WO2002087552A2 - Androgen-mediated neuroprotection and uses thereof - Google Patents

Abstract

Androgens, such as testosterone, methyltestosterone, and epitestosterone, are disclosed herein as being neuroprotective, in that they inhibit or prevent cell death or apoptosis of neural cells. A neuroprotective effect of androgens on primary cultures of human neurons with respect to serum deprivation-mediated apoptosis and amyloid beta peptide mediated neurotoxicity is described. The invention thus provides methods and uses of androgens as well as compositions and commercial packages comprising androgens for the prevention and/or treatment of neural or neurodegenerative disease and for the prevention and/or inhibition of cell death. The invention further provides methods of identifying and screening compounds for the prevention and/or treatment of neural or neurodegenerative disease.

Description

ANDROGEN-MEDIATED NEUROPROTECTION AND USES THEREOF

FIELD OF THE INVENTION

The invention relates to the prevention, treatment and study of neural disease and more particularly relates to androgen-mediated neuroprotection and its use for the prevention, treatment and study of neural disease.

BACKGROUND OF THE INVENTION

Epidemiological studies have shown that decreasing levels of estrogen are a risk factor for Alzheimer' s disease and hormone replacement therapy (HRT) offers protection against Alzheimer's disease (Paganini-Hill

1996; Schneider et al . 1996; Tang et al . 1996; Schneider et al . 1997). In animal models and cell cultures, estrogen reverses the behavioral and biochemical changes in ovariectomized rats (Simpkins et al . 1997) and enhances neuritic outgrowth and survival (Woolley and McEwen 1993, 1994; McEwen and Woolley 1994; Brinton et al . 1997; Woolley et al . 1997; McEwen et al . 1999). Estrogen acts through genomic transactivation and non- genomic pathways (reviewed by Woolley 1999) . Genomic events include up-regulation of brain-derived neurotrophic factor, nerve growth factor (NGF) , epidermal growth factor (Birge 1996) and of Bcl-2 proteins (Dubai et al . 1999; Pike 1999). Estrogen also modulates p53 activity and cell fate (Wade et al . 1999). Non-genomic events involve signal transduction, and it has been shown that estrogen activates the mitogen-activated protein kinase cascade in the cerebral cortex (Singh et al . 1999; 2000b; Toran-Allerand 2000a; 2000b) . In addition,

estrogen decreases the amount of amyloid-β peptide produced in neurons (Jaffe et al . 1994; Xu et al . 1998)

and can protect against amyloid-β peptide-mediated neurotoxicity (Goodman and Mattson 1996; Behl et al . 1997). Others propose that estrogen acts as an antioxidant, although it is unlikely that physiological levels of estrogen will have antioxidant activity (Behl et al . 1997; Moosmann and Behl 1999) .

There is therefore a need to characterize such systems and identify new compounds useful for the prevention, treatment and study of neural disease.

SUMMARY OF THE INVENTION

An object of the invention is to provide methods and compounds for the prevention and/or treatment of neural or neurodegenerative disease or dementia. Accordingly, the invention provides a method of preventing or treating a neural disease in an animal, said method comprising administering an androgen or androgen-related compound to said animal. In an embodiment, the animal is a mammal, in a further embodiment, a human. In an embodiment, the animal is male. In an embodiment, the animal is female.

In certain embodiments, the neural disease is selected from the group consisting of Alzheimer disease, Parkinson's disease, amyotropic lateral sclerosis (ALΞ), cerebellar degeneration, ischemia (stroke), traumatic injuries, prion diseases (e.g. Creutzfeldt-Jakob disease) , Huntington disease, frontal lobe dementia, vascular dementia, infection related dementia (e.g. HIV), head injury, hereditary cerebral amyloidogenesis, Down's Syndrome and cerebral hemorrhage.

In an embodiment, the neural disease is an Aβ- mediated neural disease.

In certain embodiments, the androgen is selected from the group consisting of testosterone, mibolerone, methyltestosterone and epitestosterone. In an embodiment, the androgen is testosterone. In an embodiment, the androgen-related compound is a testosterone ester, in a further embodiment, the testosterone ester is selected from the group consisting of testosterone enanthate and testosterone propionate. The invention further provides a composition for preventing or treating a neural disease in an animal, said composition comprising an androgen or androgen- related compound in admixture with a pharmaceutically suitable carrier.

The invention further provides a use of an androgen or androgen-related compound or the above-mentioned composition for preventing or treating a neural disease in an animal.

The invention further provides a use of an androgen or androgen-related compound for preparation of a medicament for preventing or treating a neural disease in an animal.

The invention further provides a commercial package comprising an androgen or androgen-related compound or the above-mentioned composition together with instructions for the prevention or treatment of a neural disease.

In certain embodiments, the neural disease is selected from the group consisting of Alzheimer disease, Parkinson's disease, amyotropic lateral sclerosis (ALS) , cerebellar degeneration, ischemia (stroke), traumatic injuries, prion diseases (e.g. Creutzfeldt-Jakob disease) , Huntington disease, frontal lobe dementia, vascular dementia, infection related dementia (e.g. HIV), head injury, hereditary cerebral amyloidogenesis, Down's

Syndrome and cerebral hemorrhage. In embodiments, the androgen is selected from the group consisting of testosterone, mibolerone, methyltestosterone and epitestosterone. In an embodiment, the androgen is testosterone. In an embodiment, the androgen-related compound is a testosterone ester, in a further embodiment, the testosterone ester is selected from the group consisting of testosterone enanthate and testosterone propionate.

The invention further provides a method for preventing or inhibiting cell death, comprising treating a cell with an androgen or androgen-related compound. In an embodiment, the cell is a neural cell. In an embodiment, the cell is a mammalian cell, in a further embodiment a human cell. In certain embodiments, the androgen is selected from the group consisting of testosterone, mibolerone, methyltestosterone and epitestosterone. In an embodiment, the androgen is testosterone. In an embodiment, the androgen-related compound is a testosterone ester, in a further embodiment, the testosterone ester is selected from the group consisting of testosterone enanthate and testosterone propionate.

The invention further provides a method of identifying or characterizing a test compound for the prevention and/or treatment of neural or neurodegenerative disease, said method comprising: contacting said test compound with a cell comprising an androgen receptor; measuring a test level of androgen-associated activity; comparing said test level of androgen- associated activity with a corresponding control level of androgen-associated activity in a corresponding cell which was not exposed to said test compound; wherein a difference between said test level and said control level indicates that the test compound may be used for prevention and/or treatment of neural or neurodegenerative disease. In an embodiment the above-mentioned cell is a neural cell. In an embodiment the above-mentioned androgen-associated activity is an inhibition or prevention of cell death or apoptosis.

The invention further provides a method of identifying and/or characterizing a mechanism and/or component associated with androgen-mediated effects on cell death, said method comprising: assessing an alteration in a cell death- as-sociated phenotype between an androgen treated cell and a corresponding untreated cell; and comparing said alteration with a corresponding alteration assessed in corresponding androgen treated versus untreated mutant cells; wherein differences in said alteration are used to identify and/or characterize a mechanism and/or component associated with androgen-mediated effects on cell death.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Chemical structures of androgen, estrogens and flutamide.

Figure 2: Androgens offer neuroprotection against serum deprivation-mediated apoptosis. (a) Time study of neuroprotection by 4 nM testosterone, epitestosterone or

methyltestosterone and 2 nm 17-β-estradiol or 17-α- estradiol treatment at 24, 48, 72 and 96 h of serum deprivation. The level of apoptosis in hormone treated neurons is expressed as percentage neuronal cell death detected by propidium iodide and TUNEL staining. Data represents the mean and SEM of experiments of 10 independent neuronal cultures for all except methyltestosterone (n= 5) . (b) Dose response effect of each hormone on neuronal protection. Data represents the mean and SEM of eight independent experiments. *p <

0.05, **p < 0.005 indicate the significance of the difference between serum deprived neurons in the absence and in the presence of hormone.

Figure 3: Mibolerone protects against serum deprivation- mediated neuronal apoptosis. Serum deprived neurons were treated with 4 nM epitestosterone, methyltestosterone or testosterone in the presence or absence of 3 nM mibolerone for 96 h. Data represents the mean and SEM from three independent neuronal preparations. *p < 0.05, **p < 0.005 indicate the significance of the difference between serum deprived neurons in the absence and in the presence of hormone.

Figure 4: Aromatase inhibitor, 4-androsten-4-OL-3, 17- dione, does not inhibit testosterone neuroprotection. Serum deprived neurons were treated in the absence or presence of 4 nM testosterone and 5 and 50 ng/mL aromatase inhibitor, 4-androsten-4-OL-3, 17-dione (Al), and kept in culture for 96 h. Neuronal apoptosis was measured as described and expressed relative to control serum deprived neurons (arbitrarily placed at 100%) .

Data represents mean and SEM of three independent experiments. *p < 0.01, **p < 0.005 indicate the significance of the difference between serum deprived neurons in the absence and in the presence of hormone/drug.

Figure 5: Flutamide prevents testosterone-mediated neuroprotection. Serum-deprived neurons treated with

4 nM androgens in the absence or presence of 2 and 20 μM flutamide for 96 h. Data represents the mean and SEM of four independent experiments. *p < 0.05, **p < 0.002 indicates the significance of the difference between neurons that are serum deprived and those treated with hormone. Comparison of hormone and flutamide treated cells were made to serum deprivation in the presence of flutamide.

Figure 6: Neuroprotection by steroid hormones against Aβι-₂-mediated toxicity of human neurons in primary culture. Results show mean and SEM of three independent experiments (200 cells/experiment) . Figure 7: Further results demonstrating estrogen- and androgen-mediated protection of human neurons against

intracellular Aβι_₄₂-mediated toxicity in primary cultures.

17-β-estradiol (βE2) , 17-α-estradiol (αE2), testosterone enanthate (Test), epitestosterone (Epi-Test) , methyl testosterone (Methyl-test) or cell impermeable BSA

conjugated-17-β-estradiol (BSA-βE2) were used. Neuronal cell death was assessed by TUNEL 24 hours after treatment. % cell death represents the number of TUNEL positive neurons over total neurons (stained with DAPI) . Each sample represents 200 microinjected cells from each of a minimum of three experiments done on different

neuron preparations. *p<0.01 relative to Aβι_₄₂- microinjected cells in absence of hormones.

Figure 8 : Effect of hormone pre-incubation on hormone- mediated protection of human neurons against

intracellular Aβι_₄₂-mediated toxicity in primary cultures. Neurons were pre-incubated with 10 nM hormones for 1 hour

prior to microinjection with Aβι-₄₂. Treatment with hormones was continued for 24 hours before measuring cell death as above. Figure 9: Effect of hormone antagonists on hormone- mediated protection of human neurons against

intracellular Aβι-₄₂-mediated toxicity in primary cultures.

The neurons were pre-treated with either hormone or hormone and antagonist (tamoxifen [TXM] for estrogens and flutamide [Flut] for androgens) for one hour prior to

microinjection with Aβι_₄₂. * p<0.01.

DETAILED DESCRIPTION OF THE INVENTION

Described herein is an assessment of the role of physiological concentrations of androgens on serum deprivation-mediated apoptosis and on intracellular amyloid beta peptide-mediated neurotoxicity of human primary CNS neuron cultures.

Applicants have determined that androgens, such as testosterone, protect neurons against cell death or apoptosis, such as that induced by serum deprivation, by acting through androgen receptors. Applicants have further determined that androgens, such as testosterone,

protect neurons from amyloid beta peptide (e.g. Aβι_₄₂)-

mediated neurotoxicity, and that estrogens (e.g. 17-β-

estradiol) protect neurons from intracellular amyloid

beta peptide (e.g. Aβi_₂) -mediated neurotoxicity. Prior to applicant's studies, much attention has

been given to the role of 17-β-estradiol against Alzheimer's disease. With regard to the aging CNS in males, men in their sixties are usually less prone to Alzheimer's disease than women of the same age (Molsa et al . 1982; Jorm et al . 1987). Androgens eventually decrease with age (Flood et al . 1995; Vermeulen 1991). Testosterone replacement therapy improves depression, and verbal and spatial memory in aging men (Sternbach 1998) . At the molecular level, testosterone is shown to increase NGF and p75-nerve growth factor receptor and to decrease

Alzheimer's amyloid-β peptide in primary rat cortical neurons (Tirassa et al . 1997; Gouras et al . 2000). A preponderance in dementia among females has been suggested (Molsa et al . 1982) .

In women, androgens are also present (although at much lower levels) and decrease with age (Rako 1998) . Decreasing androgen levels are associated with a number of post-menopausal conditions such as osteoporosis, depression, reduced muscle and bone mass and increased visceral fat (Davis 1999) . Evaluation of testosterone in neuronal cell lines failed to reveal a role (Green et al 1997) .

Testosterone propionate prevents developmental neuronal loss in the medial preoptic nucleus of males or sex-reversed female rats (Dodson and Gorski 1993) .

Androgens can increase the volume, neuron number and synapses of developing rat superior cervical ganglion

(Wright et al . 1991) . In aging, there is little evidence that androgens regulate neuronal survival. However, testosterone deficiency in males is associated with conditions that indicate CNS neuronal dysfunction such as depression, anxiety and memory loss (Sternbach 1998) .

Furthermore, replacement therapy significantly improves these symptoms.

Applicants have demonstrated that androgens offer as much neuroprotection against growth factor deprivation mediated neuronal apoptosis of CNS differentiated human

neurons as 17-β-estradiol . Applicants have further demonstrated that androgens, (e.g. testosterone) further offer as much neuroprotection against amyloid beta

peptide (e.g. Aβι-₂) -mediated neurotoxicity as 17-β- estradiol. Applicants have further demonstrated that

estrogens (e.g. 17-β-estradiol) offer neuroprotection

against intracellular amyloid beta peptide (e.g. Aβι_₄₂)- mediated neurotoxicity. Such neuroprotection is also conferred when the androgen is added prior to exposure of cells to amyloid beta peptide, thus further demonstrating a prophylactic use. Neuroprotective effects of both occur at physiological concentrations. Therefore, applicants conclude that neurons are as responsive to androgens as estrogens with respect to neuronal survival.

Applicants demonstrate herein that physiological levels of androgen, such as testosterone, protect against cell death or apoptosis, such as serum deprivation- mediated neuronal apoptosis, through interaction with androgen receptors. Applicants have confirmed the presence of androgen receptors in the human neuron cultures studied. Applicants have shown that the non- aromatizable form^' of androgen, mibolerone, induces neuroprotection similar to testosterone. Mibolerone is a highly specific synthetic androgen that binds the androgen receptor with 100-fold higher affinity than the natural androgen testosterone (Wilson and French 1976; Traish et al . 1986; Turcotte et al . 1988; Markiewicz and Gurpide 1997). Furthermore, aromatase inhibitor, 4-androsten-4-OL-3, 17-dione, does not block testosterone-mediated neuroprotection. However, the neuroprotective effect of testosterone is blocked by the pure synthetic anti-androgen, flutamide (Brogden and

Chrisp 1991; Simard et al . 1986; Namer 1988; Labrie 1993; Markiewicz and Gurpide 1997; Singh et al . 2000a). Therefore, the neuroprotective effect of testosterone is mediated through androgen receptors. It is further shown herein that the neuroprotective

effect of estrogens (e.g. 17-β-estradiol) against

intracellular amyloid beta peptide (e.g. Aβi_₄₂) -mediated neurotoxicity may be blocked by the antagonist tamoxifen, indicating that the neuroprotective effect of estrogens is mediated through estrogen receptors.

The neuroprotective effect of testosterone is 100% up to 48 h after serum deprivation. Thereafter, there is increasing neuronal apoptosis, even in the presence of testosterone, although the levels are generally 60% lower than in absence of hormone. Since the media was changed every 48 h, turnover of testosterone cannot be responsible for the less protective effect. It is more likely that cumulative insult caused by continuous serum deprivation is responsible for the inability of testosterone to neuroprotect completely in time. As shown in other systems, inhibiting cell death with one compound may provide a certain degree of treatment of neurodegenerative diseases and combination therapies including both cell death inhibitors and pro-survival factors may be used to completely suppress neuronal cell death.

The neuroprotective effect of the weak endogenous antiandrogen, epitestosterone (Nuck and Lucky 1987; Starka et al . 1989; Starka et al . 1991) is surprising. This is clearly not the case for the other anti-androgen, flutamide. Furthermore, flutamide did not antagonize the epitestosterone effect, indicating that epitestosterone, unlike mibolerone, testosterone and methyltestosterone, cannot act through the androgen receptor.

Epitestosterone is aromatized into 17-α-estradiol

(Finkelstein et al . 1981). Given that the neuroprotective effect of epitestosterone occurs only after 48 h of treatment, it is possible that the neuroprotective effect is mediated through aromatization

into 17-α-estradiol . As shown by others, and by

applicants herein, the transcriptionally inactive 17-α- estradiol is also neuroprotective (Green et al . 1997).

It is proposed that 17-α-estradiol mediates neuroprotection through signal transduction rather than through a genomic pathway.

The fact that methyltestosterone and mibolerone are not as neuroprotective as testosterone indicates that the structure of the steroid may be very important in mediating the neuroprotective effect of androgens. Comparison of the chemical structure of the three compounds shows that mibolerone and methyltestosterone share a 17-methyl group that is absent in testosterone. It is envisioned that this methyl group accounts for the lesser neuropotency of methyltestosterone and mibolerone. Like estrogen, androgens are nuclear receptor proteins that can activate gene transcription or act through signal transduction. The neuroprotective effect of estrogen is known to act through the estrogen receptor and to activate both genomic and non-genomic pathways of neuronal protection (Woolley 1999) . Through the genomic pathway, estrogen upregulates Bcl-2 levels (Dubai et al .

1999; Pike 1999) and it is envisioned that increased Bcl-

2 levels enhance neuronal protection against serum deprivation. Whether androgens regulate gene expression or activity of survival genes in a manner similar to estrogens remains to be determined but this is a likely mechanism to explain the neuroprotective nature of androgens. Applicants results in primary cultures of human neurons contrast with those observed in the estrogen responsive, human SK-N-SH, neuronal cell line (Green et al . 1997). Androgens are metabolized by these cells and can affect proliferation (Maggi et al . 1998) . However, it is not unexpected to find differences between primary neurons and neuroblastoma cell lines as these differ in many ways such as in the state of differentiation, cell growth and cell death. An advantage of the present invention is the use of primary neural cultures rather than neuronal cell lines, which eliminates the above mentioned differences and reflects more accurately the processes associated with neural cells in vivo . In vivo, androgen receptors are expressed in the temporal, frontal and hippocampal regions of the brain (Puy et al . 1995; Finley and Kritzer 1999) . Androgen receptors are selectively localized to neuronal subtypes, and immunoreactivity appears specific to pyramidal neurons in primate prefrontal cortex (Finley and Kritzer 1999) . The number of androgen receptors does not differ in male and female rat or monkey brains (Clancy et al . 1992). The neuroprotective effect of testosterone through androgen receptors observed in human neurons in culture and described herein shall also occur in the human brain.

Applicant's findings demonstrate that androgens can help in the treatment of Alzheimer disease in a manner similar to estrogen-replacement therapy in women. Symptoms associated with the decreasing levels of androgen in both men and women are alleviated by hormone replacement therapy. The content of androgens in women's HRT should be taken into consideration in epidemiologic studies on the effect of HRT against Alzheimer disease. Androgens thus provide an effective treatment for aging animals and of neuroprotection against neural disease, for example, Alzheimer disease. In an embodiment, such an animal is a mammal. In an embodiment, such animals are human. In an embodiment, such an animal is male. In an embodiment, such an animal is female.

It is envisioned that the time of treatment is to be considered in this regard, since in post-mortem human hippocampus, the amount of androgen receptors has been observed to decrease with age in the CAl region (Tohgi et al . 1995). Observations have been made with respect to HRT and the treatment of Alzheimer disease in post- menopausal women in this regard (Mulnard et al . , 2000). The invention relates to androgens and androgen- related compounds and their use in conferring protection against cell death or apoptosis. In an embodiment, such a cell is a neural cell, in which case the androgens and androgen-related compounds confer neuroprotection, i.e. the inhibition or prevention of neural cell death or apoptosis, to prevent or reduce neurodegeneration and neurotoxicity, and for the prevention and/or treatment of neural disease. Therefore, in an aspect the invention provides a method for preventing or inhibiting cell death, comprising treating or contacting a cell with, or exposing a cell to, an androgen or androgen-related compound. In an embodiment, the cell is a neural cell. In an embodiment, the cell is a mammalian cell, in a further embodiment, a human cell.

In an embodiment, the above-mentioned neural cell death or apoptosis, neurodegeneration or neurotoxicity, is caused by the presence of a level of amyloid beta peptide or an amyloid beta peptide-like compound, i.e. is amyloid beta peptide- or amyloid beta peptide-like compound-mediated. As antibodies used for detection of

for example the Aβι_₄₂ peptide recognize the C-terminus of this molecule, there is actually variability with respect to the N-terminal residue or N-terminal limit of the peptides detected as such (see Naslund, et al . 2000). Accordingly, in certain embodiments, the amyloid beta

peptide is of the structure Aβ_n.₄₂, where "n" defines the N-terminus of the peptide. In an embodiment, the amyloid

beta peptide is Aβι-₂, in further embodiments, the amyloid beta peptide is another peptide which is derived from the amyloid precursor protein. Such amyloid beta peptides have been described in for example Selkoe (1998) . A higher than normal intracellular level of amyloid beta

peptide 1-42 (or in some cases by Aβ_n-₄₂, based on the antibodies used as noted above) in neural tissue has been shown to be associated with neural disease and dementia, such as Alzheimer disease (D'Andrea et al . 2001; Gouras et al . 2000; Naslund et al . 2000) . Applicants further describe that estrogens (e.g. 17-

β-estradiol) offer neuroprotection against intracellular

amyloid beta peptide (e.g. Aβi_₄₂) -mediated neurotoxicity, a phenomenon that has not been described prior to Applicants' work described herein. Such neuroprotection is shown to be blocked by tamoxifen, indicating that it is mediated through estrogen receptors.

"Amyloid beta peptide-like compounds" refers to any compound (such as a homologous or related peptide) which possesses similar structure, features and/or activity with an amyloid beta peptide. Such a feature may be an association with a neural disease or dementia, such as Alzheimer disease. Such activity may be the ability to elicit neurotoxicity. "Androgen-related" compounds refer to any compounds which are related to an androgen by having similar structure and/or activity (i.e. an androgen-associated activity) . "Androgen-associated activity" in this case refers to any activity associated with an androgen, including binding to an androgen receptor or a fragment, homolog or variant thereof (which retains androgen binding activity) , or affecting an androgen-mediated process. Such processes may include androgen-mediated signaling pathways or androgen-mediated gene expression. Such gene expression may be measured by detection of the corresponding RNA or protein, or via the use of a suitable reporter construct comprising a transcriptional regulatory element (s) normally associated with such a gene whose expression is androgen-mediated, operably- linked to a reporter gene. A first nucleic acid sequence is "operably-linked" with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably-linked to a coding sequence if the promoter affects the transcription or expression of the coding sequences. Generally, operably-linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in reading frame. However, since for example enhancers generally function when separated from the promoters by several kilobases and intronic sequences may be of variable lengths, some polynucleotide elements may be operably-linked but not contiguous. "Transcriptional regulatory element" is a generic term that refers to DNA sequences, such as initiation and termination signals, enhancers, and promoters, splicing signals, polyadenylation signals which induce or control transcription of protein coding sequences with which they are operably-linked. The expression of such a reporter gene may be measured on the transcriptional or translational level, e.g. by the amount of RNA or protein produced. RNA may be detected by for example Northern analysis or by the reverse transcriptase-polymerase chain reaction (RT-PCR) method (see for example Sambrook et al (1989) Molecular Cloning: A Laboratory Manual (second edition) , Cold Spring Harbor Laboratory Press, Cold

Spring Harbor, New York, USA) . Protein levels may be detected either directly using affinity reagents (e.g. an antibody or fragment thereof [for methods, see for example Harlow, E. and Lane, D (1988) Antibodies : A

Labora tory Manual , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY] ; a ligand which binds the protein) or by other properties (e.g. fluorescence in the case of green fluorescent protein) or by measurement of the protein' s activity, which may entail enzymatic activity to produce a detectable product (e.g. with altered spectroscopic properties) or a detectable phenotype (e.g. alterations in cell growth). Suitable reporter genes include but are not limited to chloramphenicol acetyltransferase, beta-D galactosidase, luciferase, or green fluorescent protein.

In an embodiment, such an androgen-mediated process is neuroprotection, as determined by for example the inhibition or prevention of neural cell death or apoptosis as described in the examples below. Cell death or apoptosis may be measured by a number of methods known in the art (see for example Apoptosis Techniques and

Protocols, edited by Judes Poirier, Humana Press, Totowa,

NJ, Vol. 29 of Neuromethods series, Series editor Alan A. Boulton, Glen B. Baker, 1997) . An example of such a method of measuring cell death is TUNEL, as described in

Example 6 below.

Binding activity may be measured by contacting a test compound with an androgen receptor or fragment, homolog or variant thereof which retains binding activity. Using appropriate detection means (e.g. radiolabelling, fluorescence, reporter enzymes, ligand- binding partner systems [e.g. biotin- (strept) avidin] ) and control samples, the amount and affinity of binding of a test compound may be determined.

Androgen-related compounds also comprise compounds which share a similar structure or property (ies) with an androgen or a known androgen-related compound. Examples of androgens include but are not limited to testosterone, testosterone enanthate, methyltestosterone, epitestosterone, and mibolerone. In embodiments, androgen-related compounds include but are not limited to those compounds produced by the modification of an androgen. Such compounds include, for example, certain testosterone esters (e.g. testosterone enanthate; testosterone propionate) , which, upon hydrolysis of the ester, yield active testosterone.

The above mentioned compound could be a peptide, a polypeptide, a fragment of a polypeptide, an organic natural molecule, or a synthetic molecule. Such compounds may have agonistic or antagonistic activity.

Such compounds may further include pro-drugs that are metabolized in vivo to produce a compound as described above. Examples include the testosterone esters noted above.

Accordingly, the invention therefore provides methods of preventing or treating a neural or neurodegenerative disease or a dementia in an animal comprising administering an androgen or an androgen- related compound to an animal. In an embodiment, such an animal is a mammal. In an embodiment, the animal is a human. In an embodiment, the animal is of the male sex. In an embodiment, the animal is female. In certain embodiments, the neural or neurodegenerative disease or dementia is selected from the group consisting of

Alzheimer disease, Parkinson's disease, amyotropic lateral sclerosis (ALS) , cerebellar degeneration, ischemia (stroke), traumatic injuries, prion diseases

(e.g. Creutzfeldt-Jakob disease), Huntington disease, frontal lobe dementia, vascular dementia, infection related dementia (e.g. HIV) and head injury. The above mentioned neural or neurodegenerative disease or dementia may further comprise any condition characterized by or associated with a level of an amyloid beta peptide or an amyloid beta peptide-like compound in neural tissue, including, but not limited to, Alzheimer disease. In

embodiments, such a peptide is Aβι_₄₂. In further embodiments, such conditions include hereditary cerebral amyloidogenesis, where amyloid deposit results in the rupture of blood vessels in the brain (resulting in early death by hemorrhage), and Down's Syndrome, in which the presence of intracellular amyloid peptides precedes extracellular amyloid deposition (Gyure et al . 2001).

The invention further provides a composition for the prevention and/or treatment of a neural or neurodegenerative disease comprising an androgen or androgen-related compound in admixture with a pharmaceutically acceptable carrier.

The invention further provides a use of an androgen or androgen-related compound or the above-mentioned composition for the prevention and/or treatment of a neural or neurodegenerative disease.

The invention further provides a use of an androgen or androgen-related compound for preparation of a medicament for the prevention and/or treatment of a neural or neurodegenerative disease.

The invention further provides commercial packages comprising an androgen or an androgen-related compound or the above-mentioned composition together with instructions for the prevention and/or treatment of a neural or neurodegenerative disease.

In various embodiments, the androgens include but are not limited to testosterone, mibolerone, methyltestosterone and epitestosterone. In an embodiment, the androgen is testosterone.

In various embodiments, an androgen or androgen- related compound may be used therapeutically in formulations or medicaments to prevent or treat a neural or neurodegenerative disease. The invention provides corresponding methods of medical treatment, in which a therapeutic dose of an androgen or androgen-related compound is administered in a pharmacologically acceptable formulation. Accordingly, the invention also provides therapeutic compositions comprising an androgen or androgen-related compound and a pharmacologically acceptable excipient or carrier. The therapeutic composition may be soluble in an aqueous solution at a physiologically acceptable pH. In some embodiments, the androgens or androgen-related compounds of the invention are lipid-soluble or soluble in polar solvents (e.g. ethanol) , which, once dissolved, may be diluted in aqueous solution.

The invention provides pharmaceutical compositions (medicaments) containing (comprising) an androgen or an androgen-related compound. In one embodiment, such compositions include an androgen or androgen-related compound in a therapeutically or prophylactically effective amount sufficient to inhibit neural or neurodegenerative disease, and a pharmaceutically acceptable carrier.

A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result, such as reduction of neural or neurodegenerative disease progression. A therapeutically effective amount of androgen or androgen-related compound may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the compound to elicit a desired response in the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response. A therapeutically effective amount is also one in which any toxic or detrimental effects of the compound are outweighed by the therapeutically beneficial effects. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result, such as preventing or inhibiting neural or neurodegenerative disease onset or progression. A prophylactically effective amount can be determined as described above for the therapeutically effective amount.

For any particular subject, specific dosage regimens may be adjusted over time according to the individual need and the professional judgement of the person administering or supervising the administration of the compositions.

As used herein "pharmaceutically acceptable carrier" or "excipient" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. In one embodiment, the carrier is suitable for parenteral administration. Alternatively, the carrier can be suitable for intravenous, intraperitoneal, intramuscular, sublingual or oral administration. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the invention is contemplated.

Supplementary active compounds can also be incorporated into the compositions.

Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene 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. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or 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, monostearate salts and gelatin. Moreover, the androgen can be administered in a time release formulation, for example in a composition which includes a slow release polymer. The active compounds can be prepared with carriers that will protect the compound against rapid release, 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, polylactic acid and polylactic, polyglycolic copolymers (PLG) . Many methods for the preparation of such formulations are patented or generally known to those skilled in the art.

Sterile injectable solutions can be prepared by incorporating the active compound (e.g. androgen or androgen-related 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. In accordance with an alternative aspect of the invention, an androgen may be formulated with one or more additional compounds that enhance the solubility of the androgen.

In accordance with another aspect of the invention, therapeutic compositions of the present invention, comprising an androgen or androgen-related compound, may be provided in containers or commercial packages which further comprise instructions for use of the androgen for the prevention and/or treatment of a neural or neurodegenerative disease.

Applicants have demonstrated herein that androgens provide neuroprotective effects via different pathways and mechanisms than those conferred by estrogens. Therefore, it is envisioned that combined therapy comprising both the administration of an androgen or androgen-related compound with an estrogen or estrogen- related compound may be further effective in the prevention and/or treatment of a neural or neurodegenerative disease. Thus, in the case of women receiving hormone replacement therapy (HRT) , the additional administration of an androgen or androgen- related compound may improve the neuroprotective effects of HRT. Therefore, a further aspect the invention is a method of preventing or treating a neural or neurodegenerative disease comprising administering a prophylactically or therapeutically effective amount of an androgen or androgen-related compound and an estrogen or estrogen-related compound to an animal. The invention further provides a use of an androgen or androgen-related compound and an estrogen or estrogen-related compound for the prevention or treatment of a neural or neurodegenerative disease, as well as for the preparation of a medicament for the prevention or treatment of a neural or neurodegenerative disease. The invention further provides a composition comprising an androgen or androgen-related compound and an estrogen or estrogen- related compound in admixture with a pharmaceutically acceptable excipient or carrier. The invention further provides a commercial package comprising an androgen or androgen-related compound and an estrogen or estrogen- related compound or the above-mentioned composition together with instructions for preventing or treating a neural or neurodegenerative disease.

The invention further provides screening methods for identifying and characterizing compounds for the prevention and/or treatment of neural or neurodegenerative disease. Such compounds may include the androgen-related compounds as described above.

Accordingly, the invention further provides a method for identifying or characterizing a test compound for the prevention and/or treatment of neural or neurodegenerative disease, said method comprising: contacting said test compound with a cell comprising an androgen receptor; measuring a test level of androgen- associated activity; comparing said test level of androgen-associated activity with a corresponding control level of androgen- associated activity in a corresponding cell which was not exposed to said test compound.

A difference between the test level and the control level may indicate that the test compound may be used for prevention and/or treatment of a neural or neurodegenerative disease. The just noted difference may in various embodiments represent an increase or a decrease, depending on the activity measured.

In an embodiment, the cell is a neural cell. In an embodiment, the androgen-associated activity is neuroprotection or the inhibition or prevention of cell death or apoptosis. In an embodiment, an increase in neuroprotection or a decrease in cell death or apoptosis as a result of treatment with the test compound indicates that the test compound can be used for the prevention and/or treatment of neural or neurodegenerative disease. In an embodiment, the above-mentioned cell comprising an androgen receptor is a cell which comprises endogenous levels or expression of an androgen receptor.

The cell may also comprise an appropriate host cell comprising an exogenously introduced source of androgen receptor. Such a host cell may be prepared by the introduction of a nucleic acid encoding an androgen receptor into the host cell and providing conditions for the expression of an androgen receptor. In an embodiment, such a nucleic acid is DNA. Such host cells may be prokaryotic or eukaryotic, bacterial, yeast, amphibian or mammalian. In an embodiment, such host cells are human.

A homolog, variant and/or fragment of an androgen receptor which retains activity may also be used in the methods of the invention. Homologs include protein sequences which are substantially identical to the amino acid sequence of an androgen receptor, sharing significant structural and functional homology with an androgen receptor. Variants include, but are not limited to, proteins or peptides which differ from an androgen receptor by any modifications, and/or amino acid substitutions, deletions or additions. Modifications can occur anywhere including the polypeptide backbone, (i.e. the amino acid sequence) , the amino acid side chains and the amino or carboxy termini. Such substitutions, deletions or additions may involve one or more amino acids. Fragments include a fragment or a portion of an androgen receptor or a fragment or a portion of a homolog or variant of an androgen receptor. The above-mentioned method may be employed either with a single test compound or a plurality or library (e.g. a combinatorial library) of test compounds. In the latter case, synergistic effects provided by combinations of compounds may also be identified and characterized. The above-mentioned compounds may be used for prevention and/or treatment of neural or neurodegenerative disease or may be used as lead compounds for the development and testing of additional compounds having improved specificity, efficacy and/or pharmacological (e.g. pharmacokinetic) properties. The above-mentioned method may further be used to identify agonists or antagonists of androgen receptors. In an embodiment, the above- mentioned test compound may be selected from a group of compounds having similar structure to an androgen or and androgen-related compound and/or is known to have androgen receptor binding activity. In the case of the latter embodiment, the above-mentioned method has a greater probability of identifying a compound for prevention and/or treatment of neural or neurodegenerative disease. In certain embodiments, one or a plurality of the steps of the screening/testing methods of the invention may be automated.

The reduction of cell death/apoptosis by androgen or androgen-related compound as described herein indicates the presence of various androgen-associated mechanisms involved in cell death. Therefore, the invention further provides systems and methods for the identification and characterization of such mechanisms. Such systems and methods may comprise a comparison of androgen treated cells versus corresponding untreated cells, with respect to cell death. In an embodiment, such cells are neural cells. Such methods may be utilized for screening, wherein mutations resulting in cell death-associated phenotypes which are androgen dependent may be identified, and subsequently the mutation may be identified at the genotypic level, giving rise to the identification of a gene involved in androgen-associated cell death mechanisms. For example, if the amount or nature of neuroprotection typically conferred by an androgen is different in a certain cell, that cell harbors an alteration, such as a mutation, which gives rise to this difference. Such a mutation is thus associated with a gene involved in a mechanism of androgen-mediated protection. In embodiments, the above noted mutation renders the cell less responsive or unresponsive to androgen-mediated inhibition or prevention of cell death.

Accordingly, the invention further provides a method of identifying and/or characterizing mechanisms and/or components associated with androgen-mediated effects on cell death, said method comprising: assessing an alteration in a cell death- associated phenotype between an androgen or androgen- related compound-treated cell and a corresponding untreated cell comparing said alteration with a corresponding alteration assessed in corresponding androgen treated versus untreated mutant cells; wherein differences in said alteration are used to identify and/or characterize mechanisms and/or components associated with androgen-mediated effects on cell death. In various embodiments, the androgen is mibolerone, methyltestosterone or epitestosterone. In an embodiment, the androgen is testosterone. In an embodiment the above-mentioned cell is a neural cell. In an embodiment, the just noted difference in said alteration is a reduction or elimination of responsiveness to androgen-mediated inhibition and/or prevention of cell death. Although various embodiments of the invention are disclosed herein, many adaptations and modifications may be made within the scope of the invention in accordance with the common general knowledge of those skilled in this art. Such modifications include the substitution of known equivalents for any aspect of the invention in order to achieve the same result in substantially the same way. Numeric ranges are inclusive of the numbers defining the range. In the claims, the word "comprising" is used as an open-ended term, substantially equivalent to the phrase "including, but not limited to". The following examples are illustrative of various aspects of the invention, and do not limit the broad aspects of the invention as disclosed herein.

EXAMPLES

Example 1: Demonstration of androgen-mediated and estrogen-mediated neuroprotection against serum- deprivation-mediated apoptosis of human primary neurons. Estrogen is hypothesized to play an important role against Alzheimer's disease in women (Birge 1996). Women receiving HRT are less susceptible to Alzheimer's disease

(Tang et al . 1996). As HRT often contains androgens (Gelfand 1992) and men's susceptibility to Alzheimer's disease increases with age in parallel with reduced levels of androgens (Vermeulen 1991) . Applicants have investigated the role of testosterone enanthate, methyltestosterone and epitestosterone in neuroprotection against serum deprivation in primary cultures of human neurons.

It has been shown that these human neurons undergo a protracted form of cell death with active recombinant caspases (Zhang et al . 2000). Serum deprivation also induces a protracted cell death but there is a significant amount of neuronal cell death by serum deprivation within 24 h (p < 0.005) (Fig. 2a). The addition of physiological concentrations of 4 nM testosterone enanthate to serum-deprived neurons eliminates apoptosis completely for 24 and 48 h and significantly reduces apoptosis at 72 and 96 h of treatment. In contrast, methyltestosterone did not significantly inhibit apoptosis at 24 h, but did show a 20% reduction in apoptosis between 48 and 96 h of treatment (p < 0.05). The anti-androgen, epitestosterone also had no statistically significant effect at 24 h but reduced apoptosis by 20-40% from 48 to 96 h of treatment (p > 0.05). As shown (Behl et al . 1997; Green et al . 1997; Pike 1999; Green and Simpkins 2000), 2 nM

physiological concentrations of 17-β- (p < 0.005) and 17-

α-estradiol (p < 0.05) were also neuroprotective from 24 to 96 h of treatment.

Addition of 10 times less or between 10 and 100 times more hormone to serum-deprived neurons shows that the lower 0.2 or 0.4 nM concentration are slightly more neuroprotective than the 2 and 4 nM concentration (Fig. 2b) . Increasing the levels of hormone slightly reduced

the neuroprotective effect in 17-α-estradiol and androgen treated cultures. These results indicate that the neuroprotective effect of androgens and estrogens at physiological concentrations is receptor mediated and rules out a possible antioxidant function in neuroprotection. It is possible that the higher concentrations of hormones are slightly toxic to neurons thereby reducing the neuroprotective effect. Most importantly, the significant neuroprotective effect observed with the lowest dose of hormone indicates the strong neuropotency of these hormones. Example 2: Demonstration of direct androgen-mediated protection of neurons without aromatization into estrogens

To determine if the neuroprotective role of androgens was directly through androgen receptors or through aromatization into estrogens (Balthazart and Ball 1998), applicants first determined if androgen receptors were present in the cultures studied using non- aromatizable [³H]mibolerone in a binding assay. Results described herein show that androgen receptors are present at 8 ± 2 fmoles/mg protein. The effect of the non- aromatizable androgen, mibolerone, on neuroprotection was assessed (Fig. 3) . The results herein show that similar to testosterone, mibolerone significantly protects neurons against serum deprivation even after 96 h of serum deprivation. The neuroprotective effect of mibolerone is not as strong as that of testosterone. Similarly, methyltestosterone also shows neuroprotection at a lower level than in testosterone. Comparison of the chemical structure of these compounds (Fig. 1) indicates that the presence of the 17-methyl group decreases the neuropotency of androgens. The addition of mibolerone to testosterone, epitestosterone or methyltestosterone does not increase neuroprotection in cells treated with hormone in absence of mibolerone suggesting that both mibolerone and natural androgens act through the same receptor. The affinity of mibolerone is at least 100- fold higher than testosterone, epitestosterone or methyltestosterone (Wilson and French 1976; Turcotte et al . , 1988). Therefore, mibolerone binding to the androgen receptor would compete out the other androgens as evidenced by the levels of neuroprotection consistent with a mibolerone-specific effect. Together, these results indicate that androgens can be directly neuroprotective without being aromatized to estrogen.

To confirm that testosterone is not acting by aromatization into estrogen, a cell permeable aromatase inhibitor, 4-androsten-4-OL-3, 17-dione, was added to the testosterone-treated neurons (Fig. 4). The results show that the aromatase inhibitor does not have a significant effect on neuronal cell death by serum deprivation (p > 0.8). In the presence of testosterone, 4-androsten-4-OL- 3, 17-dione, does not prevent testosterone-mediated neuronal protection (p > 0.4). These results confirm the direct action of testosterone rather than an indirect effect through aromatization into estrogens.

To determine if activation of the androgen receptor results in the neuroprotective action of testosterone, the effect of a non-steroid pure anti-androgen, flutamide (Simard et al . 1986) on testosterone-mediated neuroprotection was assessed (Fig. 5) . Flutamide alone

at either 2 or 20 μM does not have a significant effect on neuronal survival or cell death. However, flutamide significantly abolishes testosterone-mediated neuroprotection. Together with the mibolerone and aromatase inhibitor studies, these results strongly suggest that the neuroprotective function of testosterone occurs through the androgen receptor. Flutamide also significantly inhibits methyltestosterone-mediated neuroprotection. The effect is not as significant as seen with testosterone. However, methyltestosterone is also less neuroprotective than testosterone either because of the 17-methyl group as discussed later or the lower affinity of methyltestosterone for the androgen receptor (Wiita et al . 1995). Alternatively, the slow neuroprotective effect of methyltestosterone, which is only observed after 48 h of treatment, indicates that metabolites of methyltestosterone may be produced over time and promote neuroprotection through both androgen receptor dependent (antagonized by flutamide) and androgen receptor independent (not antagonized by flutamide) mechanisms.

In contrast, flutamide could not inhibit the endogenous anti-androgen, epitestosterone-mediated neuroprotection indicating that ■ the neuroprotection of epitestosterone is independent of androgen receptors.

Together, these results confirm that the neuroprotective effect of testosterone depends on an interaction with androgen receptors and can be competed out with antagonists .

Example 3: Neuronal culture

Human fetal brain tissue (12-16 weeks) was obtained in accordance with the guidelines established by the Medical Research Council and approved by the Institutional Review Board of McGill University. Neurons were isolated and cultured as previously described (LeBlanc 1995) . To summarize, brain tissue was minced in phosphate buffered saline and dissociated with 0.25% trypsin (Gibco-BRL, Rockville, MD, USA) . The cells were subsequently treated with 10% serum and 0.1 mg/ml deoxyribonuclease I (Roche Molecular Biochemical, Indianapolis, IN, USA) and the resulting homogenate

filtered through 130- and 170- μm nylon mesh. The neurons were plated at 3 x 10⁶ cells/mL on poly-L-lysine

(Sigma Chemicals, St Louis, MO, USA) coated ACLAR™ (33C; 5 mm; Allied Chemical Corp.) coverslips and cultured in vitro for 10 days. The media contains phenol-free minimal essential media in Earle's balanced salt solution containing 0.225% sodium bicarbonate, 1 mM sodium pyruvate, 2 mM L-glutamine, 0.1% dextrose, I x antibiotic

Pen-Strep (all products from Gibco-BRL) and 5% decomplemented fetal bovine serum (HyClone, Logan. UT,

USA) . In complete serum containing media, the basal amount of testosterone is present at 9 pM and estrogen is at 18 pM.

Example 4: Neuronal treatment

The neurons were serum-deprived in the absence or

presence of 2 nM 17-α-estradiol or I7-β-estradiol, 4 nM testosterone enanthate, epitestosterone or methyltestosterone. (The concentration represents peak physiological levels in reproductive age women and men.) All hormones were purchased from Sigma (St Louis, MO, USA) except methyltestosterone obtained from USP. Testosterone enanthate was used because ester increases the duration and action of testosterone. Testosterone enanthate will not bind the androgen receptor unless the ester is hydrolyzed. The testosterone enanthate is hydrolyzed into testosterone in the neuronal cultures as evidenced by the antagonistic effect of flutamide. The chemical structure of these compounds is shown in Fig. 1. The media was changed every 48 h. Hormones were dissolved in various stock concentrations in 100% ethanol and added to the media to give final concentrations of 2 and 4 nM or the indicated dose with equivalent amounts of ethanol. Control serum-deprived neurons receive the equivalent amount of ethanol. Similarly, mibolerone, flutamide and aromatase inhibitor were dissolved in ethanol and added to the media to give final concentrations of 3 nM mibolerone (DuPont NEN, Boston.

MA, USA), 2 μM and 20 μM flutamide (Sigma, St Louis, MO, USA) , or 5 ng/mL and 50 ng/mL 4-androsten-4-OL-3, 17- dione (Sigma. St Louis, MO, USA) . At the end of the treatment, coverslips were fixed with 4% paraformaldehyde, 4% sucrose in phosphate buffered solution (Harlow and Lane 1988).

Example 5: Determination of androgen receptors

Androgen receptors were identified by incubating 6 nM [³H] mibolerone (DuPont NEN, Boston, MA. USA; Spec. Act. 85 Ci/mmol) with 6 x 10⁶ neurons to measure total binding. Non-specific binding was assessed by competing the binding of [³H] -mibolerone with a 200-fold excess cold mibolerone as previously described (Kaufman et al . 1993). Specific binding was determined by subtracting non- specific from total binding and dividing by the protein concentration as determined by the Lowry assay (Lowry et al . 1951) .

Example 6: Determination of neuronal cell death by TUNEL

Fixed neurons were pemeabilized with 0.1% Triton X- 100 in 0.1% sodium citrate. Cell death was detected by TUNEL (TdT-mediated dUTP nick-end labeling) using the Cell Death Kit I (Roche Molecular Biochemicals,

Indianapolis, IN. USA) as described by the manufacturer. All cells were counterstained with 100 ng/mL propidium iodide (Pharmingen) to allow confirmation of the apoptotic morphology of the cells and to detect the total number of cells present under fluorescence microscopy. The percentage of neuronal cell death was determined by screening five areas of each coverslip (a minimum of 500 cells) and comparing the total number of TUNEL-positive (green fluorescence) and morphologically apoptotic cells over the total number of cells (red fluorescence) present in each sample. The neuronal cell death was confirmed in representative experiments of each assay with MTT reducing assays. Briefly, neurons were plated directly into 24-well plates and studied for MTT reduction using the Cell Proliferation Kit I (MTT) as described by the manufacturer (Roche Molecular Biochemicals, Indianapolis,

IN, USA; data not shown). A two-tailed Student's t-test for unpaired samples was used for comparison between the level of neuronal cell death in serum-deprived neurons and that in serum-deprived neurons treated with hormones, p-values of <0. 05 were used as indicative of statistical significance.

Example 7 : Aβι-₄₂ -mediated neurotoxicity is protected by steroid hormones.

As presented herein, applicants have demonstrated that testosterone and estradiol protect neurons against serum deprivation-mediated apoptosis. In this study applicants have further tested if any of the androgens and estrogens could prevent Aβι_₂ -mediated neurotoxicity.

Protocol :

A toxic dose of Aβ_x_₂ and Dextran Texas Red fluorescent dye marker was microinjected into neurons and neurons were immediately treated with varying doses of 17-β-estradiol, transcriptionally inactive 17-β-estradiol analogue, testosterone enanthate, methyltestosterone and epitestosterone. Microinjected neurons were incubated for 24 hours, fixed and submitted to TUNEL analysis to assess cell death by Aβι_₄₂. The experiment was done on 100 microinjected cells/well x 2 wells/experiments in three independent neuron preparations.

As shown in Figure 6, Aβι_₄₂ induces TUNEL-positive cell death in 60-70% of neurons within 24 hours of injection. A statistically significant difference between the various groups is observed by ANOVA analysis of the data (p<0.0001, df=15) . Post-hoc analysis with Dunnett's test shows that a physiological concentration of 2 nM of 17-β-estradiol and methyltestosterone prevent Aβι-₄₂ mediated neuronal cell death by 50% (p<0.01).

Testosterone is less but still neuroprotective (p<0.05 for 2 and 4 nM and p<0.01 at 10 nM) . In contrast, epitestosterone and 17-β-estradiol do not protect against Aβι_₄₂ -mediated apoptosis.

Figure 7 illustrates further results demonstrating that estrogen and androgens protect human neurons against

intracellular Aβι_₄₂-mediated toxicity in primary cultures.

Neurons were microinjected with a lethal dose of Aβι-₄₂ and

immediately treated with 2, 4, or 10 nM 17-β-estradiol

(βE2) , 17-α-estradiol (αE2), testosterone enanthate (Test), epitestosterone (Epi-Test), methyl testosterone

(Methyl-test) or cell impermeable BSA conjugated-7-β-

estradiol (BSA-βE2) . Neuronal cell death was assessed by TUNEL 24 hours after treatment. % cell death represents the number of TUNEL positive neurons over total neurons

(stained with DAPI) . Each sample represents 200 microinjected cells from each of a minimum of three experiments done on different neuron preparations.

*p<0.01 relative to Aβι-₄₂-microinjected cells in absence of hormones.

Figure 8 illustrates further results obtained similarly to those described for figure 7, but in this case the neurons were pre-incubated with 10 nM hormones

for 1 hour prior to microinjection with Aβι-₄₂. Treatment with hormones was continued for 24 hours before measuring cell death as above. These results indicate a that these hormones may be highly neuroprotective, particularly when administered prior to Aβι_₄₂ treatment.

Figure 9 illustrates results demonstrating that estrogens and androgens protect through their respective receptors. The neurons were pre-treated with either hormone or hormone and antagonist (tamoxifen for estrogens and flutamide for androgens) for one hour and

microinjected with the Aβι_₂. Incubation of 24 hours followed by cell death measures was performed as described above. * p<0.01.

Applicants therefore conclude that both physiological concentrations of androgens and estrogen can protect against intracellular Aβ_x_₄₂ neurotoxicity. The role of estrogen against extracellular Aβ toxicity has previously been reported (Bonnefont et al . , 1998; Goodman et al . , 1996; Green et al . , 1996; Gridley et al . , 1997; Gridley et al . , 1998; Gursoy et al . , 2001; Keller et al . ,

1997; Kim et al . , 2001; Mook-Jung et al . , 1997; Svensson and Nordberg, 1999; Thomas et al . , 2001). However, prior to applicants' studies presented herein, it has not been shown that either hormone protects against "intracellular" Aβ-mediated neurotoxicity, nor has the effect of androgens against Aβ-mediated toxicity been shown. Further, applicants have demonstrated nearly complete protection, particularly in cases where hormone was added prior to Aβ introduction. Competition studies described herein with specific antagonists demonstrate that the protective activity of estrogens and androgens is manifested through their respective receptors.

All references cited above or in the References section below are herein incorporated by reference.

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Claims

WHAT IS CLAIMED IS:

1. A method of preventing or treating a neural disease in an animal, said method comprising administering an androgen or androgen-related compound to said animal.

The method of claim 1 wherein said animal is a mammal ,

The method of claim 2, wherein said mammal is a human.

The method of claim 1, wherein said animal is male,

5. The method of claim 1, wherein said neural disease is selected from the group consisting of Alzheimer disease, Parkinson's disease, amyotropic lateral sclerosis (ALS) , cerebellar degeneration, ischemia (stroke) , traumatic injuries, prion diseases, Huntington disease, frontal lobe dementia, vascular dementia, infection related dementia, head injury, hereditary cerebral amyloidogenesis, Down's Syndrome and cerebral hemorrhage.

6. The method of claim 1, wherein said androgen is selected from the group consisting of testosterone, mibolerone, methyltestosterone and epitestosterone.

7. The method of claim 6, wherein said androgen is testosterone.

8. The method of claim 1, wherein said androgen- related compound is a testosterone ester.

9. The method of claim 8, wherein said testosterone ester is selected from the group consisting of testosterone enanthate and testosterone propionate.

10. A composition for preventing or treating a neural disease in an animal, said composition comprising an androgen or androgen-related compound in admixture with a pharmaceutically suitable carrier.

11. Use of an androgen or androgen-related compound or the composition of claim 10 for preventing or treating a neural disease in an animal.

12. The use of claim 11, wherein said neural disease is selected from the group consisting of Alzheimer disease, Parkinson's disease, amyotropic lateral sclerosis (ALS) , cerebellar degeneration, ischemia (stroke) , traumatic injuries, prion diseases, Huntington disease, frontal lobe dementia, vascular dementia, infection related dementia, head injury, hereditary cerebral amyloidogenesis, Down's Syndrome and cerebral hemorrhage.

13. The use of claim 11, wherein said androgen is selected from the group consisting of testosterone, mibolerone, methyltestosterone and epitestosterone.

14. The use of claim 13, wherein said androgen is testosterone .

15. The use of claim 11, wherein said androgen- related compound is a testosterone ester.

16. The use of claim 15, wherein said testosterone ester is selected from the group consisting of testosterone enanthate and testosterone propionate.

17. Use of an androgen or androgen-related compound for preparation of a medicament for preventing or treating a neural disease in an animal.

18. A commercial package comprising an androgen or androgen-related compound together with instructions for the prevention or treatment of a neural disease.

19. A commercial package comprising the composition of claim 10 together with instructions for the prevention or treatment of a neural disease.

20. The commercial package of claim 18, wherein said neural disease is selected from the group consisting of Alzheimer disease, Parkinson's disease, amyotropic lateral sclerosis (ALS) , cerebellar degeneration, ischemia (stroke) , traumatic injuries, prion diseases, Huntington disease, frontal lobe dementia, vascular dementia, infection related dementia, head injury, hereditary cerebral amyloidogenesis, Down's Syndrome and cerebral hemorrhage.

21. The commercial package of claim 18, wherein said androgen is selected from the group consisting of testosterone, mibolerone, methyltestosterone and epitestosterone .

22. The commercial package of claim 21, wherein said androgen is testosterone.

23. The commercial package of claim 18, wherein said androgen-related compound is a testosterone ester.

24. The commercial package of claim 23, wherein said testosterone ester is selected from the group consisting of testosterone enanthate and testosterone propionate .

25. A method for preventing or inhibiting cell death, comprising treating a cell with an androgen or androgen-related compound.

26. The method of claim 25, wherein said cell is a human cell.

27. The method of claim 25, wherein said cell is a neural cell.

28. The method of claim 25, wherein said androgen is selected from the group consisting of testosterone, mibolerone, methyltestosterone and epitestosterone.

29. The method of claim 28, wherein said androgen is testosterone.

30. The method of claim 25, wherein said androgen- related compound is a testosterone ester.

31. The method of claim 30, wherein said testosterone ester is selected from the group consisting of testosterone enanthate and testosterone propionate.

32. A method of identifying or characterizing a test compound for prevention and/or treatment of neural or neurodegenerative disease, said method comprising: contacting said test compound with a cell comprising an androgen receptor; - measuring a test level of androgen- associated activity; and comparing said test level of androgen-associated activity with a corresponding control level of androgen- associated activity in a corresponding cell which was not exposed to said test compound; wherein a difference between said test level and said control level indicates that the test compound may be used for prevention and/or treatment of neural or neurodegenerative disease.

33. The method of claim 32, wherein said cell is a neural cell.

34. The method of claim 32, wherein said androgen- associated activity is an inhibition or prevention of cell death or apoptosis.

35. A method of identifying and/or characterizing a mechanism and/or component associated with androgen- mediated effects on cell death, said method comprising: assessing a first alteration in a cell death- associated phenotype between an androgen treated cell and a corresponding untreated cell; and comparing said alteration with a corresponding second alteration assessed in corresponding androgen treated versus untreated mutant cells; wherein differences in said first and second alterations are used to identify and/or characterize a mechanism and/or component associated with androgen-mediated effects on cell death.