conventional management of hyperglycemia, incidence of diabetic retinopathy remains extremely high, suggesting hyperglycemia is not lone culprit. Beyond glycemic control, photocoagulation remains only accepted treatment for diabetic retinopathy.

GH is thought to impact diabetic retinopathy by increasing IGF-1 production. Hypo- pituitary individuals with diabetes are protected from diabetic end organ damage. However, insulin in humans can acutely worsen the disease. Similarly, IGF-1R & InsR knock-out mice are resistant to proliferative retinopathy. IGF-1 (insulin) inhibits PEDF and activates VEGF (vascular epithelium growth factor). It can induces proliferation of retinal endothelium. PEDF (pigment epithelium derived factor) can maintain retinal vasculature in quiescent state, e. g. , by putting a"brake"on VEGF & other proliferative factors. Retinopathy may develop when the PEDF brake fails.

Somatostatin agonists can be used to modulate or treat diabetic retinopathy, e. g. , by lowering IGF-1 (directly & indirectly, through GH), and also by means that are independent of IGF-1. IGF-1 can function by modulate activity of receptors found in the eye, e. g. , the retina.

IGF-1 inhibits VEGF production and can maintain PEDF (the VEGF"brake"). It also has anti- fibrotic activity andr educes fibrovascular formations implicated in retinopathy Diabetic nephropathy is characterized by glomerular hyperfiltration, endothelial dysfunction, and mesangial cell hypertrophy. IGF-1 is significant in pathogenesis ; dysfunction can correlates with local IGF-1 levels. GH/IGF-1 deficient diabetic rats are protected against nephropathy, as are hypo-pituitary humans. The kidney expresses SSTR subtypes 1 & 2.

Somatostatin agonists (including octreotide) have a protective renal effect in non-obese diabetic mice Somatostatin may protect kidney by local & systemic reduction of IGF-1 Somatostatin agonists can be used to modulate or treat obesity. Hyperinsulinemia & insulin resistance are symptoms of obesity. Insulin is a primary mediator of adipogenesis through glut4, acetyl-CoA carboxylase, and fatty acid synthase enzymes. Acute glucose- stimulated insulin hypersecretion is predictive of future weight gain.

Somatostatin decreases insulin secretion in response to a meal. This decrease is mediate, at least in part, by subtype 5 mediated in rodents and subtype 2. In humans, octreotride may

normalize initial hyperglycemia in few days with lessened insulin requirement in diabetics.

Somatostatin agonists given chronically may inhibit insulin's adipogenic effect. Somatostain agonist can be used to modulate or treat subjects with hypothalamic ("ablative obesity") and non- ablative obesity.

A general strategy for targeting a somatostatin-associate disorder can include: evaluating optimal subtype specificity (ies) for the particular disease indication; and preclinical validation of compounds; identifying compound (s) for lead optimization.

Useful compounds, some of which are SSTR modulators, include: octreotide (e. g., available from Novartis), SOM 230 (e. g. , available from Novartis), cyclic hexapeptide SST1, 2,3, 5 agonist, and pegvisomant (e. g. , available from Pharmacia/Pfizer) is a pegylated GH variant and functions as a competitive GH antagonist Screening Assays A test compound can be evaluated, e. g. , to assess interaction with an SSTR protein (e. g., in a cell or in a cell-free system, e. g. , micelle) and for other properties, e. g. , ability to interact with and/or modulate the GH/IGF-1 axis or a GH/IGF-1 component. The evaluation can identify agonists of SSTR, e. g. , an SSTR of a particular subtype. Methods include in vitro and ira vivo assays. Interactions include, for example, binding a target component, altering a covalent bond in a target component, or altering a biological or physiological function of a target compound (e. g. , altering production, stability, or degradation of a target component). A test compound that modulates an SSTR (e. g. , a somatostatin agonist) can be prepared as a pharmaceutical composition (see below) and administered to a subject.

The test compounds can be obtained, for example, as described above (e. g. , based on information about an agonist of an SSTR protein) or using any of the numerous combinatorial library method. Some exemplary libraries include: biological libraries; peptoid libraries (libraries of molecules having the functionalities of peptides, but with a novel, non-peptide backbone which are resistant to enzymatic degradation but which nevertheless remain bioactive; see, e. g., Zuckermann, R. N. et al. (1994) J. Med. Chez. 37: 2678-85) ; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the'one-bead one-compound'library method; and synthetic library methods using affinity chromatography selection. These approaches can be used, for example, to produce peptide, non-peptide oligomer or small molecule libraries of compounds (see, e. g., Lam (1997) Anticancer Drug Des. 12: 145).

A biological library includes polymers that can be encoded by nucleic acid. Such encoded polymers include polypeptides and functional nucleic acids (such as nucleic acid aptamers (DNA, RNA), double stranded RNAs (e. g., RNAi), ribozymes, and so forth). The biological libraries and non-biological libraries can be used to generate peptide libraries.

Another example of a biological library is a library of dsRNAs (e. g. , siRNAs), or precursors thereof. A library of nucleic acids that can be processed or transcribed to produce double- stranded RNAs (e. g. , siRNAs) ) is also featured.

Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad. Sci. U. S. A. 90: 6909; Erb et al. (1994) Proc.

Natl. Acad. Sci. USA 91: 11422; Zuckermann et al. (1994). J. Med Chem. 37: 2678; Cho et al.

(1993) Science 261: 1303; Carrell et al. (1994) Angew. Chem. Int. Ed. Engl. 33: 2059; Carell et al.

(1994) Angew. Chem. Int. Ed. Engl. 33 : 2061 ; and Gallop et al. (1994) J. Med. Chez. 37 : 1233.

Libraries of compounds may be presented in solution (e. g. , Houghten (1992) Biotechniques 13: 412-421), or on beads (Lam (1991) Nature 354: 82-84), chips (Fodor (1993) Nature 364: 555-556), bacteria (Ladner, U. S. Patent No. 5,223, 409), spores (Ladner U. S. Patent No. 5,223, 409), plasmids (Cull et al. (1992) Proc Natl Acad Sci USA 89 : 1865-1869) or on phage (Scott and Smith (1990) Science 249: 386-390 ; Devlin (1990) Science 249: 404-406; Cwirla et al.

(1990) Proc. Natl. Acad. Sci. 87: 6378-6382; Felici (1991) jMoLBioL 222: 301-310; Ladner supra.). In many cases, a high throughput screening approach to a library of test compounds includes one or more assays, e. g. , a combination of assays. Information from each assay can be stored in a database, e. g. , to identify candidate compounds that can serve as leads for optimized or improved compounds, and to identify SARs.

Cell-Based Assays. In one embodiment, a cell-based assay is used to evaluate a test compound. The cell, for example, can be of mammalian origin, (e. g. , from a human, a mouse, rat, primate, or other non-human), or of non-mammalian origin (e. g. , Xenopus, zebrafish, or an invertebrate such as a fly or nematode). In some cases, the cell can be obtained from a transgenic organism, e. g. , an organism which includes a heterologous GH/IGF-1 axis component, (e. g. , from a mammal, e. g. , a human), e. g. , a heterologous SSTR protein. A heterologous SSTR can be expressed from a recombinant nucleic acid that is introduced into the cell.

In one example, a cell which expresses a GH/IGF-1 axis protein or polypeptide or biologically active portion thereof is contacted with a test compound, and the ability of the test compound to modulate the GH/IGF-1 axis is determined. Determining the ability of the test

compound to modulate the GH/IGF-1 axis can be accomplished by monitoring, for example, GH and/or IGF-1 levels, e. g., by radioimmunoassay. For example, the assay can include evaluate GH or IGF-1 synthesis and release. The specific example provided below can also be used, as well as similar assay that detect competition between the test compound and another compound that binds to the receptor (e. g. , somatostatin or an analogue thereof).

It is also possible to monitor an intracellular component of the GH/IGF-1 axis, e. g., abundance, activity or post-translational modification state of a PI (3) Kinase, a phosphatase (e. g., PTEN), a phosphoinositol kinase; or a serine-threonine kinase (e. g., an AKT kinase). Changes in post-translational modification can be monitored using modification specific antibodies, changes in electrophoretic mobility, and mass spectroscopy, for example.

Another exemplary cellular assay includes contacting a hormone responsive cell with a hormone (e. g. , somatostatin, GH or IGF-1) in the presence of the test compound and evaluating a parameter (e. g. , a qualitative or quantitative property) of the cell (e. g. , expression of one or a profile of genes, abundance of one or more proteins, and so forth). Alteration of the parameter relative to a control cell or a reference parameter (e. g. , a reference value) indicates that the test compound can modulate the responsiveness of the cell.

Still other cell-based assays including contacting cells with the test compound and evaluating resistance to a stress, for example, hypoxia, DNA damage (genotoxic stress), or oxidative stress. For example, it is possible to determine whether hypoxia-mediated cell death is attenuated by the test compound.

Cell-Free Assays. In addition to cell-based assays, cell-free assays can also be used. In one example, the ability of the test compound to modulate interaction between a first GH/IGF-1 axis component and a second axis component is evaluated, e. g. , interaction between GH and the GH receptor or GHRH and the GHRH receptor. This type of assay can be accomplished, for example, by coupling one of the components, with a radioisotope or enzymatic label such that binding of the labeled component to the other GH/IGF-1 axis component can be determined by detecting the labeled compound in a complex. A GH/IGF-1 axis component can be labeled with 125I, 35S, 14C, or 3H, either directly or indirectly, and the radioisotope detected by direct counting of radioemmission or by scintillation counting. Alternatively, a component can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.

Competition assays can also be used to evaluate a physical interaction between a test compound and a target. For example, Pong et al.. (1996) Mol Endocrinol 10: 57 describes an assay which detects the displacement of a radiolabeled MK-0677 molecule from pituitary membranes.

In yet another embodiment, a cell-free assay is provided in which a GH/IGF-1 axis protein or biologically active portion thereof is contacted with a test compound and the ability of the test compound to bind to the GH/IGF-1 axis protein or biologically active portion thereof is evaluated. Preferred biologically active portions of the GH/IGF-1 axis proteins to be used in assays of the present invention include fragments which participate in interactions with non- GH/IGF-1 axis molecules, e. g. , an ectodomain of a cell surface receptor, a cytoplasmic domain of a cell surface receptor, a kinase domain, and so forth.

Soluble and/or membrane-bound forms of isolated proteins (e. g. , GH/IGF-1 axis components and their receptors or biologically active portions thereof) can be used in the cell- free assays of the invention. When membrane-bound forms of the protein are used, it may be desirable to utilize a solubilizing agent. Examples of such solubilizing agents include non-ionic detergents such as n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N- methylglucamide, decanoyl-N-methylglucamide, Triton X-100, Triton X-114, Thesit, Isotridecypoly (ethylene glycol ether) n, 3- [ (3-cholamidopropyl) dimethylamminio]-l-propane sulfonate (CHAPS), 3- [ (3-cholamidopropyl) dimethylamminio]-2-hydroxy-1-propane sulfonate (CHAPSO), or N-dodecyl=N, N-dimethyl-3-amnonio-1-propane sulfonate. In another example, the axis component can reside in a membrane, e. g. , a liposome or other vesicle.

Cell-free assays involve preparing a reaction mixture of the target protein (e. g. , the GH/IGF-1 axis component) and the test compound under conditions and for a time sufficient to allow the two components to interact and bind, thus forming a complex that can be removed and/or detected.

The interaction between two molecules can also be detected, e. g. , using a fluorescence assay in which at least one molecule is fluorescently labeled. One example of such an assay includes fluorescence energy transfer (FET or FRET for fluorescence resonance energy transfer) (see, for example, Lakowicz et al., U. S. Patent No. 5,631, 169; Stavrianopoulos, et al., U. S.

Patent No. 4,868, 103). A fluorophore label on the first,'donor'molecule is selected such that its emitted fluorescent energy will be absorbed by a fluorescent label on a second,'acceptor' molecule, which in turn is able to fluoresce due to the absorbed energy. Alternately, the'donor' protein molecule may simply utilize the natural fluorescent energy of tryptophan residues.

Labels are chosen that emit different wavelengths of light, such that the'acceptor'molecule label may be differentiated from that of the'donor'. Since the efficiency of energy transfer between the labels is related to the distance separating the molecules, the spatial relationship between the molecules can be assessed. In a situation in which binding occurs between the molecules, the fluorescent emission of the'acceptor'molecule label in the assay should be maximal. A FET binding event can be conveniently measured through standard fluorometric detection means well known in the art (e. g. , using a fluorimeter).

Another example of a fluorescence assay is fluorescence polarization (FP). For FP, only one component needs to be labeled. A binding interaction is detected by a change in molecular size of the labeled component. The size change alters the tumbling rate of the component in solution and is detected as a change in FP. See, e. g. , Nasir et al. (1999) Comb Chem HTS 2 : 177- 190; Jameson et al. (1995) Methods Enzymol 246: 283; Seethala et al.. (1998) Anal Biochem.

255: 257. Fluorescence polarization can be monitored in multiwell plates, e. g. , using the TECAN POLARIONTM reader. See, e. g. , Parker et al. (2000) Journal ofBiomolecular Screening 5: 77- 88 ; and Shoeman, et al. (1999) 38,16802-16809.

In another embodiment, determining the ability of the GH/IGF-1 axis component protein to bind to a target molecule can be accomplished using real-time Biomolecular Interaction Analysis (BIA) (see, e. g. , Sjolander, S. andUrbaniczky, C. (1991) Anal. Che7al. 63: 2338-2345 and Szabo et al. (1995) Curr. Opin. Struct. Biol. 5: 699-705). "Surface plasmon resonance"or "BIA"detects biospecific interactions in real time, without labeling any of the interactants (e. g., BIAcore). Changes in the mass at the binding surface (indicative of a binding event) result in alterations of the refractive index of light near the surface (the optical phenomenon of surface plasmon resonance (SPR) ), resulting in a detectable signal which can be used as an indication of real-time reactions between biological molecules.

In one embodiment, the axis component is anchored onto a solid phase. The axis component/test compound complexes anchored on the solid phase can be detected at the end of the reaction, e. g. , the binding reaction. For example, the axis component can be anchored onto a solid surface, and the test compound, (which is not anchored), can be labeled, either directly or indirectly, with detectable labels discussed herein.

It may be desirable to immobilize either the GH/IGF-1 axis component or an anti- GH/IGF-1 axis component antibody to facilitate separation of complexed from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay.

Binding of a test compound to a GH/IGF-1 axis component protein, or interaction of a GH/IGF-1

axis component protein with a second component in the presence and absence of a candidate compound, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix. For example, glutathione-S-transferase/GH/IGF-1 axis component fusion proteins or glutathione-S-transferase/target fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtiter plates, which are then combined with the test compound or the test compound and either the non-adsorbed target protein or GH/IGF-1 axis component protein, and the mixture incubated under conditions conducive to complex formation (e. g. , at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of GH/IGF-1 axis component binding or activity determined using standard techniques.

Other techniques for immobilizing either a GH/IGF-1 axis component protein or a target molecule on matrices include using conjugation of biotin and streptavidin. Biotinylated GH/IGF-1 axis component protein or target molecules can be prepared from biotin-NHS (N- hydroxy-succinimide) using techniques known in the art (e. g. , biotinylation kit, Pierce Chemicals, Rockford, IL), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical).

In order to conduct the assay, the non-immobilized component is added to the coated surface containing the anchored component. After the reaction is complete, unreacted components are removed (e. g. , by washing) under conditions such that any complexes formed will remain immobilized on the solid surface. The detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the previously non-immobilized component is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the previously non-immobilized component is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface, e. g. , using a labeled antibody specific for the immobilized component (the antibody, in turn, can be directly labeled or indirectly labeled with, e. g. , a labeled anti-Ig antibody).

In one embodiment, this assay is performed utilizing antibodies reactive with a GH/IGF-1 axis component protein or target molecules but which do not interfere with binding of the

GH/IGF-1 axis component protein to its target molecule. Such antibodies can be derivatized to the wells of the plate, and unbound target or the GH/IGF-1 axis component protein trapped in the wells by antibody conjugation. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the GH/IGF-1 axis component protein or target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the GH/IGF-1 axis component protein or target molecule.

Alternatively, cell free assays can be conducted in a liquid phase. In such an assay, the reaction products are separated from unreacted components, by any of a number of standard techniques, including but not limited to: differential centrifugation (see, for example, Rivas, G., and Minton, A. P. , (1993) Trends Biochem Sci 18: 284-7); chromatography (gel filtration chromatography, ion-exchange chromatography); electrophoresis (see, e. g. , Ausubel, F. et al., eds. Current Protocols in Molecular Biology 1999, J. Wiley: New York. ) ; and immunoprecipitation (see, for example, Ausubel, F. et al., eds. (1999) Current Protocols in Molecular Biology, J. Wiley: New York). Such resins and chromatographic techniques are known to one skilled in the art (see, e. g. , Heegaard, N. H. , (1998) JMol Recognit 11: 141-8 ; Hage, D. S. , and Tweed, S. A. (1997) J Chromatogr B Biomed Sci Appl. 699: 499-525). Further, fluorescence energy transfer may also be conveniently utilized, as described herein, to detect binding without further purification of the complex from solution.

In a preferred embodiment, the assay includes contacting the GH/IGF-1 axis component protein or biologically active portion thereof with a known compound which binds a GH/IGF-1 axis component to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a GH/IGF-1 axis component protein, wherein determining the ability of the test compound to interact with the GH/IGF-1 axis component protein includes determining the ability of the test compound to preferentially bind to the GH/IGF-1 axis component or biologically active portion thereof, or to modulate the activity of a target molecule, as compared to the known compound.

The target products of the invention can, n vivo, interact with one or more cellular or extracellular macromolecules, such as proteins. For the purposes of this discussion, such cellular and extracellular macromolecules are referred to herein as"binding partners. "Compounds that disrupt such interactions can be useful in regulating the activity of the target product. Such compounds can include, but are not limited to molecules such as antibodies, peptides, and small molecules. The preferred targets/products for use in this embodiment are the GH/IGF-1 axis

components. In an alternative embodiment, the invention provides methods for determining the ability of the test compound to modulate the activity of a GH/IGF-1 axis component protein through modulation of the activity of a downstream effector of a GH/IGF-1 axis component target molecule. For example, the activity of the effector molecule on an appropriate target can be determined, or the binding of the effector to an appropriate target can be determined, as previously described.

To identify compounds that interfere with the interaction between the target product and its cellular or extracellular binding partner (s), a reaction mixture containing the target product and the binding partner is prepared, under conditions and for a time sufficient, to allow the two products to form complex. In order to test an inhibitory agent, the reaction mixture is provided in the presence and absence of the test compound. The test compound can be initially included in the reaction mixture, or can be added at a time subsequent to the addition of the target and its cellular or extracellular binding partner. Control reaction mixtures are incubated without the test compound or with a placebo. The formation of any complexes between the target product and the cellular or extracellular binding partner is then detected. The formation of a complex in the control reaction, but not in the reaction mixture containing the test compound, indicates that the compound interferes with the interaction of the target product and the interactive binding partner.

Additionally, complex formation within reaction mixtures containing the test compound and normal target product can also be compared to complex formation within reaction mixtures containing the test compound and mutant target product. This comparison can be important in those cases wherein it is desirable to identify compounds that disrupt interactions of mutant but not normal target products.

These assays can be conducted in a heterogeneous or homogeneous format.

Heterogeneous assays involve anchoring either the target product or the binding partner onto a solid phase, and detecting complexes anchored on the solid phase at the end of the reaction. In homogeneous assays, the entire reaction is carried out in a liquid phase. In either approach, the order of addition of reactants can be varied to obtain different information about the compounds being tested. For example, test compounds that interfere with the interaction between the target products and the binding partners, e. g. , by competition, can be identified by conducting the reaction in the presence of the test substance. Alternatively, test compounds that disrupt preformed complexes, e. g. , compounds with higher binding constants that displace one of the components from the complex, can be tested by adding the test compound to the reaction mixture after complexes have been formed. The various formats are briefly described below.

In a heterogeneous assay system, either the target product or the interactive cellular or extracellular binding partner, is anchored onto a solid surface (e. g. , a microtiter plate), while the non-anchored species is labeled, either directly or indirectly. The anchored species can be immobilized by non-covalent or covalent attachments. Alternatively, an immobilized antibody specific for the species to be anchored can be used to anchor the species to the solid surface.

In order to conduct the assay, the partner of the immobilized species is exposed to the coated surface with or without the test compound. After the reaction is complete, unreacted components are removed (e. g. , by washing) and any complexes formed will remain immobilized on the solid surface. Where the non-immobilized species is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the non-immobilized species is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e. g. , using a labeled antibody specific for the initially non-immobilized species (the antibody, in turn, can be directly labeled or indirectly labeled with, e. g. , a labeled anti-Ig antibody). Depending upon the order of addition of reaction components, test compounds that inhibit complex formation or that disrupt preformed complexes can be detected.

Alternatively, the reaction can be conducted in a liquid phase in the presence or absence of the test compound, the reaction products separated from unreacted components, and complexes detected; e. g. , using an immobilized antibody specific for one of the binding components to anchor any complexes formed in solution, and a labeled antibody specific for the other partner to detect anchored complexes. Again, depending upon the order of addition of reactants to the liquid phase, test compounds that inhibit complex or that disrupt preformed complexes can be identified.

In an alternate embodiment of the invention, a homogeneous assay can be used. For example, a preformed complex of the target product and the interactive cellular or extracellular binding partner product is prepared in that either the target products or their binding partners are labeled, but the signal generated by the label is quenched due to complex formation (see, e. g., U. S. Patent No. 4,109, 496 that utilizes this approach for immunoassays). The addition of a test substance that competes with and displaces one of the species from the preformed complex will result in the generation of a signal above background. In this way, test substances that disrupt target product-binding partner interaction can be identified.

In another embodiment, modulators of a GH/IGF-I axis component gene expression are identified. For example, a cell or cell free mixture is contacted with a candidate compound and the expression of the GH/IGF-I axis component mRNA or protein evaluated relative to the level

of expression of GH/IGF-I axis component mRNA or protein in the absence of the candidate compound. When expression of the GH/IGF-I axis component mRNA or protein is greater in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of GH/IGF-I axis component mRNA or protein expression. Alternatively, when expression of the GH/IGF-I axis component mRNA or protein is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of the GH/IGF-I axis component mRNA or protein expression. The level of the GH/IGF-I axis component mRNA or protein expression can be determined by methods for detecting GH/IGF-I axis component mRNA or protein.

Animal Models Still other methods for evaluating a test compound include organismal based assays, e. g., using a mammal (e. g. , a mouse, rat, primate, or other non-human), or other animal (e. g., Xenopus, zebrafish, or an invertebrate such as a fly or nematode). In some cases, the organism is a transgenic organism, e. g. , an organism which includes a heterologous GH/IGF-1 axis component, (e. g. , from a mammal, e. g. , a human). The test compound can be administered to the organism once or as a regimen (regular or irregular). A parameter of the organism is then evaluated, e. g. , an age-associated parameter or a parameter of the GH/IGF-1 axis. Test compounds that are indicated as of interest result in a change in the parameter relative to a reference, e. g. , a parameter of a control organism. Other parameters (e. g. , related to toxicity, clearance, and pharmacokinetics) can also be evaluated.

In some embodiment, the test compound is evaluated using an animal that has a particular disorder, e. g. , a disorder described herein, e. g. , an age-related disorder, a geriatric disorder, a neoplastic disorder, a non-neoplastic disorder, a metabolic disorder, an immunological disorder, a neurological disorder, a dermatological disorder, a dermatological tissue condition, or a cardio- vascular disorder. These disorders can also provide a sensitized system in which the test compound's effects on physiology can be observed. Exemplary disorders include: denervation, disuse atrophy; metabolic disorders (e. g. , disorder of obese and/or diabetic animals such as db/db mouse and ob/ob mouse); cerebral, liver ischemia; cisplatin/taxol/vincristine models; various tissue (xenograph) transplants; transgenic bone models; Pain syndromes (include inflammatory and neuropathic disorders); Paraquat, genotoxic, oxidative stress models; pulmonary obstruction (e. g. , asthma models); and tumor models. In a preferred embodiment, the animal model is an animal that has an altered phenotype when calorically restricted. For example, F344 rats provide

a useful assay system for evaluating a test compound. When calorically restricted, F344 rats have a 0 to 10% incidence of nephropathy. However, when fed ad libitum, they have a 60 to 100% incidence of nephropathy. See Table 2.

Table 1: F344 rats-Frequency of nephropathy.

Months Ad lib CR 6 0% 0% 12 60% 0% 18 100% 0% 24100% 0% Additional animals are listed in Table 2: Exemplary Animal Models: Table 2: Exemplary Animal Models Mean Lifespan (months) Model Ad lib CR Predisposition SH Rat 18 30 Hypertension SA Mouse 10 15 Amyloid NZB Mouse 12 16 SLE kdlkd Mouse 8 18 Nephritis MRL/1 Mouse 6 >15 Autoimmune oblob Mouse 14 26 Diabetes To evaluate a test compound, it is administered to the animal (e. g. , an F344 rat or an animal listed in Table 3), and a parameter of the animal is evaluated, e. g. , after a period of time.

The animal can be fed ad libitum or normally (e. g. , not under caloric restriction, although some parameters can be evaluated under such conditions). Typically, a cohort of such animals is used for the assay. Generally, a test compound can be indicated as favorably altering lifespan regulation in the animal if the test compound affects the parameter in the direction of the phenotype of a similar animal subject to caloric restriction. Such test compounds may cause at least some of the lifespan regulatory effects of caloric restriction, e. g. , a subset of such effects, without having to deprive the organism of caloric intake.

In one embodiment, the parameter is an age-associated or disease associated parameter, e. g. , a symptom of the disorder associated with the animal model (e. g. , the disorder in the "Predisposition column of Table 3). For example, the test compound can be administered to the

SH Rat, and blood pressure is monitored. A test compound that is favorably indicated can cause an amelioration of the symptom relative to a similar reference animal not treated with the compound. In a related embodiment, the parameter is a parameter of the GH/IGF-1 axis. In some embodiment, a parameter relevant to a disorder or to aging can include: antioxidant levels (e. g, . antioxidant enzyme levels or activity), stress resistance (e. g. , paraquat resistance), core body temperature, glucose levels, insulin levels, thyroid-stimulating hormone levels, prolactin levels, and leutinizing hormone levels.

Still other in vivo models and organismal assays include evaluating an animal for a metabolic parameter, e. g. , a parameter relevant to an insulin disorder. Exemplary metabolic parameters include: glucose concentration, insulin concentration, leptin concentration, ghrelin concentration, corticosterone concentration, and insulin sensitivity. Another set of metabolic parameters are parameters associated with the function of the growth hormone (GH)/insulin-like growth factor (IGF-1) axis, e. g. , GH concentration, IGF-1 concentration, GHS concentration, and so forth.

In assessing whether a test compound is capable of inhibiting the GH/IGF-1 axis a number of parameters or biomarkers can be monitored or evaluated. Exemplary parameters include: (i) presence or abundance of active forkhead transcription factors in nervous tissues, which can be detected by analysis of the phosphorylation states of the forkhead proteins FOXO1, 3a, and 4; (ii) presence or abundance of gene transcripts or gene product in the cell or organism that depends on IGF-1 and HSF-1 (exemplary products include the heat shock proteins hsp27, hsp40, hsp70, hsp90, and lisp 104) ; (iii) resistance of the cell or organism to stress; (iv) one or more metabolic parameters of the cell or organism (exemplary parameters include blood IGF-1 concentration, circulating insulin levels, blood glucose levels; fat content; core body temperature and so forth); (v) proliferative capacity of the cell or a set of cells present in the organism; and (vi) physical appearance or behavior of the cell or organism.

Test compounds can be administered to animals over extended periods of time to determine if the changes in pharmacodynamic markers and transcription factor activation can be sustained. In addition, animals can be monitored for signs of overt toxicity, such as excessive weight loss (cachexia), lethargy, change in coat, ptosis, lowered body temperature, urticaria and conjunctivitis.

In another embodiment, a marker associated with the GH/IGF-1 axis is evaluated in a subject organism of a screening assay (or a treated subject). Although these markers may not be

age-associated, they may be indicative of a physiological state that is altered when the GH/IGF-1 axis is modulated.

Cellular models derived from cells of an animal described herein or analogous to an animal model described herein can be used for a cell-based assay.

Exemplary animal models that can be used to evaluate aspects of Alzheimer's disease and neurodegenerative disorders that are caused at least in part by polyglutamine aggregation are provided below.

Cells and animals for evaluating the effect of a compound on ALS status include a mouse which has an altered SOD gene, e. g. , a SOD1-G93A transgenic mouse which carries a variable number of copies of the human G93A SOD mutation driven by the endogenous promoter, a SOD1-G37R transgenic mouse (Wong et al., Neuron, 14 (6): 1105-16 (1995) ) ; SOD1-G85R transgenic mouse (Bruijn et al., Neuron, 18 (2): 327-38 (1997) ) ; C. elegant strains expressing mutant human SOD1 (Oeda et al., Hum Mol Genet. , 10: 2013-23 (2001)) ; and a Drosophila expressing mutations in Cu/Zn superoxide dismutase (SOD). (Phillips et al., Proc. Natl. Acad.

Sci. U. S. A. , 92: 8574-78 (1995) and McCabe, Proc. Natl. Acad. Sci. U. S. A. , 92: 8533-34 (1995)).

Exemplary animal models of Parkinson's disease include primates rendered parkinsonian by treatment with the dopaminergic neurotoxin 1-methyl-4 phenyl 1,2, 3,6-tetrahydropyridine (MPTP) (see, e. g. , US Appl 20030055231 and Wichmann et al. , Ann. N. Y. Acad. Sci. , 991: 199- 213 (2003); 6-hydroxydopamine-lesioned rats (e. g., Lab. Anim. Sci. , 49: 363-71 (1999) ) ; and transgenic invertebrate models (e. g. , Lakso et al. , J. Neurochem. , 86: 165-72 (2003) and Link, Mech. Ageing Dev. , 122: 1639-49 (2001)).

Exemplary molecular models of Type II diabetes include: a transgenic mouse having defective Nix-2. 2 or Nkx-6.1 ; (US 6,127, 598); Zucker Diabetic Fatty fa/fa (ZDF) rat. (US 6569832); and Rhesus monkeys, which spontaneously develop obesity and subsequently frequently progress to overt type 2 diabetes (Hotta et al., Diabetes, 50: 1126-33 (2001); and a transgenic mouse with a dominant-negative IGF-I receptor (KR-IGF-IR) having Type 2 diabetes- like insulin resistance.

Exemplary animal and cellular models for neuropathy include: vincristine induced sensory-motor neuropathy in mice (US Appl 5420112) or rabbits (Ogawa et al. , Neurotoxicology, 21: 501-11 (2000) ) ; a streptozotocin (STZ) -diabetic rat for study of autonomic neuropathy (Schmidt et al., Am. J. Pathol. , 163: 21-8 (2003) ) ; and a progressive motor neuropathy (pmn) mouse (Martin et al. , Genomics, 75: 9-16 (2001) ). In one embodiment, the animal is a transgenic mouse that can express (in at least one cell) a human Huntingtin protein, a portion thereof, or

fusion protein comprising human Huntingtin protein, or a portion thereof, with, for example, at least 36 glutamines (e. g. , encoded by CAG repeats (alternatively, any number of the CAG repeats may be CAA) in the CAG repeat segment of exon 1 encoding the polyglutamine tract).

An example of such a transgenic mouse strain is the R6/2 line (Mangiarini et al. Cell 87 : 493-506 (1996) ). The R6/2 mice are transgenic Huntington's disease mice, which over-express exon one of the human HD gene (under the control of the endogenous promoter). The exon 1 of the R6/2 human HD gene has an expanded CAG/polyglutamine repeat lengths (150 CAG repeats on average). These mice develop a progressive, ultimately fatal neurological disease with many features of human Huntington's disease. Abnormal aggregates, constituted in part by the N- terminal part of Huntingtin (encoded by HD exon 1), are observed in R6/2 mice, both in the cytoplasm and nuclei of cells (Davies et al. Cell 90: 537-548 (1997) ). For example, the human Huntingtin protein in the transgenic animal is encoded by a gene that includes at least 55 CAG repeats and more preferably about 150 CAG repeats.

These transgenic animals can develop a Huntington's disease-like phenotype. These transgenic mice are characterized by reduced weight gain, reduced lifespan and motor impairment characterized by abnormal gait, resting tremor, hindlimb clasping and hyperactivity from 8 to 10 weeks after birth (for example the R6/2 strain; see Mangiarini et al. Cell 87: 493- 506 (1996) ). The phenotype worsens progressively toward hypokinesia. The brains of these transgenic mice also demonstrate neurochemical and histological abnormalities, such as changes in neurotransmitter receptors (glutamate, dopaminergic), decreased concentration of N- acetylaspartate (a marker of neuronal integrity) and reduced striatum and brain size.

Accordingly, evaluating can include assessing parameters related to neurotransmitter levels, neurotransmitter receptor levels, brain size and striatum size. In addition, abnormal aggregates containing the transgenic part of or full-length human Huntingtin protein are present in the brain tissue of these animals (e. g. , the R6/2 transgenic mouse strain). See, e. g. , Mangiarini et al. Cell 87: 493-506 (1996), Davies et al. Cell 90: 537-548 (1997), Brouillet, Functional Neurology 15 (4): 239-251 (2000) and Cha et al. Proc. Natl. Acad. Sci. USA 95: 6480-6485 (1998).

To test the effect of the test compound or known compound described in the application in an animal model, different concentrations of test compound are administered to the transgenic animal, for example by injecting the test compound into circulation of the animal.

In one embodiment, a Huntington's disease-like symptom is evaluated in the animal. For example, the progression of the Huntington's disease-like symptoms, e. g. as described above for the mouse model, is then monitored to determine whether treatment with the test compound

results in reduction or delay of symptoms. In another embodiment, disaggregation of the Huntingtin protein aggregates in these animals is monitored. The animal can then be sacrificed and brain slices are obtained. The brain slices are then analyzed for the presence of aggregates containing the transgenic human Huntingtin protein, a portion thereof, or a fusion protein comprising human Huntingtin protein, or a portion thereof. This analysis can includes, for example, staining the slices of brain tissue with anti-Huntingtin antibody and adding a secondary antibody conjugated with FITC which recognizes the anti-Huntingtin's antibody (for example, the anti-Huntingtin antibody is mouse anti-human antibody and the secondary antibody is specific for human antibody) and visualizing the protein aggregates by fluorescent microscopy.

Alternatively, the anti-Huntingtin antibody can be directly conjugated with FITC. The levels of Huntingtin's protein aggregates are then visualized by fluorescent microscopy.

Somatostatin Agonists Somatostatin and somatostatin agonists can be used to downregulate the GH/IGF-1 axis.

As used herein a"somatostatin agonist"is a compound that has at least one biological function of somatostatin and that can alter regulation of the GH/IGF-1 axis.

One useful somatostatin agonist is L-054,522. See, e. g. , Pasternak et al. (1999) Bioorganic & Medicinal Chemistry Letters which also provides L-054,522 related compounds with improved bioavailability ; and Yang et al. (1998) ProcNatAcadUSA 95 : 10836. L-054,522 binds to human SST2 with an apparent Kd of 0.01 nM and is highly selective. One exemplary L- 054,522 compound has the following structure: Other useful somatostatin agonists include BIM-23244, BIM-23197, BIM-23268, octreotide, TT-232, butreotide, lanreotide, and vapreotide. The compound SOM230 is described, e. g. , in Eur J Endocrinol. 2002 May; 146 (5): 707-16. Octreotide and lanreotide are currently approved for treatment of acromegaly. These bind the receptors on the anterior pituitary gland and function to lower the production and secretion of GH.

Somatostatin is a hypothalamic factor that, among other biological functions, suppresses the secretion of GH from the anterior pituitary. It is produced by a large number of tissues. Due to its rapid degradation and clearance, somatostatin is not a truly circulating hormone. It is produced locally to its site of function, presumably to prevent inappropriate activation of receptors in tissues throughout the body. In developing drugs that mimic somatostatin, a key goal is to increase its stability thus extending its circulating half-life. In one embodiment, a somatostatin analog has local tissue specificity. For example, it may bind a subset of the five distinct receptor subtypes that bind to somatostatin, particularly the SST2 or SST5 receptors.

EP01492 (Cortistatin 8), is a somatostatin antagonist which has been shown to inhibit feeding in animal studies. EP01492 is an 8 amino acid peptide somatostatin analogue. See generally, e. g. , WO 03/004518 and WO 02/08250.

Additional exemplary somatostatin agonists are described in U. S. 6,342, 479 Somatostatin agonists which can be used to practice the therapeutic method of the present invention include, but are not limited to, those covered by the formulae or those specifically recited in the publications set forth below: EP Application No. P5 164 EU (Inventor: G. Keri); Van Binst, G. et al. Peptide Research 5: 8 (1992); Horvath, A. et al. Abstract, "Conformations of Somatostatin Analogs Having Antitumor Activity", 22nd European peptide Symposium, Sep. 13-19,1992, Interlaken, Switzerland; PCT Application WO 91/09056 (1991); EP Application 0 363 589 A2 (1990); U. S.

Pat. No. 4,904, 642 (1990); U. S. Pat. No. 4,871, 717 (1989); U. S. Pat. No. 4,853, 371 (1989); U. S.

Pat. No. 4,725, 577 (1988); U. S. Pat. No. 4, 684, 620 (1987) U. S. Pat. No. 4,650, 787 (1987); U. S.

Pat. No. 4,603, 120 (1986); U. S. Pat. No. 4,585, 755 (1986); EPApplication 0 203 031 A2 (1986); U. S. Pat. No. 4,522, 813 (1985) ; U. S. Pat. No. 4,486, 415 (1984); U. S. Pat. No. 4, 485, 101 (1984); U. S. Pat. No. 4,435, 385 (1984); U. S. Pat. No. 4,395, 403 (1983) ; U. S. Pat. No. 4,369, 179 (1983); U. S. Pat. No. 4,360, 516 (1982); U. S. Pat. No. 4,358, 439 (1982); U. S. Pat. No. 4,328, 214 (1982) ; U. S. Pat. No. 4,316, 890 (1982); U. S. Pat. No. 4,310, 518 (1982); U. S. Pat. No. 4,291, 022 (1981) ; U. S. Pat. No. 4,238, 481 (1980); U. S. Pat. No. 4,235, 886 (1980); U. S. Pat. No. 4,224, 190 (1980); U. S. Pat. No. 4,211, 693 (1980); U. S. Pat. No. 4,190, 648 (1980) ; U. S. Pat. No. 4,146, 612 (1979); and U. S. Pat. No. 4,133, 782 (1979).

Examples of somatostatin agonists include, but are not limited to, the following somatostatin analogs which are disclosed in the above-cited references:

Tyr-D-Trp-Lys-Val-Cys-Thr-NH2 ; H-D-Phe-Cys-Tyr-D-Trp-Lys-Abu-Cys-Thr-NH2 ; H-D-Phe- Cys-Tyr-D-Trp-Lys-Abu-Cys-p-Nal-NH2 ; H-pentafluoro-D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys- Thr-NH2 ; Ac-D-ß-Nal-Cys-pentafluoro-Phe-D-Trp-Lys-Val-Cys-Thr-NH2 ; H-D-ß-Nal-Cys-Tyr- D-Trp-Lys-Val-Cys-ß-Nal-NH2 ; H-D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-p-Nal-NH2 ; H-D-ß-Nal- Cys-Tyr-D-Trp-Lys-Abu-Cys-Thr-NH2 ; H-D-p-Cl-Phe-Cys-Tyr-D-Trp-Lys-Abu-Cys-Thr-NH2 ; Ac-D-p-Cl-Phe-Cys-Tyr-D-Trp-Lys-Abu-Cys-Thr-NH2 ; H-D-Phe-Cys-p-Nal-D-Trp-Lys-Val- Cys-Thr-NH2 ; H-D-Phe-Cys-Tyr-D-Trp-Lys-Cys-Thr-NH2 ; cyclo (Pro-Phe-D-Trp-N-Me-Lys- Thr-Phe) ; cyclo (Pro-Phe-D-Trp-N-Me-Lys-Thr-Phe) ; cyclo (Pro-Phe-D-Trp-Lys-Thr-N-Me-Phe) ; cyclo (N-Me-Ala-Tyr-D-Trp-Lys-Thr-Phe) ; cyclo (Pro-Tyr-D-Trp-Lys-Thr-Phe) ; cyclo (Pro-Phe- D-Trp-Lys-Thr-Phe) ; cyclo (Pro-Phe-L-Trp-Lys-Thr-Phe) ; cyclo (Pro-Phe-D-Trp (F)-Lys-Thr- Phe) ; cyclo (Pro-Phe-Trp (F)-Lys-Thr-Phe) ; cyclo (Pro-Phe-D-Trp-Lys-Ser-Phe) ; cyclo (Pro-Phe- D-Trp-Lys-Thr-p-Cl-Phe) ; cyclo (D-Ala-N-Me-D-Phe-D-Thr-D-Lys-Trp-D-Phe) ; cyclo (D-Ala-N- Me-D-Phe-D-Val-Lys-D-Trp-D-Phe) ; cyclo (D-Ala-N-Me-D-Phe-D-Thr-Lys-D-Trp-D-Phe) ; cyclo (D-Abu-N-Me-D-Phe-D-Val-Lys-D-Trp-D-Tyr) ; cyclo (Pro-Tyr-D-Trp-t-4-AchxAla-Thr- Phe) ; cyclo (Pro-Phe-D-Trp-t-4-AchxAla-Thr-Phe) ; cyclo (N-Me-Ala-Tyr-D-Trp-Lys-Val-Phe) ; cyclo (N-Me-Ala-Tyr-D-Trp-t-4-AchxAla-Thr-Phe) ; cyclo (Pro-Tyr-D-Trp-4-Amphe-Thr-Phe) ; cyclo (Pro-Phe-D-Trp-4-Amphe-Thr-Phe) ; cyclo (N-Me-Ala-Tyr-D-Trp-4-Amphe-Thr-Phe) ; cyclo (Asn-Phe-Phe-D-Trp-Lys-Thr-Phe-Gaba) ; cyclo (Asn-Phe-Phe-D-Trp-Lys-Thr-Phe-Gaba- Gaba) ; cyclo (Asn-Phe-D-Trp-Lys-Thr-Phe) ; cyclo (Asn-Phe-Phe-D-Trp-Lys-Thr-Phe-NH (CH2) 4 CO) ; cyclo (Asn-Phe-Phe-D-Trp-Lys-Thr-Phe-p-Ala) ; cyclo (Asn-Phe-Phe-D-Trp-Lys-Thr-Phe- D-Glu)-OH ; cyclo (Phe-Phe-D-Trp-Lys-Thr-Phe) ; cyclo (Phe-Phe-D-Trp-Lys-Thr-Phe-Gly) ; cyclo (Phe-Phe-D-Trp-Lys-Thr-Phe-Gaba) ; cyclo (Asn-Phe-Phe-D-Trp-Lys-Thr-Phe-Gly) ; cyclo (Asn-Phe-Phe-D-Trp (F)-Lys-Thr-Phe-Gaba) ; cyclo (Asn-Phe-Phe-D-Trp (NO2)-Lys-Thr- Phe-Gaba) ; cyclo (Asn-Phe-Phe-Trp (Br)-Lys-Thr-Phe-Gaba) ; cyclo (Asn-Phe-Phe-D-Trp-Lys- Thr-Phe (I)-Gaba) ; cyclo (Asn-Phe-Phe-D-Trp-Lys-Thr-Tyr (But)-Gaba) ; cyclo (Bmp-Lys-Asn- Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-Pro-Cys)-OH ; cyclo (Bmp-Lys-Asn-Phe-Phe-D-Trp-Lys-Thr- Phe-Thr-Pro-Cys)-OH ; cyclo (Bmp-Lys-Asn-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-Tpo-Cys)-OH ; cyclo (Bmp-Lys-Asn-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-MeLeu-Cys)-OH ; cyclo (Phe-Phe-D-Trp- Lys-Thr-Phe-Phe-Gaba) ; cyclo (Phe-Phe-D-Trp-Lys-Thr-Phe-D-Phe-Gaba) ; cyclo (Phe-Phe-D- Trp (5F)-Lys-Thr-Phe-Phe-Gaba) ; cyclo (Asn-Phe-Phe-D-Trp-Lys (Ac)-Thr-Phe-NH-- (CH2) 3-- CO) ; cyclo (Lys-Phe-Phe-D-Trp-Lys-Thr-Phe-Gaba) ; cyclo (Lys-Phe-Phe-D-Trp-Lys-Thr-Phe- Gaba) ; cyclo (Orn-Phe-Phe-D-Trp-Lys-Thr-Phe-Gaba) ; and H-Cys-Phe-Phe-D-Trp-Lys-Thr-Phe- Cys-NH2 (BIM-23268).

Pharmaceutical Compositions and Administration A compound that modulates the GH/IGF-1 axis, e. g. , an agonist of an SSTR protein, can be incorporated into a pharmaceutical composition for administration to a subject, e. g. , a human, a non-human animal, e. g., an animal patient (e. g. , pet or agricultural animal) or an animal model (e. g. , an animal model for aging or a metabolic disorder (e. g. , a disorder of the GH/IGF-1 axis, a pancreatic or insulin related disorder, or other disorder described herein). Such compositions typically include the a small molecule (e. g. , a small molecule that is an SSTR agonist), nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier. As used herein the language"pharmaceutically acceptable carrier"includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions.

A pharmaceutical composition is formulated to be compatible with its intended route of administration. 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. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and

liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

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.

For administration by inhalation, the compounds are 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.

Systemic administration 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.

The 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.

In one embodiment, the active compounds are prepared with carriers that will protect the compound 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. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to particular cells, e. g. , a pituitary cell) 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, 811.

The"treating"refers to administering a therapy in amount, manner, and/or mode effective to improve a condition, symptom, or parameter associated with a disorder or to prevent progression of a disorder, to either a statistically significant degree or to a degree detectable to one skilled in the art. An effective amount, manner, or mode can vary depending on the subject and may be tailored to the subject. By preventing progression of a disorder, a treatment can prevent deterioration of a disorder or onset of an additional symptom in an affected or diagnosed subject.

It is advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated ; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

Toxicity and therapeutic efficacy of such 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. Compounds which exhibit high therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

The data obtained from the 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 therapeutic compound can be a small molecule including, e. g., peptides, peptidomimetics (e. g. , peptoids), amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organic or inorganic compounds (i. e., including heteroorganic and organometallic compounds) having a molecular weight less than about 10,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds.

Exemplary doses include milligram or microgram amounts of the small molecule per kilogram of subject or sample weight (e. g. , about 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 5 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram. It is furthermore understood that appropriate doses of a small molecule may depend upon a number of factors, such as the potency of the small molecule with respect to the expression or activity to be modulated (e. g. , affinity for target compound and efficacy) and pharmacokinetic properties.

When one or more of these small molecules is to be administered to an animal (e. g. , a human) in order to modulate expression or activity of a polypeptide or nucleic acid of the invention, a physician, veterinarian, or researcher may, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained. In addition, it is understood that the specific dose level for any particular animal subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the degree of expression or activity to be modulated.

The nucleic acid molecules that modulate SSTR activity can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U. S. Patent 5,328, 470) or by stereotactic injection (see e. g. , Chen et al. Proc. Natl. Acad. Sci. USA 91: 3054-3057,1994). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e. g. , retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.

The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

A therapeutic compound can be provided in a kit. The kit includes (a) the modulator, e. g. , a composition that includes the therapeutic compound, and (b) informational material. The informational material can be descriptive, instructional, marketing or other material that relates to the methods described herein and/or the use of the therapeutic compound for the methods described herein. For example, the informational material describes methods for administering the therapeutic compound to alter lifespan regulation or at least one symptom of aging or an age related disease.

In one embodiment, the informational material can include instructions to administer the therapeutic compound in a suitable manner, e. g. , in a suitable dose, dosage form, or mode of administration (e. g. , a dose, dosage form, or mode of administration described herein). In another embodiment, the informational material can include instructions for identifying a suitable subject, e. g. , a human, e. g. , an adult human. For example, the human is an adult, e. g. , an adult with normal or reduced GH/IGF-1 axis activity for the adult's age, or with abnormal axis

activity (e. g. , above average activity for the adult's age). The informational material of the kits is not limited in its form. In many cases, the informational material, e. g. , instructions, is provided in printed matter, e. g. , a printed text, drawing, and/or photograph, e. g. , a label or printed sheet.

However, the informational material can also be provided in other formats, such as Braille, computer readable material, video recording, or audio recording. In another embodiment, the informational material of the kit is a link or contact information, e. g. , a physical address, email address, hyperlink, website, or telephone number, where a user of the kit can obtain substantive information about the therapeutic compound and/or its use in the methods described herein. Of course, the informational material can also be provided in any combination of formats.

In addition to the therapeutic compound, the composition of the kit can include other ingredients, such as a solvent or buffer, a stabilizer or a preservative, and/or a second agent for treating a condition or disorder described herein, e. g. an age-related disorder. Alternatively, the other ingredients can be included in the kit, but in different compositions or containers than the therapeutic compound. In such embodiments, the kit can include instructions for admixing the therapeutic compound and the other ingredients, or for using the therapeutic compound together with the other ingredients.

The therapeutic compound can be provided in any form, e. g. , liquid, dried or lyophilized form. It is preferred that the therapeutic compound be substantially pure and/or sterile. When the therapeutic compound is provided in a liquid solution, the liquid solution preferably is an aqueous solution, with a sterile aqueous solution being preferred. When the therapeutic compound is provided as a dried form, reconstitution generally is by the addition of a suitable solvent. The solvent, e. g. , sterile water or buffer, can optionally be provided in the kit.

The kit can include one or more containers for the composition containing the therapeutic compound. In some embodiments, the kit contains separate containers, dividers or compartments for the composition and informational material. For example, the composition can be contained in a bottle, vial, or syringe, and the informational material can be contained in a plastic sleeve or packet. In other embodiments, the separate elements of the kit are contained within a single, undivided container. For example, the composition is contained in a bottle, vial or syringe that has attached thereto the informational material in the form of a label. In some embodiments, the kit includes a plurality (e. g. , a pack) of individual containers, each containing one or more unit dosage forms (e. g. , a dosage form described herein) of the therapeutic compound. For example, the kit includes a plurality of syringes, ampules, foil packets, or blister

packs, each containing a single unit dose of the therapeutic compound. The containers of the kits can be air tight and/or waterproof.

The compositions can be administered to a subject, e. g. , an adult subject, particularly a healthy adult subject or a subject having an age-related disease. In the latter case, the method can include evaluating a subject, e. g. , to characterize a symptom of an age-related disease or other disease marker, and thereby identifying a subject as having a neurodegenerative disease, a disease associated with protein misfolding or protein aggregation, an age-related disease or being pre-disposed to such a disease.

EXAMPLE: Somatostatin Receptor Binding Assays The human SSTR-1, SSTR-2, SSTR-3, SSTR-4, and SSTR-5 cDNA clones have been described (SSTR-1 and SSTR-2 in Yamada, Y. , et al. , Proc. Natl. Acad. Sci. USA. , 89: 251-255 (1992); SSTR-3 in Yamade, et al. , Mol. Endocrinol. 6: 2136-2142 (1993); and SSTR-4 and SSTR-5 in Yamada, et al. , Biochem. Biophys. Res. Commun. 195: 844-852 (1993) ) and are also available from American Type Culture Collection (ATCC, Rockville, Md. ) (ATCC Nos. 79044 (SSTR-1), 79046 (SSTR-2), and 79048 (SSTR-3) ). Based on the restriction endonuclease maps, the entire coding region of each SSTR cDNA may be excised by suitable restriction endonuclease digestion (Maniatis, T. , et al., Molecular Cloning--A Laboratory Manual, CSHL, 1982). Restriction endonucleases are available from New England Biolabs (Beverly, Mass. ).

This cDNA fragment can be inserted into the mammalian expression vector, pCMV (Russell, D., et al. , J. Biol. Chem. , 264: 8222-8229 (1989) ), using standard molecular biology techniques (see e. g., Maniais, T. , et al., Molecular Cloning,--A Laboratory Manual, Cold Spring Harbor Laboratory, 1982) to produce the expression plasmid, pCMV-human SSTR-1 through pCMV- human SSTR-5. Other mammalian expression vectors include pcDNAl/Amp (Invitrogen, Sandlesy, Calif.). The expression plasmids were introduced into the suitable bacterial host, E.

Coli HB101 (Stratagene, La Jolla, Calif. ) and plasmid DNAs, for transfection, were prepared on Cesium Chloride gradients.

CHO-K1 (ovary, Chinese hamster) cells can be obtained from ATCC (ATCC No. CCL 61). The cells are grown and maintained in Ham's F12 media (Gibco BRL, Grand Island, N. Y.) supplemented with 10% fetal bovine serum under standard tissue culture conditions. For transfection, the cells are seeded at a density 106/60-cm plate (Baxter Scientific Products, McGaw Park, 111.). DNA mediated transfection are carried out using the calcium phosphate co- precipitation method (Ausubel, F. M. , et al., Current Protocols in Molecular Biology, John Wiley

& Sons, 1987). The plasmid pRSV-neo (ATCC; ATCC No. 37198) can be included as a selectable marker at 1/10 the concentration of the expression plasmid. CHO-K1 clonal cell lines that have stably inherited the transfected DNA can be selected for growth in Ham's F12 media containing 10% fetal bovine serum and 0.5 mg/ml of G418 (Sigma). The cells can be ring-cloned and expanded in the same media for analysis.

Expression of the human SSTR-1 through SSTR-5 receptors in the CHO-K1 cells can be detected by Northern blot analysis of total RNA prepared from the cells (Sambrook, J. E. , et al., Molecular Cloning--A Laboratory Manual, Ed. 2, Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. , 1989) and by receptor binding using [125I-Tyr11]somatostatin-14 as a ligand.

Transfected cell lines expressing the human SSTR receptors are clonally expanded in culture and used in the following SSTR binding protocol.

Crude membranes are prepared by homogenization of the transfected cells in 20 ml of ice-cold 50 mM Tris-HCl with a POLYTRONTM homogenizer (setting 6,15 sec). Buffer can be added to obtain a final volume of 40 ml, and the homogenate was centrifuged in a Sorval SS-34 rotor at 39,000 g for 10 min at 0-4 °C. The resulting supernatant can be decanted and discarded.

The pellet can be rehomogenized in ice-cold buffer, diluted, and centrifuged as before. The final pellet can be resuspended in the 10 mM Tris HCl and held on ice for the receptor binding assay.

Aliquots of the membrane preparation were incubated for 30 min at 30°C with 0.05 nM [125 I-Tyr11] somatostatin-14 (2000 Ci/mmol; Amersham Corp. , Arlington Heights, 111.) in 50 mM HEPES (pH 7.4) containing a test somatostatin agonist of various concentrations (e. g., 10. sup. - 11 to 10. sup. -6), 10 mg/ml bovine serum albumin (fraction V) (Sigma Chemical Co. , St. Louis, Mo.), MgCl2 (5 mM), Trasylol (200 KIU ml), bacitracin (0.02 mg/ml), and phenylmethylsulphonyl fluoride (0.02 mg/ml). The final assay volume can be 0.3 ml. The incubations are terminated by rapid filtration through GF/C filters (pre-soaked in 0.3% polyethylenimine for 30 min) using a Brandel filtration manifold. Each tube and filter are then washed three times with 5 ml aliquots of ice-cold buffer. Specific binding can be calculated as the total [121 I-Tyrl l] somatostatin-14 bound minus that bound in the presence of 1000 nM. The Ki values for the tested somatostatin agonists were calculated by using the following formula: Ki=IC50/ [1+ (LC/LEC) ] where IC50 is the concentration of test somatostatin agonist required to inhibit 50 percent of the specific binding of the radioligand [125 I-Tyr11] somatostatin-14, LC is the concentration of the radioligand (0.05 nM), and LEC is the equilibrium dissociation constant of the radioligand (0.16 nM).

EXAMPLE General SSTR agonist compound assessment can include one or more of the following: 1) evaluating blood IGF-1 (e. g. , levels) and other pharmacodynamic markers, e. g. as discussed below, when administered acutely. (For example, is blood IGF-1 levels decreased?) 2) evaluate activity of forkhead or HSF-1 transcription factors. (For example, is the decreased IGF-1 accompanied by increased transcription factor activity?) 3) evaluate duration the effect of the tested compound 4) evaluate behavioral deficits and life span (for example, using the R6/2 huntingtin mutant mouse (normal lifespan = 15 weeks) ) In one implementation, compounds can be selected that have one or more of the following properties: decrease blood IGF-1 levels, increase forkhead and/or HSF-1 activity, provide effects that are sustained over a period of weeks; and extend lifespan.

Compounds Two to four compounds each that show 50-1000 fold selectivity towards one or more the five SSTR subtypes (including compounds that are selective to one subtype relative to another) are assessed for their ability to lower blood IGF-1 concentration and increase forkhead activity in selected target tissues. In addition, compounds are tested that have mixed activities on SSTR receptor subtypes (e. g., SSTR1, 2; SSTR2, 3; SSTR2, 5; SSTR1, 2,3, 5, and other combinations).

Pharmacodynamic measures Exemplary pharmacodynamic markers include: IGF-1; IGF-1 BP, GH, blood insulin, oral glucose tolerance test, blood glucose, VEGF, ghrelin, FFA; TG; cholesterols, cortisol, Prolactin; TSH, body weight/composition.

All measurements can be carried out initially in eight mice per treatment group. Mice can receive injections of compounds at 1-20 mg/kg, i. p. , (dose based on existing information or by estimate) once or twice daily for 7 days. At that time body weights can be recorded for all animals; cardiac puncture can be performed in anesthetized mice to collect blood. Two animals in each group can be transcardially perfused with 10% paraformaldehyde and the brains removed and stored in 30% sucrose/saline at 4°C. For the remaining animals (n=6) brains can be removed, subregions dissected (see below) and stored at-80°C for Western blot analysis (see below). Also, wet weights can be determined for major organs including muscle, kidney, heart,

adrenal and white fat. Each blood sample can be assayed utilizing either radioimmunoassay or enzyme linked immunoassay methods for: GH, IGF-1, insulin, leptin, and ghrelin. Blood chemistry profiles can measure the following: glucose, corticosterone, free fatty acids, cholesterol (e. g. , hdl/ldl ratios), and triglycerides For those compounds that produce the desired effects on blood hormone and chemistry profiles, a more in-depth analysis of electrolytes, serum enzyme markers, BUN/creatinine and various other metabolites (n-20) can be performed, e. g. , to obtain pharmacodynamic and potential toxicology profiles of select compounds.

Also for a select number of compounds, oral glucose tolerance tests (insulin sensitivity) can be performed by repeating the acute dosing regimen (7 days).

Forkhead and HSF 1 Analysis Compounds that appropriately decrease components of the GH/IGF-1/insulin axis can be further analyzed immunochemically for activation of forkhead and HSF-1 transcription factors in nervous tissues. Frozen brain regions (striatum, cortex, and hippocampus) can be dissected, homogenized and analyzed for expression and phosphorylation status of the forkhead proteins FOXO1, 3a, and 4 using pan-and phospho-specific antibodies by Western blots. HSF-1 activity in the homogenates can be assayed by determining expression of heat shock proteins (hsp27, hsp40, hsp70, hsp90, hsp104) by Western blotting. Sub-cellular localization and cell type- specific expression of the transcription factors can be determined immunohistochemically with paraformaldehyde-fixed brain regions using pan-and phospho-specific anti-forkhead antibodies, and antibodies specific for the heat shock proteins (as above).

Individually or collectively, these tests can address one or more of the following: 1. whether SSTR agonist compounds decrease IGF-1 (and associated markers), 2. which SSTR subtype specificity (ies) is optimal for achieving the desired IGF-1 decrease and, 3. whether IGF-1 decrease is accompanied by increased forlchead/HSF-1 transcriptional activity.

Approximately three compounds can be tested simultaneously for assessment of blood hormone levels, blood chemistries and immunochemical analyses of brain samples.

Immunochemical analyses can be carried out only with brain tissue from animals receiving compounds that significantly decrease IGF-1 levels and other components of the GH/IGF-l/Insulin axis.

Sets of compounds can be overlapped with in-life and analytical phases, and evaluation of-15-20 compounds will take approximately 2.5-5 months for all analytical assessment.

Compounds that decrease plasma IGF-1 by >60% and increased forkhead/HSF-1 transcriptional activity in the 1 week treatment protocols can be selected.

Sustained activity of candidate co7npounds A compound that fulfills the above criteria (decreased blood levels of the GH/IGF-1/insulin axis components and increased forkhead/HSF-1 transcription factor activity) can be administered to animals for periods of 5-15 weeks to determine if the changes in pharmacodynamic markers and transcription factor activation can be sustained. In addition, animals can be monitored for signs of overt toxicity, such as excessive weight loss (cachexia), lethargy, change in coat, ptosis, lowered body temperature, urticaria and conjunctivitis.

Compounds that fail to sustain activity or that induce a pattern of toxicity can be discarded.

To examine whether select compounds can sustain their effects, various dosing regimens (e. g. , qd, bid) and treatment durations (5-15 weeks) can be employed. In these studies, animals can be monitored for overt toxicities, as well. Compounds that maintain appropriate modulation of the GH/IGHF-1/insulin axis and augmentation of forkhead and/or HSF-1 activity over the 15 week test period can be tested in an animal model of neurodegenerative disease, namely Huntington's disease.

Animal Disease Models A hallmark of Huntington disease is protein aggregates, likely as a result of misfolded mutant protein. A Huntington's disease mouse model expressing a mutant form of the huntingtin gene (R 6/2) displays protein aggregates within the CNS, loss of motor function and premature death at-15 weeks of age.

Compounds that: 1) lower blood levels of GH/IGF-1/insulin axis components and 2) increase forkhead/HSF-1 signaling for sustained periods can be tested for lifespan prolongation, improvement of motor behavior, and diminution of histopathology hallmarks (aggregates/inclusions) in the R6/2 strain compared to wild type or other corresponding controls.

Lifespans of animals can be determined by Kaplan-Meyer plots (death as endpoint); motor behavior is assessed by rotorod performance (latency to fall off the apparatus); huntingtin protein aggregates are measured qualitatively using anti-huntingtin antibodies in perfusion-fixed brain samples.

Other animals that can be used include animal models of Parkinson's disease (cc- synuclein over-expression), Alzheimer's disease (ß-amyloid/Tau transgenic mice) and ALS (SOD mutant mice), our initial studies will focus only on Huntington's Disease.

A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.