CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of, and incorporates by reference, U.S. Provisional Patent Application No. 60/685, 189, filed May 27, 2005, and U.S. Provisional Patent Application No. 60/711,828, filed August 26, 2005.

FIELD

This application relates to compounds and agents of use in the treatment of neurological injury and disease.

BACKGROUND

Following traumatic or mechanically induced axonal degeneration in the peripheral nervous system, axonal regeneration often ensues, resulting in functional recovery. However, the rate of axonal elongation (3-4 mm/day) is slow, and sometimes does not result in recovery of full neurological function. If neurological function is restored, recovery usually occurs in weeks or months, depending upon the distance between the site of injury and the target tissue. Therapies that speed regeneration over long distances would be highly beneficial to patients and would significantly reduce health care costs.

Certain neurological conditions result from dysfunction of neurons in the peripheral or central nervous systems that is caused by chronic disease or injury. Chronic disease processes can permanently and progressively damage the nervous system, and (particularly in the central nervous system) usually result in permanent loss of function. Such loss of neurological function is a major cause of physical incapacitation and death throughout the world.

Neurons in the central and peripheral nervous systems degenerate as a normal function of human development and aging. Pathological neuron degeneration, however, is a serious condition seen in several neurological disorders. Neuronal degeneration can be specific or diffuse, and can lead to sensory, motor, and cognitive impairments. Neurodegenerative disorders encompass a range of seriously debilitating conditions including Parkinson's disease, amyotrophic lateral sclerosis (ALS, "Lou Gehrig's disease"), multiple sclerosis, Huntington's disease, Alzheimer's disease, Pantothenate kinase associated neurodegeneration (PKAN, formerly Hallervorden-Spatz syndrome), multiple system atrophy, diabetic retinopathy, multi-infarct dementia, macular degeneration, and the like. These conditions are characterized by a gradual but relentless worsening of the patient's condition over time. These disorders affect a large population of humans, especially older adults.

Many advances have been made in years past in gaming a better understanding of Parkinson's disease, Alzheimer's disease, and Huntington's disease. The primary cause of cognitive dysfunction for all three disorders has been directly linked to neuron degeneration, usually in specific areas of the brain.

Parkinson's disease is linked to degeneration of neurons in the substantia nigra, while Alzheimer's disease is in some part due to loss of pyramidal neurons in the limbic cortex (Braak, E. & Braak, H., 1999, In: V.E. Koliatsos & R.R. Ratan (eds.), Cell Death and Diseases of the Nervous System, Totowa, NJ: Humana Press, pp. 497-508). Huntington's disease's cognitive deficits are produced by degeneration of cells in the caudate nucleus of the striatum. However, although the symptoms and progression of these diseases are well characterized, there is a need for compounds that can be used to treat these disorders.

Many of the compounds previously shown to stimulate nerve regeneration have undesired side- effects, such as immunosuppression (FK506 and analogs that retain immunosuppressant activity) or androgenic or estrogenic stimulation. There is therefore a need to provide additional nerve growth stimulating compounds, particularly compounds that are well tolerated by subjects who take them.

SUMMARY

Disclosed herein are methods for promoting nerve regeneration and nerve outgrowth. In one embodiment, the methods involve the use of a compound represented by Formula I, below.

In Formula I, Q is C or N; X, is O, S, N, NH, CH, CH ₂ , or CO;

X ₂ is hydrogen, a halogen, O, CN, OR, SR, C(O)R, -S(O) ₂ R, NHOR, C(O)NR ₂ , or is optionally substituted perhaloalkyl, alkyl, alkenyl, alkynyl, aryl, alicyclic, arylalkyl, aryloxyalkyl, alkoxyalkyl, or heterocyclic;

X ₃ is selected from H, a halogen, CN, N ₃ , O, NR ₂ , NH ₂ , NHSO ₂ R, C(O)N(R) ₂ ,, NRNR ₂ , NROR, OH, OR, SR, SH, or is optionally substituted lower alkyl, amidine, guanidine, or perhaloalkyl;

X ₄ is hydrogen, C(O)R, S(O) ₂ R, C(O)NHR, C(O)OR, or is optionally substituted, including with one or more heteroatoms such as a halogen, N, O, or S, alkyl, alkenyl, alkynyl, aryl, alicyclic, aryloxy, alkoxy, alkylcarbonyl, arylalkyl, aryloxyalkyl, alkoxyalkyl, amino, alkylamino, alkylaminoalkyl,

alkylcarbonylaminoalkyl, arylamino, arylamino alkyl, alkylamino aryl, alkylcarbonyoxylalkyl, hydroxyalkyl, heterocyclic, haloalkyl, perhaloalkyl, or group with hydrophobic character; X ₅ is CF ₂ , S, SO, SO ₂ , O, CO, CH ₂ , CHF, NH, optionally substituted alkyl, alkenyl, or alkynyl, or is NR', where R' is alkyl; X ₆ and R are independently selected from H, C(O)R, -C(O)OR, C(O)NR ₂ , C(S)OR, C(S)NR ₂ , PO ₃ R, SO ₂ R, or optionally substituted alkyl, cycloalkyl, arylalkyl, aryl, heteroaryl, or heterocyclic; and the rings may include one or more internal double bonds or attached hydrogen atoms, such as to satisfy valence requirements, for example, when a ring is a pyrimidine, such as when the compound is a purine. In additional embodiments, the methods involve the use of a compound -that is represented by

Formula II, below.

In Formula II, X ₂ , X ₃ , X ₄ , X ₅ , and X ₆ are selected as described above for compound I and n may be O or 1 and is selected to satisfy valence requirements depending on the number of double bonds included in the rings.

In further embodiments, the method includes the use of a compound represented by Formula III, below.

In Formula III, X ₂ is hydrogen or a halogen;

X ₄ is hydrogen, C(O)R, S(O) ₂ R, C(O)NHR, C(O)OR, or is optionally substituted, including with one or more heteroatoms such as a halogen, N, O, or S, alkyl, alkenyl, alkynyl, aryl, alicyclic, aryloxy, alkoxy, alkylcarbonyl, arylalkyl, aryloxyalkyl, alkoxyalkyl, amino, alkylamino, alkylaminoalkyl, alkylcarbonylaminoalkyl, arylamino, arylamino alkyl, alkylamino aryl, alkylcarbonyoxylalkyl, hydroxyalkyl, heterocyclic, haloalkyl, perhaloalkyl, or group with hydrophobic character; X ₅ is CH ₂ , CF ₂ , S, SO, SO ₂ , O, NH, or NR', where R' is alkyl;

X ₇ is a halogen or optionally substituted alkyl, alkoxy, halogenated alkoxy, hydroxyalkyl, pyrollyl, or aryloxy; and X ₈ is one or more substituents on the aryl group, however Xg represents at least one substituent in the 5'- position selected from a halogen, optionally substituted alkyl, alkoxy, halogenated alkoxy, hydroxyalkyl, pyrollyl, or aryloxy, or has the formula -0-(CH ₂ ) _m -0-, where m is an integer from O to 2 and one of the oxygen atoms is bonded to the aryl ring at the 5 '-position, provided that, when X ₈ represents only one substituent, X ₇ and X ₈ are different.

In several non-limiting examples, the method includes the use of compounds having the preceding general Formula III and X ₅ is CH ₂ or S, X ₇ is a halogen, X ₈ provides substituents at the 3, 4, and 5 positions or the 4 and 5 positions, and X ₄ is a Ci to C _]0 alkynyl, a Ci to Ci ₀ alkyl, a C ₁ to Ci ₀ alkylamino alkyl, or a Ci to Ci ₀ alkoxyalkyl, optionally substituted with heteroatoms, such as N or O.

The foregoing and other features and advantages will become more apparent from the following detailed description of several embodiments, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic diagram illustrating the receptor-hsp-90 heterocomplex assembly of the steroid receptor complex. FIG. 2 is a bar graph showing that compounds V(a) and V(b) stimulated neurite elongation in

SH-S Y5 Y cells. Data shown are after 168 hours of no treatment (NT) and treatment with NGF only, FK506, V(a) at three different concentrations, and V(b) at three different concentrations.

FIG. 3 is a bar graph showing that compound V(d) stimulated neurite elongation in SH-SY5Y cells. Data shown are representative of six experiments: no treatment (NT), NGF only, FK506, and compound V(d) at three different concentrations.

FIGS. 4A-4B are, respectively, a bar graph and a chart showing that compounds V(a), and V(e)- V(h) stimulated neurite elongation in SH-SY5Y cells. Data shown are representative of eight experiments. FIG. 4A is a bar graph showing mean neurite lengths in SH-SY5Y cells at 168 hours shown for no treatment (NT), NGF only, FK506, and 10 nM concentrations of V(a) and V(e)-V(h). Figure 4B is a cumulative histogram showing the distribution of the neurite length for the eight experiments. The shift to the right for the cells treated with V(a) or V(e)-V(h) is comparable to cells treated with NGF.

DETAILED DESCRIPTION

/. Abbreviations

IM: intramuscular IP: intraperitoneal

IV: intravenous

HIV: human immunodeficiency virus

NGF: nerve growth factor

EGF: epidermal growth factor FKBP : FK506 binding protein

PBS: phosphate buffered saline

SQ: subcutaneous

FBS: fetal bovine serum

HS: human serum Hsp-90: heat shock protein

PD: Parkinson's disease

SRC: steroid receptor complex

//. Terms Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The term "comprises" means "includes." In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

In order to facilitate review of the various embodiments of this disclosure, the following explanations of specific terms are provided:

Chemical Terms "Halogenated" means that one or more hydrogen atoms of an organic radical have been replaced with a halogen.

"Optionally substituted" compounds are those that have one or more hydrogen or carbon atoms replaced by another group, such as a halogen or a heteroatom such as N, O, or S or an aryl, alkoxy, aryloxy, aminoalkyl, alkyl, cycloalkyl, heterocycloalkyl, alkylhalos, hydroxy, amino, alkoxy, thio, and combinations thereof.

"Alkyl" refers to a cyclic, branched, or straight chain alkyl group containing only carbon and hydrogen, and unless otherwise mentioned, typically contains one to twelve carbon atoms. This term is further exemplified by groups such as methyl, ethyl, n-propyl, isobutyl, t-butyl, pentyl, pivalyl, heptyl, adamantyl, and cyclopentyl. Unless otherwise specified, "lower alkyl" groups have from 1 to 5 carbon atoms.

"Haloalkyl" refers to an alkyl that is substituted with one or more halogens, which maybe the same or different.

"Alkenyl" refers to a cyclic, branched, or straight chain alkyl group which contains one or more carbon-carbon double-bonds and, unless otherwise mentioned, typically contains one to twelve carbon atoms. This term is further exemplified by groups such as ethenyl, n-propenyl, and butenyl.

"Alkynyl" refers to a cyclic, branched, or straight chain alkyl group which contains one or more carbon-carbon triple-bonds and, unless otherwise mentioned, typically contains one to twelve carbon atoms. This term is further exemplified by groups such as ethynyl, propynyl, and butynyl.

"Perhaloalkyl" refers to an alkyl with all of its hydrogens replaced by halogens, such as, for example, carbon tetrachloride and perfluoroethyl.

"Alicyclic" refers to compounds which have both aliphatic and cyclic portions. "Arylalkyl" refers to compounds which have an alkyl group which is substituted with one or more aryl groups.

"Aryloxyalkyl" refers to compounds which have an alkyl group substituted with one or more aryloxy groups.

"Alkoxyalkyl" refers to compounds which have an alkyl group substituted with one or more alkoxy groups.

"Alkylamino" refers to an amino radical which is bonded to an alkyl group, for example, methylamino, ethylamino, and propylamino. "Arylamino" refers to an amino radical which is bonded to an aryl group, for example, phenylamino.

"Arylamino alkyl" refers to an alkyl group substituted with at least one arylamino group.

"Alkylamino aryl" refers to an aryl group substititued with at least one alkylamino group.

"Alkylamino alkyl" refers to an alkyl group substituted with at least one alkylamino group.

"Hydroxyalkyl" refers to an alkyl group substituted with one or more hydroxy groups. "Alkylcarbonyl" refers to an alkyl group substituted with one or more C(O) groups, such as aldehydes and ketones.

"Alkylcarbonylaminoalkyl" refers to an organic radical having aminoalkyl and alkylcarbonyl substituents.

"Alkylcarbonyoxylalkyl" refers to an organic radical having an alkylcarbonyl group bonded to an alkoxy group.

"Pyrollyl" refers to a pyrrole group.

"Heteroalkyl" refers to an alkyl as described above in which one or more hydrogen or carbon atom of the alkyl is replaced by a heteroatom such as N, O, P, B or S. An alkyl substituted with a heterocycloalkyl, substituted heterocycloalkyl, heteroaryl, substituted heteroaryl, alkoxy, aryloxy or amino is included within "heteroalkyl." Illustrative heteroalkyls include cyano, benzoyl, 2-pyridyl, 2- furyl and the like.

"Cycloalkyl" refers to a saturated or unsaturated cyclic non-aromatic hydrocarbon radical having a single ring or multiple condensed rings. Illustrative cycloalkyls include cyclopentyl, cyclohexyl, bicyclooctyl and the like. "Heterocycloalkyl" or "heterocycle" refers to a cycloalkyl radical as described above in which one or more of the carbon atoms of the cyclic radical is replaced by a heteroatom such as N, O, P, B or S. Illustrative hetercycloalkyls include, for example, piperazinyl, morpholinyl, tetrahydropyranyl, tetrahydrofuranyl, piperidinyl, pyrrolindinyl, oxazolinyl and the like.

"Aryl" refers to an aromatic substituent that may be a single aromatic ring or multiple aromatic rings that are fused together, linked covalently, or linked to a common group such as a methylene or ethylene moiety. The common linking group may also be a carbonyl as in benzophenone or oxygen as in diphenylether or nitrogen in diphenylamine. The aromatic ring(s) may include phenyl, naphthyl, biphenyl, diphenylether, diphenylamine and benzophenone among others. In particular examples, aryls have between 1 and 20 carbon atoms. "Heteroaryl" refers to aromatic rings in which one or more carbon atoms of the aromatic ring(s) are replaced by a heteroatom(s) such as N, O, P, B or S. Heteroaryl refers to structures that may be a single aromatic ring, multiple aromatic rings or one or more aromatic rings coupled to one or more nonaromatic rings. Illustrative heteroaryls include, for example, thiophene, pyridine, isoxazole, phthalidimide, pyrazole, indole, furan and the like. "Aryloxy" refers to an aryl ether, for example, phenoxy or benzyloxy.

"Alkoxy" refers to an -OZ radical wherein Z is selected from alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, and combinations thereof. Illustrative alkoxy radicals include methoxy, ethoxy, benzyloxy, and t-butoxy. A related term is "aryloxy" wherein Z is selected from aryl, substituted aryl, heteroaryl, substituted heteroaryl, and combinations thereof. Illustrative alkoxy radicals include phenoxy, substituted phenoxy, 2-pyridinoxy, 8- quinalinoxy and the like.

"Arylalkyl" refers to an alkyl radical in which an H atom is replaced by an aryl group, for example, benzyl or 2-phenylethyl.

"Amino" refers to the group -NZ ¹ Z ² wherein each of Z ¹ and Z ² is independently selected from hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkoxy, aryloxy, and combinations thereof.

"Thio" refers to the group -SZ ¹ Z ² wherein each of Z ¹ and Z ² is independently selected from hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkoxy, aryloxy, and combinations thereof.

"Halogen" refers to fluoro, bromo, chloro, and iodo substituents.

"R" denotes, unless otherwise specified, any atom or group of atoms, such as substituents found on organic compounds.

Other Terms

"Administration of and "administering" a compound or composition should be understood to mean providing a compound, a prodrug of a compound, or a pharmaceutical composition as described herein. An "animal" is a living multicellular vertebrate organism, a category that includes, for example, mammals and birds. A "mammal" includes both human and non-human mammals. "Subject" includes both human and animal subjects.

"Axonal growth" or "axonal regeneration" as used herein refer both to the ability of an axon to grow and to the ability of an axon to sprout. An axon sprout is defined as a new process that extends from an existing or growing axon. (See, e.g., Ma et al., Nat. Neurosci. 2:24-30, 1999).

"Dosage" means the amount delivered in vivo to a subject of a compound, a prodrug of a compound, a pharmaceutical agent, a drug, or a pharmaceutical composition as described herein.

A "prodrug" is a drug precursor, which may be converted to a drug by metabolic processes after the prodrug is administered to a subject. A "neurodegenerative disorder" is an abnormality in the nervous system of a subject, such as a mammal, in which neuronal integrity is threatened. Without being bound by theory, neuronal integrity

can be threatened when neuronal cells display decreased survival or when the neurons can no longer propagate a signal. Specific, non-limiting examples of a neurodegenerative disorder are Alzheimer's disease, Pantothenate kinase associated neurodegeneration, Parkinson's disease, Huntington's disease (Dexter et al., Brain 114:1953-1975, 1991), HIV encephalopathy (Miszkziel et al., Magnetic Res. Imag. 15:1113-1119, 1997), and amyotrophic lateral sclerosis.

Alzheimer's disease manifests itself as pre-senile dementia. The disease is characterized by confusion, memory failure, disorientation, restlessness, speech disturbances, and hallucination in mammals (Medical, Nursing, and Allied Health Dictionary, 4th Ed., 1994, Editors: Anderson, Anderson, Glanze, St. Louis, Mosby). Parkinson's disease is a slowly progressive, degenerative, neurological disorder characterized by resting tremor, loss of postural reflexes, and muscle rigidity and weakness (Medical, Nursing, and Allied Health Dictionary, 4th Ed., 1994, Editors: Anderson, Anderson, Glanze, St. Louis, Mosby).

Amyotrophic lateral sclerosis is a degenerative disease of the motor neurons characterized by weakness and atrophy of the muscles of the hands, forearms, and legs, spreading to involve most of the body and face (Medical, Nursing, and Allied Health Dictionary, 4th Ed., 1994, Editors: Anderson, Anderson, Glanze, St. Louis, Mosby).

Pantothenate kinase associated neurodegeneration (PKAN, MIM# 234200, formerly Hallervorden-Spatz syndrome) is an autosomal recessive neurodegenerative disorder associated with brain iron accumulation. Clinical features include extrapyramidal dysfunction, onset in childhood, and a relentlessly progressive course (Dooling et al., Arch. Neurol. 30:70-83, 1974). PKAN is a clinically heterogeneous group of disorders that includes classical disease with onset in the first two decades, dystonia, high globus pallidus iron with a characteristic radiographic appearance (Angelini et al., J. Neurol. 239:A\1-A25, 1992), and often either pigmentary retinopathy or optic atrophy.

A "neurodegenerative-related disorder" is a disorder, such as speech disorders, that is associated with a neurodegenerative disorder. Specific non-limiting examples of a neurodegenerative related disorder include, but are not limited to, palilalia, tachylalia, echolalia, gait disturbance, perseverative movements, bradykinesia, spasticity, rigidity, retinopathy, optic atrophy, dysarthria, and dementia.

"Nerve" encompasses a single bundle of nerve fibers or a plurality of bundles of nerve fibers. "Nerve regeneration" refers to axonal regeneration and restoration of connectivity within neural networks after nerve injury or damage. For example, nerve regeneration can include complete axonal nerve regeneration, including vascularization and reformation of the myelin sheath. More specifically, when a nerve is severed, a gap is formed between the proximal and distal portions of the injured nerve. In order for the nerve axon to regenerate and reestablish nerve function, it must navigate and bridge the gap. Nerve regeneration involves the proximal end forming neurite growth cones that navigate the gap and enter endoneural tubes on the distal portion, re-connecting the neural network. Thus, a necessary action for nerve regeneration is sufficient neurite elongation as well as a sufficient rate of neurite

elongation. In certain examples, the desirable neurite elongation is significantly greater than that achieved with nerve growth factor alone in cell cultures as described below. For instance, the neurite elongation may be at least about 200 μm, and more particularly about 200 μm to about 1000 μm, in treated cells at 168 hours. With respect to nerve regeneration in animals, a functional improvement may be observed, for example, with at least about a 15% increase in the rate of neurite elongation, more particularly at least about a 30% rate increase, relative to the rate of neurite elongation for untreated nerve injuries.

"Inhibiting" or "treating" a disease refers to inhibiting the full development of a disease or condition, for example, in a subject who is at risk for a disease such as an autoimmune disease, graft- versus-host disease, or rejection of a transplanted tissue or organ. "Treatment" refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop. As used herein, the term "ameliorating," with reference to a disease or pathological condition, refers to any observable beneficial effect of the treatment. The beneficial effect can be evidenced, for example, by a delayed onset of clinical symptoms of the disease in a susceptible subject, a reduction in severity of some or all clinical symptoms of the disease, a slower progression of the disease, a reduction in the number of relapses of the disease, an improvement in the overall health or well-being of the subject, or by other parameters well known in the art that are specific to the particular disease.

The "peripheral nervous system" (PNS) is the part of an animal's nervous system other than the central nervous system. Generally, the PNS is located in the peripheral parts of the body and includes cranial nerves, spinal nerves and their branches, and the autonomic nervous system. The "central nervous system" (CNS) is the part of the nervous system of an animal that contains a high concentration of cell bodies and synapses and is the main site of integration of nervous activity. In higher animals, the CNS generally refers to the brain and spinal cord.

A "pharmaceutical agent" or "drug" refers to a chemical compound or composition capable of inducing a desired therapeutic or prophylactic effect when properly administered to a subject.

"Pharmaceutically acceptable salts" include those formed from cations such as sodium, potassium, aluminum, calcium, lithium, magnesium, zinc, and from bases such as ammonia, ethylenediamine, N-methyl-glutamine, lysine, arginine, ornithine, choline, N ₅ N ¹ - dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, diethylamine, piperazine, tris(hydroxymethyl)aminomethane, and tetramethylammonium hydroxide.

These salts may be prepared by standard procedures, for example by reacting the free acid with a suitable organic or inorganic base. Any chemical compound recited in this specification may alternatively be administered as a pharmaceutically acceptable salt thereof. "Pharmaceutically acceptable salts" also include the free acid, base, and zwitterionic forms. Descriptions of suitable pharmaceutically acceptable salts can be found in Handbook of Pharmaceutical Salts, Properties, Selection and Use, Wiley VCH (2002).

"Therapeutically-active" refers to an agent, compound or composition capable of inducing a desired therapeutic or prophylactic effect when properly administered to a subject. In this case, the desired therapeutic effect is nerve regeneration.

"Therapeutically-effective amount" or "nerve regeneration promoting-amount" is an amount sufficient to achieve a statistically significant promotion of nerve cell regeneration compared to a control. Nerve cell regeneration can be readily assessed using an in vitro assay, such as the assays described in the Examples below. Alternatively, nerve regeneration can be determined in an in vivo assay or by direct or indirect signs of nerve cell regeneration in a subject. Preferably, the increase in nerve regeneration is at least 10%, preferably at least 30%, and most preferably 50% or more compared to a control. A "steroid receptor complex," or SRC, is a multiprotein complex associated with any steroid receptor, including, but not limited to, the progesterone receptor, glucocorticoid receptor, estrogen receptor, androgen receptor, and mineralocorticoid receptor.

A "nerve growth promoting agent," or NGPA, is a substance that binds to a polypeptide component of a steroid receptor complex and promotes nerve regeneration, without limitation to a particular mechanism of action. Such polypeptide components include, but are not limited to, hsp-90 and FKBP-52. Certain NGPAs interfere with the interaction of the bound polypeptide with another polypeptide in the steroid receptor complex. The neurotrophic agents include compounds that either physically disrupt association of the mature SRC (either by inhibiting association or promoting dissociation of the SRC), or inhibit interaction of components (such as p23, FKBP-52 or hsp-90) of the SRC.

"Neurotrophic" refers to a substance that promotes nerve growth.

DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS

Methods are disclosed herein for increasing neurite outgrowth and/or promoting nerve regeneration in vivo and in vitro. In one embodiment, the methods include administering an effective amount of a compound according to Formula I, or a pharmaceutically acceptable salt thereof, which is represented by Formula I set forth below.

In Formula I, Q is C or N; X ₁ is O, S, N, NH, CH, CH ₂ , or CO;

X ₂ is hydrogen, a halogen, O, CN, OR, SR, C(O)R, -S(O) ₂ R, NHOR, C(O)NR ₂ , or is optionally substituted perhaloalkyl, alkyl, alkenyl, alkynyl, aryl, alicyclic, arylalkyl, aryloxyalkyl, alkoxyalkyl, or heterocyclic;

X ₃ is selected from H, a halogen, CN, N ₃ , O, NR ₂ , NH ₂ , NHSO ₂ R, C(O)N(R) ₂ ,, NRNR ₂ , NROR, OH, OR, SR, SH, or is optionally substituted lower alkyl, amidine, guanidine, or perhaloalkyl; X ₄ is hydrogen, C(O)R, S(O) ₂ R, C(O)NHR, C(O)OR, or is optionally substituted, including with one or more heteroatoms such as a halogen, N, O, or S, alkyl, alkenyl, alkynyl, aryl, alicyclic, aryloxy, alkoxy, alkylcarbonyl, arylalkyl, aryloxyalkyl, alkoxyalkyl, amino, alkylamino, alkylaminoalkyl, alkylcarbonylaminoalkyl, arylamino, arylamino alkyl, alkylamino aryl, alkylcarbonyoxylalkyl, hydroxyalkyl, heterocyclic, haloalkyl, perhaloalkyl, or group with hydrophobic character; X ₅ is CF ₂ , S, SO, SO ₂ , O, CO, CH ₂ , CHF, NH, optionally substituted alkyl, alkenyl, or alkynyl, or is NR', where R' is alkyl;

X ₆ and R are independently selected from H, C(O)R, -C(O)OR, C(O)NR ₂ , C(S)OR, C(S)NR ₂ , PO ₃ R, SO ₂ R, or optionally substituted alkyl, cycloalkyl, arylalkyl, aryl, heteroaryl, or heterocyclic; and the rings may include one or more internal double bonds or attached hydrogen atoms, such as to satisfy valence requirements, for example, when a ring is a pyrimidine, such as when the compound is a purine.

In certain embodiments, X ₅ is not S, such as being selected from CH ₂ , CO, CF ₂ , NH, O, optionally substituted alkyl, alkenyl, or alkynyl, and NR'. For example, X ₅ is selected from CH ₂ , CF ₂ , and CO. In a particular example, X ₅ is CH ₂ . In further embodiments, X ₅ includes a sulfur atom, such as being selected from S, SO, and SO ₂ . In at least certain embodiments, the identity of X ₅ affects the activity of the compound according to Formula I.

In further embodiments, X ₄ is hydrogen, C(O)R, S(O) ₂ R, C(O)NHR, C(O)OR, or is optionally substituted, including with one or more heteroatoms such as a halogen, N, O, or S, alkyl, alkenyl, alkynyl, aryl, alicyclic, arylalkyl, aryloxyalkyl, alkoxyalkyl, alkylaminoalkyl, alkylcarbonylaminoalkyl, alkylcarbonyoxylalkyl, hydroxyalkyl, heterocyclic, haloalkyl, perhaloalkyl, or group with hydrophobic character. In yet further embodiments, X ₄ is a C ₁ to C ₁₀ alkynyl, a C ₁ to C ₁₀ alkyl, a C ₁ to C ₁₀ alkylamino alkyl, or a C ₁ to C ₁₀ alkoxyalkyl, optionally substituted with heteroatoms, such as N or O.

In additional embodiments, the methods involve the use of a compound represented by Formula II, below.

In Formula II, X ₂ , X ₃ , X ₄ , X ₅ , and X ₆ are selected as described above for compound I and n may be 0 or 1 and is selected to satisfy valence requirements depending on the number of double bonds included in the rings.

In further embodiments, the methods include the use of a compound represented by Formula III, below.

In Formula III, X ₂ is hydrogen or a halogen; X ₄ is hydrogen, C(O)R, S(O) ₂ R, C(O)NHR, C(O)OR, or is optionally substituted, including with one or more heteroatoms such as a halogen, N, O, or S, alkyl, alkenyl, alkynyl, aryl, alicyclic, aryloxy, alkoxy, alkylcarbonyl, arylalkyl, aryloxyalkyl, alkoxyalkyl, amino, alkylamino, alkylaminoalkyl, alkylcarbonylaminoalkyl, arylamino, arylamino alkyl, alkylamino aryl, alkylcarbonyoxylalkyl, hydroxyalkyl, heterocyclic, haloalkyl, perhaloalkyl, or group with hydrophobic character; X ₅ is CH ₂ , CF ₂ , S, SO, SO ₂ , O, NH, or NR', where R' is alkyl;

X ₇ is a halogen or optionally substituted alkyl, alkoxy, halogenated alkoxy, hydroxyalkyl, pyrollyl, or aryloxy; and

X ₈ is one or more substituents on the aryl group, however X ₈ represents at least one substituent in the 5'- position selected from a halogen, optionally substituted alkyl, alkoxy, halogenated alkoxy, hydroxyalkyl, pyrollyl, or aryloxy, or has the formula -O-(CH ₂ ) _m -O-, where m is an integer from O to 2 and one of the oxygen atoms is bonded to the aryl ring at the 5'-position, provided that, when X ₈ represents only one

substituent, X ₇ and Xg are different. In several non-limiting examples, the method includes the use of compounds have the preceding general Formula III and X ₅ is CH ₂ or S, X ₇ is a halogen, X ₈ provides substituents at the 3, 4, and 5 positions or the 4 and 5 positions, and X ₄ is a C ₁ to Ci ₀ alkynyl, a C ₁ to C ₁₀ alkyl, a C ₁ to C ₁₀ alkylamino alkyl, or a Ci to C ₁₀ alkoxyalkyl, optionally substituted with heteroatoms, such as N or O.

In certain embodiments, X ₅ is not S, such as being selected from CH ₂ , CO, CF ₂ , NH, O, and NR'. For example, X ₅ is selected from CH ₂ , CF ₂ , and CO. In a particular example, X ₅ is CH ₂ . In further embodiments, X ₅ includes a sulfur atom, such as being selected from S, SO, and SO ₂ . In at least certain embodiments, the identity of X ₅ affects the activity of the compound according to Formula III. In further embodiments, X ₄ is hydrogen or is an optionally substituted, including with one or more heteroatoms such as a halogen, N, O, or S, C ₁ to C ₁₀ alkyl, alkenyl, alkynyl, alkoxyalkyl, or alkylamino alkyl group. In yet further embodiments, X ₄ is is hydrogen or is an optionally substituted, including with one or more heteroatoms such as a halogen, N, O, or S, C ₁ to C ₁₀ alkyl, alkenyl, alkynyl, or alkoxyalkyl group. In several examples, the methods include the use of one or more of the compounds shown below:

Where X ₂ is a halogen or hydrogen, and X ₇ is a halogen. Particular examples include the compounds shown below:

In more particular examples, the methods include the use of one or more of compounds V(a)-

VG).

These compounds may be prepared by various methods known in the art, such as those described in PCT publication WO 03/037860 and PCT publication WO 02/36075, the entire contents of which are hereby expressly incorporated by reference. In particular, WO 02/036075 specifically discloses a method for preparing V(c) and V(d).

In further examples, the methods include the use of a compound according to Formula II selected from an illustrative species of Table 1, which is adapted from PCT publication WO 03/037860 or one of the compounds disclosed in PCT publication WO 02/36075.

TABLE l

Methods are described for promoting nerve regeneration and/or neurite outgrowth. The methods include administering an effective amount of one or more of the compounds set forth in Formulas I, II, III, or rV(a)-IV(g), and/or compounds V(a)-V(j). m certain embodiments, the methods include administering to the subject a therapeutically effective amount of a composition that includes one or a combination of compounds of Formula I ₅ or a purine. Compounds having a formula set forth as Formula II, III, or rV(a)-IV(g), and compounds V(a)-V(j) are purines, purine derivatives, or purine analogs (hereinafter collectively referred to as a "purine").

Steroid receptors are part of a superfamily of molecules that regulate gene expression by direct interaction with the upstream region of specific structural genes. It is essential to hormone action that a receptor must be able to assume both an active and an inactive state. This regulation is accomplished by association of the receptor (the steroid ligand binding component) with a multimeric complex of chaperone proteins, such as heat shock proteins (hsρ-90), p23, and FKBP-52, which form the steroid « receptor complex (SRC). When the steroid receptor binds its ligand, the receptor is activated, the chaperone proteins of the SRC are dissociated, and a DNA binding domain of the receptor is exposed for interaction with gene regulatory sequences. Members of the steroid receptor family that are regulated in this fashion include mineralocorticoids (such as aldosterone), glucocorticoids (such as dexamethasone), progestins (such as progesterone), androgens (such as testosterone), and estrogens (including estrogen, P- estriol and -estradiol).

A model of steroid receptor complex assembly is shown in FIG. 1. The mature SRC (top) consists of the receptor protein (which is able to bind steroid), FKBP-52, hsp-90, hsp-70 (according to some reports, hence the dotted line), and p23. Without being bound by theory, the compounds of use, such as the compounds of Formula I (indicated in FIG. 1 as "I"), Formula II, or the purines of Formula III, bind to a component of the SRC, such as hsp-90. These compounds can disrupt formation or encourage dissociation of the mature SRC. A dissociated SRC (the lower structure of FIG. 1) can lead to axonal protection and/or neurite outgrowth. Thus, a compound of use binds to a component of the steroid receptor complex, such as hsp-90. In one example, the binding of the compound causes dissociation of hsp-90 from the steroid receptor complex and/or prevents association of hsp-90 with the steroid receptor complex.

A method is provided for inducing neurite outgrowth from a nerve cell, either in vivo or in vitro. The method includes contacting the cell with an effective amount of a composition that includes at least one compound of Formula I, Formula II, Formula III, or Formula IV(a)-IV(g), and/or compound V(a)- V(j) or a pharmaceutically acceptable salt thereof. The cell can be in vivo or in vitro. In a further example, the compound or the pharmaceutically acceptable salt thereof is administered in conjunction with nerve growth factor (NGF).

In certain methods, nerve cell growth is induced by administering an effective amount of one or more compounds according to Formula I, II, III, or IV(a)-IV(g), for example, V(a)-V(j), that promote disassembly or disrupts assembly or function of a steroid receptor complex, for example of a mature steroid receptor complex (such as by inhibiting association or promoting dissociation). In particular embodiments, the compound is administered to disrupt association of a p23 component of the steroid receptor complex with an hsp-90 component or disrupt association of FKBP-52 with hsp-90 or inhibit interaction of hsp-90 with the steroid receptor complex, rn other embodiments, the compound is administered to competitively bind with ATP at an amino terminal ATP binding site of hsp-90. In certain examples, the compound binds to the SRC, or a component thereof, with a dissociation constant (IQ) of less than about 1 x 10 ^"6 M. The method can also include administering a second neurotrophic factor, other than the compound that disrupts association of the steroid receptor complex, such as another neurotrophic agent. The compound can be incorporated into a pharmaceutical composition, which can also include another neurotrophic factor, such as NGF, IGF-I , FGF, FGF, PDGF, BDNF, CNTF, GDNF, NT-3, NT 4/5, and mixtures thereof, or a steroid hormone that is a ligand of the steroid receptor complex (such as an estrogen, an androgen or a corticosteroid such as dexamethasone).

Methods are disclosed herein for increasing neurite outgrowth and/or promoting nerve regeneration in a subject. In one example, the subject has a degenerative disorder of the nervous system. In another embodiment, the subject has a partially or completely transected nerve, rn additional examples, the subject has a partial transaction of the spinal cord or a peripheral nerve.

The subject can be any subject of interest. The method is useful in the treatment of animals (including mammals such as humans) having a neurological condition associated with neuronal dysfunction caused by disease or injury to neurons in either the central or peripheral nervous systems. Compounds or compositions are administered in a therapeutically effective neurotrophic amount, such as an amount sufficient to bind to the mature steroid receptor complex (to disrupt association of the mature steroid receptor complex) and promote neurite outgrowth from neurons. The method can also be used in association with procedures such as a surgical nerve graft, or other implantation of neurological tissue, to promote healing of the graft or implant, and promote incorporation of the graft or implant into adjacent tissue. For administration to a subject, a therapeutically effective dose of a pharmaceutical composition containing one or more compounds of Formula I, Formula II, Formula III, Formula rV(a)-IV(g), or Formula V(a)-V(j), or the pharmaceutically acceptable salt thereof, can be included in a pharmaceutically acceptable carrier. In one example, the pharmaceutical compositions are prepared and administered in dose units. Solid dose units include tablets, capsules and suppositories. For treatment of a subject, different daily doses may be necessary depending on activity of the compound, manner of administration, nature and severity of the disorder, age, and body weight of the patient. Under certain circumstances, however, higher or lower daily doses may be appropriate. The administration of the daily dose can be carried out both by single administration in the form of an individual dose unit or several smaller dose units and also by multiple administrations of subdivided doses at specific intervals. The pharmaceutical compositions are in general administered topically, intravenously, orally, intracranially, intramuscularly, parenterally or as implants, but even rectal use is possible in principle. Suitable solid or liquid pharmaceutical preparation forms are, for example, granules, powders, tablets, coated tablets, (microcapsules, suppositories, syrups, drops, emulsions, suspensions, creams, aerosols, drops or injectable solution in ampoule form and also preparations with protracted release of active compounds, in whose preparation excipients and additives and/or auxiliaries such as disintegrants, binders, coating agents, swelling agents, lubricants, flavorings, sweeteners or solubilizers are customarily used as described above. The pharmaceutical compositions are suitable for use in a variety of drug delivery systems. For a brief review of present methods for drug delivery, see Langer, Science 249:1527- 1533, 1990. The pharmaceutical compositions can be administered locally or systemically.- In one embodiment, a therapeutically effective dose is the quantity of a compound that is sufficient to promote neurite outgrowth or nerve regeneration. In a specific example, a therapeutically effective amount is an amount sufficient to increase the neurite outgrowth and/or nerve regeneration induced by administration of nerve growth factor (NGF). In another specific example, a therapeutically effective amount is an amount necessary to prevent, to cure, or at least partially arrest the symptoms of a neurodegenerative disorder and its complications. Amounts effective for this use will, of course, depend on the severity of

the disease and the weight and general state of the patient, as well as the absorption, inactivation, and excretion rates of the therapeutically-active compound or component, the dosage schedule, and amount administered as well as other factors known to those of skill in the art. It also should be apparent to one skilled in the art that the exact dosage and frequency of administration will depend on the particular compounds administered, the particular condition being treated, the severity of the condition being treated, the age, weight, general physical condition of the particular patient, and other medication the individual may be taking. Typically, dosages used in vitro may provide useful guidance in the amounts useful for in vivo administration of the pharmaceutical composition, and animal models maybe used to determine effective dosages for treatment of particular disorders. Various considerations are described, e.g., in Gilman et al., eds., Goodman And Gilman's: The Pharmacological Bases of Therapeutics, 8th ed., Pergamon Press, 1990; and Remington 's Pharmaceutical Sciences, 17th ed., Mack Publishing Co., Easton, Pa., 1990, each of which is herein incorporated by reference.

In one example, the compound can be administered as a single dose per day, or it can be divided into at least two unit dosages for administration over a 24-hour period, or it may be a single continuous dose for a longer period of time, such as 1-10 weeks. Treatment may be continued as long as necessary to achieve the desired results. For instance, treatment may continue for about 3 or 4 weeks up to about 12-24 months. The compound can also be administered in several doses intermittently, such as every few days (for example, at least about every two, three, four, five, or ten days) or every few weeks (for example at least about every two, three, four, five, or ten weeks). The compound, such as but not limited to a compound according to Formulas I-III. IV(a)-IV(g), or V(a)-V(j) or a pharmaceutical salt thereof, can be formulated into therapeutically-active pharmaceutical compositions that can be administered to a subject parenterally or orally. Parenteral administration routes include, but are not limited to, subcutaneous injections (subcutaneous (SQ and depot SQ), intravenous (IV), intramuscular (IM and depot EVI), intrasternal injection or infusion techniques, intranasal (inhalation), intrathecal, transdermal, topical, and ophthalmic.

The compound or pharmaceutically acceptable salt thereof can be mixed or combined with a suitable pharmaceutically acceptable carrier to prepare pharmaceutical compositions. Pharmaceutically acceptable carriers include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (such as human serum albumin), buffers (such as phosphates), glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene- polyoxypropylene-block polymers, polyethylene glycol, and wool fat, for example. Liposomal suspensions may also be suitable as pharmaceutically acceptable carriers. Upon mixing or addition of the agent(s), the resulting mixture may be a solution, suspension, emulsion, or the like. These may be

prepared according to methods known to those skilled in the art. The form of the resulting mixture depends upon a number of factors, including the intended mode of administration and the solubility of the agent in the selected carrier or vehicle, hi one example, the effective concentration is sufficient for lessening or ameliorating at least one symptom of the disease, disorder, or condition treated and maybe empirically determined.

Pharmaceutical carriers or vehicles suitable for administration of the compound or pharmaceutically acceptable salts thereof include any such carriers known to be suitable for the particular mode of administration. In addition, the active materials can also be mixed with other active materials that do not impair the desired action, or with materials that supplement the desired action, or have another action. The agents may be formulated as the sole pharmaceutically active ingredient in the composition or may be combined with other active ingredients.

Methods for solubilizing may be used where the agents exhibit insufficient solubility in a carrier. Such methods are known and include, but are not limited to, using cosolvents such as dimethylsulfoxide (DMSO), using surfactants such as Tween®, and dissolution in aqueous sodium bicarbonate. The compound can be prepared with carriers that protect them against rapid elimination from the body, such as time-release formulations or coatings. Such carriers include controlled release formulations, such as, but not limited to, microencapsulated delivery systems. The therapeutically-activecompound is included in the pharmaceutically acceptable carrier in an amount sufficient to exert a therapeutically useful effect in the absence of undesirable side effects on the subject treated. The therapeutically effective concentration may be determined empirically by testing the compounds in known in vitro and in vivo model systems for the treated condition.

Injectable solutions or suspensions can be formulated, using suitable non-toxic, parenterally- acceptable diluents or solvents, such as mannitol; 1,3-butanediol; water; saline solution; Ringer's solution or isotonic sodium chloride solution; or suitable dispersing or wetting and suspending agents, such as sterile, bland, fixed oils, including synthetic mono- or diglycerides, and fatty acids, including oleic acid; a naturally occurring vegetable oil such as sesame oil, coconut oil, peanut oil, cottonseed oil, and the like; polyethylene glycol; glycerine; propylene glycol; or other synthetic solvent; antimicrobial agents such as benzyl alcohol and methyl parabens; antioxidants such as ascorbic acid and sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates, and phosphates; and agents for the adjustment of tonicity such as sodium chloride and dextrose. Parenteral preparations can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass, plastic, or other suitable material. Buffers, preservatives, antioxidants, and the like can be incorporated as required. Where administered intravenously, suitable carriers include physiological saline, phosphate buffered saline (PBS), and solutions containing thickening and solubilizing agents such as glucose, polyethylene glycol, polypropyleneglycol, and mixtures thereof. Liposomal suspensions including tissue-targeted liposomes may also be suitable as pharmaceutically acceptable carriers.

For topical application, the compound of use maybe made up into a solution, suspension, cream, lotion, or ointment in a suitable aqueous or non-aqueous vehicle. Additives can also be included, e.g., buffers such as sodium metabisulphite or disodium edeate; preservatives such as bactericidal and fungicidal agents, including phenyl mercuric acetate or nitrate, benzalkonium chloride or chlorhexidine, and thickening agents, such as hypromellose.

If the compound is administered orally as a suspension, the pharmaceutical compositions can be prepared according to techniques well known in the art of pharmaceutical formulation and may contain microcrystalline cellulose for imparting bulk, alginic acid or sodium alginate as a suspending agent, methylcellulose as a viscosity enhancer, and sweeteners/flavoring agents. Oral liquid preparations can contain conventional additives such as suspending agents, e.g., sorbitol, syrup, methyl cellulose, glucose syrup, gelatin, hydrogenated edible fats; emulsifying agents, e.g., lecithin, sorbitan monooleate, or acacia; non-aqueous vehicles (including edible oils), e.g., almond oil, fractionated coconut oil, oily esters such as glycerine, propylene glycol, or ethyl alcohol; preservatives such as methyl or propyl p-hydroxybenzoate or sorbic acid; and, if desired, conventional flavoring or coloring agents. The pharmaceutical compositions also can be administered in the form of a tea. As immediate release tablets, these compositions can contain microcrystalline cellulose, dicalcium phosphate, starch, magnesium stearate and lactose and/or other excipients, binders, extenders, disintegrants, diluents, and lubricants.

If oral administration is desired, the compound or pharmaceutically acceptable salt thereof can be provided in a composition that protects it from the acidic environment of the stomach. For example, the composition can be formulated in an enteric coating that maintains its integrity in the stomach and releases the active compound in the intestine. The composition can also be formulated in combination with an antacid or other such ingredient.

Oral compositions will generally include an inert diluent or an edible carrier and can be compressed into tablets or enclosed in gelatin capsules. For the purpose of oral therapeutic administration, the active compound or compounds can be incorporated with excipients and used in the form of tablets, capsules, or troches. Pharmaceutically compatible binding agents and 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, but not limited to, gum tragacanth, acacia, corn starch, sorbitol, polyvinylpyrrolidone or gelatin; a filler such as microcrystalline cellulose, starch, calcium phosphate, glycine or lactose; a disintegrating agent such as, but not limited to, alginic acid and corn starch; a lubricant such as, but not limited to, magnesium stearate, talc, polyethylene glycol, or silica; a gildant, such as, but not limited to, colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; disintegrants such as potato starch; and dispersing or wetting agents such as sodium lauryl sulfate; and a flavoring agent such as peppermint, methyl salicylate, or fruit flavoring.

When the dosage unit form is a capsule, it can contain, in addition to material of the above type, a liquid carrier such as a fatty oil. In addition, dosage unit forms can contain various other materials, which modify the physical form of the dosage unit, for example, coatings of sugar and other enteric agents. The compounds can also be administered as a component of an elixir, suspension, syrup, wafer, chewing gum, or the like. A syrup may contain, in addition to the active compounds, sucrose or glycerin as a sweetening agent and certain preservatives, dyes and colorings, and flavors.

When administered orally, the compounds can be administered in usual dosage forms for oral administration. These dosage forms include the usual solid unit dosage forms of tablets and capsules as well as liquid dosage forms such as solutions, suspensions, and elixirs. When the solid dosage forms are used, they can be of the sustained release type so that the compounds need to be administered only once or twice daily.

As noted above, the compound or compounds, such as, but not limited to, one or more compunds according to Formulas I-III, rV(a)-IV(g), or V(a)-V(j), may optionally be co-administered with at least one other neurotrophic agent such as the neurotrophins (nerve growth factor (NGF) and NT-3), brain derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), insulin growth factors (IGFs), FK506, or FK506 analogs, such as an FKBP12-binding analog of FK506. In one example, the compound or pharmaceutically acceptable salt thereof is administered in conjunction with nerve growth factor. The administration can be simultaneous or sequential.

The methods disclosed herein can be useful whenever neurite outgrowth is sought, for example following any acute or chronic nervous system injury resulting from physical transection/trauma, contusion/compression or surgical lesion, vascular pharmacologic insults including hemorrhagic or ischemic damage, or from neurodegenerative or other neurological diseases wherein neurite regeneration is desired.

In one embodiment, the subject has a neurodegenerative disorder such as Alzheimer's disease, Pantothenate kinase associated neurodegeneration, Parkinson's disease, Huntingdon's disease, human immunodeficiency virus (HTV) encephalopathy, or amyotrophic lateral sclerosis. In another embodiment, the subject has a "neurodegenerative-related disorder" such as palilalia, tachylalia, echolalia, gait disturbance, perseverative movements, bradykinesia, spasticity, rigidity, retinopathy, optic atrophy, dysarthria, or dementia. The methods cawalso be used in association with procedures such as a surgical nerve graft, or other implantation of neurological tissue, to promote healing of the graft or implant, and promote incorporation of the graft or implant into adjacent tissue. According to another aspect, the compositions could be coated or otherwise incorporated into a device or biomechanical structure designed to promote nerve regeneration. In one embodiment, a transection of a peripheral nerve or a spinal cord injury can be treated by administering a nerve regenerative stimulating amount of the compound, such as but not limited to one or

more compounds according to Formulas I-III, IV(a)-IV(g), or V(a)-V(j), to a mammal and grafting to the peripheral nerve or spinal cord an allograft (Osawa et al., J. Neurocytol. 19:833-849, 1990; Buttemeyer et al., Ann. Plastic Surgery 35:396-401, 1995) or an artificial nerve graft (Madison and Archibald, Exp. Neurol. 128:266-275, 1994; Wells et al., Exp. Neurol. 146:395-402, 1997). The transection can be a partial or a complete transection. The space between the transected ends of the peripheral nerve or spinal cord can be filled with a non-cellular gap-filling material such as collagen, methyl cellulose, etc., or cell suspensions that promote nerve cell growth, such as Schwann cells (Xu et al., J. Neurocytol. 26:1-16, 1997), olfactory cells, and sheathing cells (Li et al., Science 277:2000-2002, 1997). The compound can be included together with such cellular or non-cellular gap-filling materials. In a further embodiment, the compound or pharmaceutically acceptable salt thereof is provided to the site of injury in a biocompatible, bioresorbable carrier capable of maintaining the compound at the site and, where necessary, means for directing axonal growth from the proximal to the distal ends of a severed neuron. For example, means for directing axonal growth can be required where nerve regeneration is to be induced over an extended distance, such as greater than 10 mm. Many carriers capable of providing these functions are envisioned. For example, useful carriers include substantially insoluble materials or viscous solutions prepared as disclosed herein comprising laminin, hyaluronic acid or collagen, or other suitable synthetic, biocompatible polymeric materials such as polylactic, polyglycolic, or polybutyric acids and/or copolymers thereof. In one example, the carrier includes an extracellular matrix composition derived, for example, from mouse sarcoma cells. In a certain examples, the compound or pharmaceutically acceptable salt thereof is disposed in a nerve guidance channel which spans the distance of the damaged pathway. The channel acts both as a protective covering and a physical means for guiding growth of a neurite. Useful channels include a biocompatible membrane, which may be tubular in structure, having a dimension sufficient to span the gap in the nerve to be repaired, and having openings adapted to receive severed nerve ends. The membrane can be made of any biocompatible, nonirritating material, such as silicone or a biocompatible polymer, such as polyethylene or polyethylene vinyl acetate. The casing also may be composed of biocompatible, bioresorbable polymers, including, for example, collagen, hyaluronic acid, polylactic, polybutyric, and polyglycolic acids. In one embodiment, the outer surface of the channel is substantially impermeable. The disclosure is illustrated by the following non-limiting Examples.

EXAMPLES

Example 1 Material and Methods Primary Neuronal Cultures: The substantia nigra or striatal region is dissected from 4 day-old

Sprague-Dawley rats and incubated in minimum essential medium (MEM) containing 20 U/ml papain for 2 hours at 37°C. The tissue is then triturated using fire-polished Pasteur pipettes in MEM supplemented with 10% fetal bovine serum, 0.45% glucose, 5 pg/ml insulin, 0.5 mM glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. Cells are plated on 18 mm diameter poly-D-lysine-coated glass coverslips (Fisher 12-545-84 18cir-lD) at a density of 75,000 cells per coverslip for immunofluorescence imaging or in 12-well or 6-well tissue culture plates for immunoblotting studies. Neurobasal medium (GibcoBRL, Carlsbad, CA) containing B27 supplement (GibcoBRL) and 500 nM L-glutamine is added one hour after initial plating. To obtain a near pure neuronal culture, cytosine arabinoside (AraC) 10 μM is added after 1 day of culturing and for 2 days to stop the proliferation of the astrocytes. After 3 days, the medium is changed to remove AraC. The cultures are maintained in a humidified atmosphere with 5% CO ₂ for at least 10 days before experiments.

Cell preparation for microscopy: Neuroblastoma cells and rat striatal neurons are grown on glass coverslips and treated with poly-D-Lysine. Cells are fixed in 4% paraformaldehyde/Tris buffered saline (TBS, 50 mM Tris, 8 % NaCl, pH 7.4) for 15 minutes, permeabilized, and blocked in 5% goat serum and 0.5% Triton X-100 in TBS for 1 hour at room temperature. The coverslips are then mounted with the ProLong™ antifade kit (Molecular Probes) and dried in the dark. The samples are examined by an Olympus CX640 fluorescence microscope, or scanned with a Leica TCS SP confocal laser scanning microscope. Images are digitally captured and recorded with computer. The confocal images are deconvolved using Power HazeBuster™ imaging program (VayTek, Inc., Fairfield, VA). Cell stimulation, immunoblotting. and immunoprecipitation: Cells or neurons are grown in 6- well or 12-well plates to 80-90% confluence. The cells are starved in serum-free DMEM overnight, and then incubated with receptor agonists at 37°C for the indicated durations. In some experiments, signaling inhibitors are added 45 minutes before simulation. Incubation is terminated by placing the tissue culture cluster on ice and rapidly aspirating the medium, followed by the addition of ice-cold RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 % NP-40, 0.5 % deoxycholate, 0.1 % SDS, 1 mM NaVO3, and protease inhibitor (Calbiochem, San Diego, CA)) and incubation for 15 minutes with shaking. After centrifugation (14,000 x g at 4°C for 15 minutes), the supernatant is collected and the protein concentration is measured and adjusted using RIPA ⁺ buffer. Samples (30 μl) with equal amounts of protein mixed with Laemmli loading buffer are denatured at 70 ⁰ C for 10 minutes and separated by SDS- PAGE. Alternatively, for coprecipitation experiments, solubilized cell lysates are centrifuged at 20,000 x

g for 15 minutes at 4°C, and the supernatants are collected and precleared with protein G-Sepharose beads (Sigma).

Analysis of neurite length in SH-SY5Y neuroblastoma cells: SH-SY5Y neuroblastoma cells develop axonal-like processes on treatment with NGF. For analysis of process length, cells (20 fields/well) are randomly photographed at 96 and 168 hours. Neurite lengths are measured on photographic prints using a Houston Instrument HI-PAD digitizing tablet connected to a computer with appropriate software (Bioquant IV); only processes more than twice the cell body length are measured. Data from identically treated wells are combined. Mean values and histograms are constructed from these data; each histogram is constructed from measurement of 90 to 160 cells. Histograms are compared using a Mann- Whitney U test (α = 0.05), which makes no assumptions about the shape of the distribution.

Dopamine uptake: NS20Y cells or neuron cultures are plated on poly-L-lysine (for NS20Y) or poly-D-lysine (for neuron culture)-coated 24-well plates and grown to confluence. The medium is decanted and cells are prepared for [ ³ H]doρamine uptake. Cells are incubated in the presence or absence ofmazindol (10 μM to determine nonspecific uptake) for at least 10 minutes prior to addition of [ ³ H]dopamine. The uptake assay (final volume 0.5 ml) is initiated by the addition of 50 nM

[ ³ H]dopamine and 2 μM dopamine in Krebs-HEPES buffer. Pargyline (monoamine oxidase (MAO) inhibitor) and tropolone (catechol-O-methyl transferase (COMT) inhibitor) are included in the buffer to prevent metabolic degradation of dopamine. Assays are carried out at 37°C for 10 minutes. Experiments are terminated by aspiration and treatment with 250 μl of 0.1 M HCl. Radioactivity remaining in each well is determined using liquid scintillation spectrometry. Experiments will be conducted with triplicate determinations.

Tyrosine hydroxylase histoimmunostaining and quantitative morphology: On day 21 as described in Example 5, mice are euthanized via perfusion with fixative agent (2.5% glutaraldehyde, 0.5% paraformaldehyde, 0.1% picric acid, and 2 mM NaVO ₃ in 0.1M HEPES buffer, pH 7.3) performed under anesthesia with mouse cocktail. The brains are processed for immunohistochemical studies. Sections (30 μm) are incubated with a monoclonal anti-tyrosine hydroxylase (TH, 1:1,000 dilution, Sigma) for 48 hours at 4°C. Biotinylated secondary antibodies followed by avidin-biotin complex are used. Immunoreactivity is visualized by incubation in 3,3'-diaminobenzidine/glucose/glucose oxidase. Total numbers of TH-positive neurons in SNc region are counted with a microscope. Striatal OD of TH immunostaining, determined by the Scion Imageprogram (Scion Corp., Frederick, Maryland, USA), is used as an index of striatal density of TH innervation.

Example 2 Neurite outgrowth stimulated in vitro by V(a)-V(j)

As disclosed herein, compounds V(a)-V(h) induced neurite growth in SH-SY5Y neuroblastoma cells and protected neuronal cells from neurotoxins. To test this, SH-SY5Y human neuroblastoma cells were used, which can be induced into neuron-like phenotype by NGF treatment. This is an established method for measuring neurite growth-promoting activity in vitro (see Gold et al., Neurosignals. 13:122- 129, 2004; Gold et al., Exp. Neurol. \A1:269-2T&, 1997, which are incorporated herein by reference). As shown in FIGS. 2-4, SH-SY5Y cells developed long axonal-like processes upon exposure for 168 hours to NGF (10 ng/ml) that were dramatically increased in length by FK506 (100 nM, a positive control) and compounds V(a) and V(b) (FIG. 2) at 1 nM, 10 nM, and 100 nM concentrations, compound V(d) (FIG. 3) at 1 μM, 10 nM, and 100 nM concentrations, and compounds V(a) and V(e)-V(h) (FIG. 4) at 10 nM concentrations.

Example 3 Nerve regeneration stimulated by V(a)-V(j) assessed by rat sciatic nerve crush test

The effects of systematic administration of a selected purine compound on nerve regeneration and functional recovery are analyzed following a crush injury to the rat sciatic nerve. Briefly, the right sciatic nerve of anaesthetized rats is exposed, and the nerve crushed twice using forceps at the level of the hip. Following the sciatic nerve crush, the compound of interest is administered to the rats, such as by subcutaneous injection or oral administration. The compound can be administered with another nerve growth promoting agent, such as nerve growth factor (NGF).

Functional recovery is assessed by determining the number of days following nerve crush until the animal demonstrates onset of an ability to right its foot and move its toes, and the number of days until the animal demonstrates an ability to walk on its hind feet and toes. Nerve regeneration is also assessed by sampling tissues from the sciatic nerve at known (0.5 cm) distances from the crush site and examining the number of myelinated fibers and the size of axons by light microscopy. The axons are also examined by electron microscopy. Axonal areas of both myelinated and unmyelinated fibers are determined by tracing the axolemma using a digitizing tablet connected to a computer with appropriate software. Cumulative histograms are constructed from these data and mean values and standard errors are calculated to assess the effect of administration of the test compound on axonal areas.

Example 4

Mechanisms of neuroprotection of dopaminergic neurons in vitro induced by one of compounds

V(a)-V(j) The dopaminergic neurons that have been cultured on poly-D-lysine treated coverslips or 12- well cell culture plates for 10 days are treated with vehicle or a compound of interest, such as one of

compounds V(a)-V(j) at 1 nM, 10 nM, 100 nM, or 1 μM for 7 days. MPP ⁺ at 20 μM (a concentration that has been shown to be effective in SNc neuron cultures) is added 1 day before or after the treatment with one of compounds V(a)-V(j). Half of the medium is changed with fresh medium containing the selected compound at day 4. To count the TH-positive neurons, the neurons on coverslips are fixed, stained for tyrosine hydroxylase (TH) at the end of treatment with the selected purine and imaged with a conventional or confocal fluorescence microscope. At the same time, a duplicate set of neurons in 12- well plate is incubated with [ ³ H]dopamine to measure the level of dopamine uptake. Immunoblotting is used to detect TH and β-tubulin in cell lysates from the neurons.

TH-positive neurons and associated dopamine uptake is almost absent from MPP ⁺ -treated cultures in the absence of a compound, such as a compound selected from V(a)-V(j), while those treated with a selected compound either before or after MPP ⁺ treatment show increased survival rates. Neurons with a selected compound added before MPP ⁺ treatment survive better than those with a selected compound added after MPP ⁺ treatment.

Example 5

Assessment of neuroprotection and neuroregeneration and side effects induced by a compound, such as compounds V(a)-V(j) in MPTP-treated C57BL/6 mice and primates

The MPTP-treated animal model has provided the best available tool to date for pathogenesis studies and the assessment of efficacy of new therapeutic interventions of PD. Systemic administration of MPTP in animals replicates most of the clinical Parkinsonian symptoms and the biochemical and pathologic hallmarks of the disease, including the selective loss of dopaminergic neurons in the SNc and severe reductions in the concentrations of dopamine, noradrenaline, and serotonin in the striatum. Thus, to predict the efficacy of compounds V(a)-V(j) (or another compound disclosed herein) in treating dopaminergic neuron loss in PD patients, one of compounds V(a)-VQ) (or the other compound) is tested in MPTP-treated C57BL/6 mice, a mouse model of PD (Sunstrom et al., Brain Res. 528:181-188, 1990). Possible side effects of compounds V(a)-V(j) (or the other compound) are assessed by monitoring behavior abnormalities and histopathologic changes in the important organs of the mice.

For example, to assess the neuroprotective activity of compounds V(a)-V(j), C57BL/6 mice are divided into 5 groups (4 mice/group), and receive different treatments as outlined in Table 2.

Table 2. Treatment strategies for different mice groups. All injections will be daily.

Gro Day l Day 2 to day 9 Day 10 to day 17 Day 21 up#

1 test*, i.p. 5 MPTP, s.c, 30 mg/kg+ purine, Saline euthanasia mg/kg i.p. 5 mg/kg

2 saline MPTP, s.c, 30 mg/kg purine, i.p. 5 euthanasia mg/kg

3 saline MPTP, s.c, 30 mg/kg Saline euthanasia

4 test* i.p. 5 mg/kg purine, i.p. f j mg/kg Saline euthanasia

5 saline saline Saline euthanasia

* administration of compound of interest

At day 21, the mice are euthanized via perfusion with fixative agent performed under anesthesia. The brain is sectioned and the number of TH-positive neurons in SNc, the dopaminergic nerve innervation density in striatum will be compared by immunohistochemistry staining for TH. The histopathologic changes in the SNc and striatum of mice are evaluated by routine Hematoxilin and Eosin (H&E) staining. Any behavior abnormalities and histopathologic changes in all brain areas, as well as livers, hearts, blood vessels, lungs, eyes, spleens, lymph nodes, muscles, etc. associated with purine treatment are carefully monitored for the possible side effects of purine in this strain of mice. Finally, the concentration of the administered purine distributed in the CNS is measured.

It has been shown that the MPTP -treatment procedure produces an almost complete destruction (i.e. ~ 95-99%) of TH-positive nerves in C57BL/6 mice (unpublished result). Based on the in vitro effects of the compound of interest, such as compounds V(a)-V(j), it is demonstrated that a compound, such as compounds V(a)-V(j) induce regenerative sprouting from damaged nigrostriatal dopaminergic neurons and also reduce the loss of dopaminergic cell bodies. The density of TH staining in the SNc is sharply decreased in MPTP -treated mice, but is reversed by the treatment with one of compounds V(a)- V(j). In the SNc, obvious histopathologic changes are seen by H&E staining in MPTP-only treated mice (which is alleviated by treatment with compounds V(a)-V(j)). For the first experiment, 5 mg/kg of the selected compound is used in vivo.

An additional effective experimental model of Parkinson's disease is the MPTP-treated primate (Jenner et al., Parkinsonism.Relat Disord. 9:131-137, 2003). These primates, when treated with MPTP, develop most Parkinsonian symptoms including bradykinesia, rigidity, and postural abnormalities. In addition, MPTP-treated primates are responsive to all commonly used anti-Parkinsonian agents and display treatment-associated motor complications such as dyskinesia, wearing-off, and on-off phenomena. Thus the MPTP-treated primate provides a useful model for preclinical testing of the antiparkinsonian activity of a compound such as, but not limited to, compounds V(a)-V(j). Thus, similar studies can be conducted in primates, using methods known in the art.

Example 6 Assays for identifying nerve growth promoting compounds

Assays can be used to determine if a compound of interest, such as a compound of Formulas I- rV, and/or compounds V(a)-V(j), binds to a polypeptide that is a component of a steroid receptor complex, such as hsp-90 and thus is of use in promoting neurite outgrowth and/or nerve regeneration. Immunoassays can also be performed using conventional immunoassay methodologies and antibodies that are specific for steroid receptor complex components, such as antibodies that specifically bind hsp-90 (Sanchez et al., /. Biol. Chem. 260:12398-12401, 1985; Catelli et al., EMBOJ. 4:3131-3135, 1985; Schuh et al., J. Biol. Chem. 260: 14292-14296, 1985).

An additional assay for detecting binding to hsp-90 is described, for example, by Whitesell et al. (Proc. Natl. Acad. ScL USA 91:8324-8328, 1994). Commercial hsp-90 (StressGen Biotechnologies, Victoria, BC) dissolved in 20 μg/mL of TNESV buffer (50 mM Tris-HCl, pH 7.4/1% Nonidet P-40/2 mM EDTA/100 mM NaCl/1 mM orthovanadate/1 mM phenylmethylsulfonyl fluoride/20 μg leupeprin per mL/20 μg of aprotinin per ml) and the test compound are incubated for 45 minutes at 4 ⁰ C with geldanamycin immobilized on a conventional solid support, e.g., geldanamycin-coupled agarose beads (Whitesell et al., Proc. Natl. Acad. Sd. USA 91 :8324-8328, 1994). The beads are then washed with TNESV buffer and bound hsp-90 is eluted by heating in reducing loading buffer, and can be analyzed by SDS/PAGE and silver staining (Bio-Rad). Alternatively, if the hsp-90 is labeled, the assay can be performed for the bound label instead of the free label. Test compounds that compete with geldanamycin for binding to hsp-90 inhibit the binding of solubilized hsp-90 to the beads.

A qualitative assay for receptor transformation, which involves dissociation of hsp-90 from the receptor complex, is conversion of a receptor complex to a state that binds polyanions such as phosphocellulose (Kalimi et al., J. Biol. Chem. 250:1080-1086, 1975; Atger and Milgrom, Biochem. 15:4298-4304, 1976), ATP-Sepharose (Toft et al., J. Steroid Biochem. 7:1053-1059, 1976; Miller and Toft, Biochem. 17:173-177, 1978), and carboxymethol-Sephadex (Milgrom et al, Biochem. 12:5198- 5205, 1973; Parchman and Litwack, Arch. Biochem. Biophys. 183:374-382, 1977).

Assays can be used to determine if a compound of interest, such as a compound of Formulas I- IV, and/or compounds V(a)-V(j), disrupts the steroid receptor complex (SRC) and thus is of use in promoting neurite outgrowth and/or nerve regeneration. Detection of disruption of the SRC can be assessed by assays known in the art. For example, disruption of the SRC by release of ρ23 can be assessed by the techniques disclosed in Whitesell and Cook, MoI. Endocrinol. 10:705-715, 1996, where benzoquinone ansamycin binding to hsp-90 was shown to result in complete loss of p23 protein from glucocorticoid receptor immunoprecipitates. For example, as disclosed in Whitesell and Cook, affinity precipitation is performed by lysing cells in TNESV buffer (50 mM Tris-HCl, pH 7.4%/l% Nonidet P-40/2 mM EDTA/100 mM NaCl/lmM

orthovanadate/1 itiM phenylmethysulfonyl fluoride/20 μg/ml leupeptin/20 μg/ml aprotinin) and lysates (0.75 mg of total protein per precipitation) incubated with geldanamycin-coupled beads. Bound proteins are eluted by heating in reduced loading buffer and analyzed by SDS-PAGE followed by Coomassie blue staining. Immnoprecipitation from cell lysates is performed using a specific monoclonal antibody BuGR- 2, and protein G Sepharose beads (Pharmacia). For experiments involving co-precipitation of GR with heteroprotein complex components, cells are lysed in detergent-free hypotonic buffer with 10 mM sodium molbydate. Immunoblot detection of proteins in total cells lysates, geldanamycin affinity precipitates, and glucocorticoid receptor immunoprecipitates are performed after SDS-PAGE and electrophoretic transfer of proteins to nitrocellulose. BuGR-2 hybridoma supernatant (1 :40) is used for detection of rodent derived GR while a peptide-derived rabbit polyclonal antibody (1 :250; PAl -512, Affinity Bioreagents; Golden, CO) is used for the human GR. Hsp-90 and hsp-70 are detected with antibodies AC88 and N27F3-4 respectively (1 :5000; StressGen; Victoria, BC, Canada). Ascites containing antibody JJ3 (1:1000) is used to blot forp23. Polyclonal rabbit anti-ubiquitin antiserum (1 :500; Sigma Chemical Co.) is used to detect ubiquitinated proteins after blots are autoclaved for 20 minutes to fully denature ubiquitinated proteins and enhance their detection. Detection is achieved using appropriate peroxidase-conjugated secondary antibodies (1:20,000) and chemiluminescent substrate (Kierkegaard and Perry Laboratories, Gaithersburg, MD).

Loss of dexamethasone binding activity can be determined with a binding assay in which HeLa cells (2 x 10 ⁵ /well, 24-well plate) are treated with various concentrations of geldanamycin for varying periods of time in complete medium at 37 ⁰ C. At the end of the treatment interval, medium is aspirated and monolayers washed twice with ice-cold PBS containing 1 % BSA and 0.1% sodium azide (binding buffer). Monolayers are then incubated for 60 minutes on ice with 1.0 μCi/well (48 nM) [ ³ H]dexamethasone (Amersham, 82 Ci/mmol) in binding buffer with or without 5mM non-radioactive dexamethasone. After this binding interval, wells are washed four times with cold binding buffer and . then extracted with 0.5 ml ethanol for 30 minutes. The ethanol solution is transferred to scintillation vials and evaporated to dryness before standard liquid scintillation counting. Specific binding is calculated as the counts per minute bound in the absence of excess nonradioactive dexamethasone less the counts per minute bound in its presence. Similar measurements can be made with estrogen and other steroid receptors, to detect disruption in ligand binding, by substituting those steroids for dexamethasone. A loss of detectable hsp-90 (disappearance of the hsp-90 band) from receptor immunoprecipitates can indicate loss of hsp in association with disruption of the SRC.

It will be apparent that the precise details of the methods or compositions described may be varied or modified without departing from the spirit of the described invention. We claim all such modifications and variations that fall within the scope and spirit of the claims below.