WO2005009359A2 - Agents, compositions and methods for enhancing neurological function - Google Patents

Abstract

Neuro-enhancing agents, compositions and methods are disclosed herein. Preferred neuro-enhancing agents of the present invention include progesterone and metabolites of progesterone, such as 3α-hydroxy-5α-pregnan-20-one (THP). These agents yield neuro-enhancing effects on neural cells that include neural progenitor and/or stem cells, whereby the agents stimulate mitosis of neural progenitor cells, stimulate neurite growth and organization, protect against neural loss, or one or more of these neural processes. Thus, the neuro-enhancing agents, compositions and methods disclosed herein are useful to reverse or prevent neurological disease or defects associated with neural loss or degeneration., such as Alzheimer's disease, neurological injuries, including injuries resulting from radiation therapy, and age-related neurological decline, including impairments in memory and learning.

Description

AGENTS, COMPOSITIONS AND METHODS FOR ENHANCING NEUROLOGICAL FUNCTION

RELATED APPLICATIONS

This application is related to and claims priority from U.S. provisional patent application, Serial No.: 60/487,688, filed on July 15, 2003, which is incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

The mammalian nervous system includes a peripheral nervous system (PNS) and a central nervous system (CNS), including the brain and spinal cord, and is composed of two principal classes of cells, namely neurons and glial cells. The glial cells fill the spaces between neurons, nourishing them and modulating their function. During development, differentiating neurons from the central and peripheral nervous systems send out axons that grow and make contact with specific target cells. In some cases, axons must cover enormous distances with some growing into the periphery, whereas others are confined within the central nervous system. J-n mammals, this stage of neurogenesis is thought to be complete during the embryonic phase of life. Further, neuronal cells are generally thought not to multiply once they have fully differentiated.

A host of neuropathies, including neurodegenerative diseases, have been identified that affect the nervous system of mammals. These neuropathies, which may affect neurons themselves or associated glial cells, may result from cellular metabolic dysfunction, infection, injury, exposure to toxic agents, autoimmunity, malnutrition, ischemia or may be due to age-related neurological changes. In some cases, the neuropathy is thought to induce cell death directly, hi other cases, the neuropathy may induce sufficient tissue necrosis to stimulate the body's immune/inflammatory system and the immune response to the initial injury then destroys neural pathways. Also, neuronal tissue may be lost as a result of physical insult or trauma. Loss of neurons, either directly or indirectly, was thought to be irreversible in the adult human brain, as it was long held that the generation of new neurons did not occur in the mature brain. In most brain regions, the generation of neurons is generally confined to a discrete developmental period. However, notable exceptions are found in the dentate gyrus and the subventricular zone of several species where it has been shown that new neurons are generated well into the postnatal and adult period. Granule neurons are generated throughout life from a population of continuously dividing neural progenitor cells residing in the sub granular zone of the dentate gyrus in the rodent brain. "Newborn" neurons generated from these neural progenitor cells migrate into the granule cell layer, differentiate, extend axons and express neuronal marker proteins. The mechanisms and appropriate stimuli that promote the generation of new neurons, however, are largely unknown.

Attempts to counteract the effects of acute or neurodegenerative lesions of the brain and/or spinal cord have primarily involved implantation of embryonic neurons in an effort to compensate for lost or deficient neural or neurological function. However, human fetal cell transplantation research is severely restricted. Administration of neurotrophic factors, such as nerve growth factor and insulin-like growth factor, also has been suggested to stimulate neuronal growth within the CNS.

To date, however, no satisfactory agents or treatment methods exists to repair, or counteract, the neuronal damage associated with neuropathies, such as Parkinson's disease and Alzhemier's disease, neurological injury or neurological age-related decline or impairment. Accordingly, there is a need for new treatment modalities directed to improving the adverse neurological conditions associated with neuropathies, neurological injuries and age-related neurological decline or impairment. SUMMARY OF THE INVENTION The present invention provides for therapeutic agents, compositions and treatment methods for neuro-enhancement in an individual who has undergone or is undergoing loss in neurological function due to neurological disease, neurological injury or age- related neurological decline or impairment. One aspect of the invention is directed to methods for neuro-enhancement in an individual, who has undergone or is undergoing loss in neurological function. The methods generally include contacting the neural cells of the individual with one or more neuro-enhancing agents disclosed herein, thereby leading to an improvement or a restoration of neurological function. A particular embodiment of the methods disclosed herein for enhancing neurological function in an individual includes contacting the neural cells of the individual neuro-enhancing agents comprising 3α-hydroxy-5α-pregnan-20-one or a substantially equivalent variant molecule.

The methods for enhancing neurological function in an individual can be practiced in-vivo and/or ex-vivo. Both the in-vivo and ex-vivo methods generally include contacting a population of neural cells, which include neural progenitor cells or neural stem cells. In-vivo administration includes contacting the neural cells of an individual with one or more neuro-enhancing agents of the present invention via injection, infusion, implantation, inhalation, oral delivery, topical delivery or the like. On the other hand, ex- vivo administration includes contacting apαpulations of neural cells with-one-or more neuro-enhancing agents of the present invention ex-vivo to yield an expanded population of neural cells. Following ex-vivo expansion of neural cells, the expanded population of neural cells is directly administered to one or more central nervous regions of the individual. A preferred method for direct administration of the expanded population of neural cells is via intracerebroventricular infusion. These ex-vivo methods may further include directly administering the neuro-enhancing agents to the individual in-vivo before, after, or before and after the expanded population of neural cells is administered to the individual. Other aspects of the invention are therapeutic methods for enhancing neurological function in an individual with a neurological disease, neurological injury or age-related neuronal decline or impairment. These therapeutic methods include administering to an individual an effective amount of one or more neuro-enhancing agents disclosed herein, which generally include 3α -hydroxy-5α -pregnan-20-one or a substantially equivalent variant molecule, over a period of time effective to stimulate neural mitosis, to prevent neuronal loss, or to both stimulate neural mitosis and to prevent neuronal loss. The administration of the effective amounts of these agents to the individual over the effective period of time leads to enhancement of neurological function in the individual. Effective administration periods depend on the particular neurological disease or defect being targeted. Generally effective administration periods are about one month or longer, but can be about six months to about one year or longer. Effective therapeutic amounts of the neuro-enhancing agents also will depend on the neurological disease or defect being targeted, but generally range from about 10 mg to 1000 mg. These amounts can be administered on a daily basis. Target neurological dysfunctions and disease states include Alzheimer's disease, neurological injuries, including those following radiation therapy for brain-related cancers, and age-related memory decline and age-related learning impairments

Another aspect of the invention provides for therapeutic, neuro-enhancing agents and comμositions that improve or restore neural or neurological function by inducing or stimulating the generation of new neurons, protecting against neuronal loss, stimulating or inducing neurite outgrowth and organization or protecting against loss of neurites and neural networks, or one or more of these processes. The neuro-enhancing agents and compositions provided in the present invention are generally progesterone variants, including progesterone, precursors of progesterone, progesterone metabolites and progesterone derivatives in its metabolic pathway. In a preferred embodiment, the present invention includes therapeutic agents and compositions comprising a naturally occurring metabolite of progesterone, namely 3 -hydroxy-5α -pregnan-20-one, also known as tetrahydroprogesterone (THP). Other preferred embodiments of the neuro- enhancing agents and compositions of the invention include substituted 3 beta- phenylethynyl derivatives of 3α-hydroxy-5α-pregnan-20-one. Still other preferred embodiments of the neuro-enhancing agents and compositions of the invention include variant molecules of 3α-hydroxy-5α-pregnan-20-one that exhibit substantially equivalent neuro-enhancing activity as 3α-hydroxy-5α-pregnan-20-one.

Variant molecules of 3α -hydroxy-5α -pregnan-20-one include progesterone, and progesterone-like molecules, which are either natural metabolites of progesterone or synthetic variants of progesterone, and exhibit substantially equivalent neuro-enhancing activity as 3α-hydroxy-5α-pregnan-20-one. As defined in the present invention, the process of neuro-enhancement resulting from the use of the agents, compositions and treatment methods of the invention includes the stimulation or induction of neural mitosis leading to the generation of new neurons, i.e., exhibiting a neurogenic effect, stimulation or induction of neurite growth and organization, prevention or retardation of neural loss, including a decrease in the rate of neural loss, i.e., exhibiting a neuroprotective effect, or one or more of these modes of action. Substantially equivalent neuro-enhancing activity is defined as approximately 30% to approximately 300% of the neurogenic activity of 3α-hydroxy-5α-pregnan-20-one.

Still other aspects of the present invention include pharmaceutical composition for enhancing neurological function in an individual. These pharmaceutical compositions include one or rriore neuro-enhancing agents, preferably including 3α-hydroxy-5α- pregnan-20-one or a substantially equivalent variant molecule contained in a pharmaceutically acceptable carrier, diluent or stabilizer. Preferably the carrier, diluent or stabilzer maintains the active form of the neuro-enhancing agents and is suitable for administration to the individual. A preferred embodiment of the pharmaceutical compositions of the invention is suitable for oral administration to the individual. The pharmaceutical compositions of the invention generally include about 10 mg or greater of the pharmaceutically active form of 3α-hydroxy-5α-pregnan-20-one or a substantially equivalent variant molecule. Generally these pharmaceutical compositions include about 0.1 mg to about 100 mg of pharmaceutically active form of 3α-hydroxy-5α-pregnan-20- one or a substantially equivalent variant molecule, but active doses may include 500 mg or greater of one or more neuro-enhancing agents. Effective doses of active forms of the neuro-enhancing agents of the present invention, of course, can be adjusted to suit the particular neurological disease or defect being targeted.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 depicts a graphic representation of the effect of 3α-hydroxy-5α-pregnan-20-one administration on the percent of the total number of hippocampal neural cells exhibiting a mitotic appearance, i.e., exhibiting a doublet form cell body indicative of mitosis; data are expressed as percent the total number of neurons exhibiting mitotic phenotype, mean ± SEM, **p<.01, ***ρ<.001.

Figure 2 depicts show the effect of THP administration on hippocampal neurons relative to the number of control neuron with a mitotic appearance, where mitotic appearance was defined as a double cell body indicative of mitosis; data are expressed as percent of mean ± SEM, **p<.01, ***ρ<.001.

Figure 3 depicts a hippocampal neuron that was treated with THP and immunostained with antibodies for the nuclear proliferation marker, ki-67 antigen, which appears in yellow; the figure also depicts mitosis of the neuron, as derived from a population of hippocampal neurons; in the figure the cytoplasms of the donor and daughter cells have not yet completely s-eparated.

Figure 4 shows that THP increases expression of cell division of control protein 2 (cdc2) in hippocampal neurons; neurons were collected following 24hrs of THP exposure; forty μg protein of the total cell lysate was loaded and separated by 12% SDS-gel using antibody specifically against cdc2 and analyzed.

Figure 5 reveals that THP increases the total number of hippocampal neurons where neurons were treated AP for 1 day and cell number determined by Coulter counter and verified by microscopic hemocytometer cell counting analysis. Figure 6 shows that THP can increase neuron number as assessed in MuLN-GFP infected mouse neurons; the effect of THP on HT-22 cells proliferation was detected on MuLN infected cells; left panel shows the FACS profile of vehicle; right panel shows the FACS profile of THP treated MuLV-GFP infected cells; the accompanying table (Table 1) summarizes the FACS results. V = vehicle; THP (250 nM).

Figure 7 shows the dose response of 3α-hydroxy-5 -pregnan-20-one administration on ³H-thymidine incorporation in hippocampal neural cells as measured by ³H-thymidine incoiporation.

Figure 8 graphically depicts the steroid specificity of 3α -hydroxy-5α -pregnan-20-one (THP), as compared to other structurally and chemically similar steroids by measuring the THP-induced ³H-thymidine incorporation in hippocampal neural cells.

Figure 9 depicts the time course 3 -hydroxy-5α-pregnan-20-one-induced ³H-thymidine incoiporation in hippocampal neural cells at 1 hour, 8 hour and 24 hour time intervals.

Figure 10 shows the effect of 3α-hydroxy-5α-pregnan-20-one administration on rat neural stem/ progenitor cell growth.

Figure 11 shows that THP promotes the proliferation of human neural stem cells; fetal cortex derived neural stem cells were treated with varying concentrations of APα [1- 1000 nM] or with bFGF [20 ng/ml] + heparin [ 5 μg/ml] as a THP positive control.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention is directed to therapeutic, neuro-enhancing agents, compositions and treatment methods for enhancing neurological function in an individual, preferably the individual is a mammal, and more preferably the mammal is a human who has lost some amount of neurological function as a result of neurological disease, neurological injury or age-related neurological decline or impairment. Thus, individuals who can be treated by the neuro-enhancing agents, compositions and methods of this invention include living organisms, e.g. mammals, susceptible to age- and/or disease-related neural loss, or neural loss resulting from injury. Generally neural loss implies any neural loss at the cellular level, including loss in neurites, neural organization or neural networks. Examples of subjects include humans, dogs, cats, rats, and mice. Lower mammal models using, for example, rats or mice can be used to predict modes of general brain aging and associated neuronal loss in higher mammals, such as humans.

As defined in the present invention, the process of neuro-enhancement resulting from the use of the agents, compositions and treatment methods of the invention includes the stimulation or induction of neural mitosis leading to the generation of new neurons, i.e., exhibiting a neurogenic effect, prevention or retardation of neural loss, including a decrease in the rate of neural loss, i.e., exhibiting a neuroprotective effect, or one or moreof these modes of action. The term "neuroprotective effect" is intended to include prevention, retardation, and/or termination of deterioration, impairment, or death of an individual's neurons, neurites and neural networks, hi accordance with the present invention, an agent, composition or method possesses neuro-enhancing properties if administration of the agent, composition or method to an individual leads to an improvement, or enhancement, of neurological function in an individual with a neurological disease, neurological injury, or age-related neuronal decline or impairment.

The neuro-enhancing agents, composition and treatment methods of the present invention generally reverse neural loss and/or prevent neural loss in an individual with a neurological disease, neurological injury, or age-related neuronal decline or impairment, thus yielding an improvement in neurological function. Improvements in neurological function can be assessed by numerous tests known in the art of medicine, neurology, psychology, or the like. Some of these tests assess changes in largely behavioral characteristics, such as deficits in memory, spatial relations, visual motor processing, quantitative skills, attentive ability, and the like, which are indicative of changes in an individual's neural cells and or neural connections and associated with a loss of neurological function. Whereas other tests aid in the visualization of specific morphological changes in the brain, such as magnetic resonance imaging (MRI), computed tomography scans (CT) and the like, which result from underlying changes in an individual's neural cells and/or neural connections and are also associated with changes in neurological function.

Given these types of tests as examples, administration of the agents, compositions and treatment methods of the invention to an individual with a neurological disease, neurological injury, or age-related neuronal decline or impairment yields improvements in the behavioral characteristic being measured or improvements in the neurological morphology being measured, any of which is indicative of overall improvements in neurological function. Other tests can be applied, however, that assess whether a given change is indicative of a change in neurological function at the level of an individual's neural cells and/or neural connections.

An individual with a neurological disease, neurological injury, or age-related neuronal decline or impairment has experienced or is experiencing loss in neurological function resulting generally from neural loss. Neural loss can be the result of any condition of a neuron, including neurites, in which its normal function is compromised. Neural deterioration can be the result of any condition which compromises neural function which is likely to lead to neural loss, Neural function can be compromised by, for example, altered biochemistry, physiology, or anatomy of a neuron, including its neurite. Deterioration of a neuron may include membrane, dendritic, or synaptic changes which are detrimental to normal neuronal functioning. The cause of the neuron deterioration, impairment, and/or death may be unknown. Alternatively, it may be the result of age-, injury- and/or disease-related neurological changes which occur in the nervous system of an individual.

When neural loss is described herein as "age-related", it is intended to include neural loss resulting from known and unknown bodily changes of an individual that are associated with aging. When neural loss is described herein as "disease-related", it is intended to include neural loss resulting from known and unknown bodily changes of an individual which are associated with disease. When neural loss is described herein as "injury-related", it is intended to include neural loss resulting from known and unknown bodily changes of an individual which are associated with injury or trauma. It should be understood, however, that these terms are not mutually exclusive and that, in fact, many conditions that result in the loss of neural cells and/or neural com ections can be related to age, disease and/or injury.

Some of the more common age-related neuropathies associated with neural loss and changes in neural morphology include, for example, Alzheimer's disease, Pick's disease, Parkinson's disease, vascular disease, Huntington's disease, and Age- Associated Memory Impairment. In Alzheimer's patients, neural loss is most notable in the hippocampus, frontal, parietal, and anterior temporal cortices, amygdala, and the olfactory system. The most prominently affected zones of the hippocampus include the CA1 region, the subiculum, and the entorhinal cortex. Memory loss is considered the earliest and most representative cognitive change because the hippocampus is well known to play a crucial role in memory. Pick's disease is characterized by severe neural degeneration in the neocortex of the frontal and anterior temporal lobes which is sometimes accompanied by death of neurons in the striatum. Parkinson's disease can be identified by the loss of neural cells in the substantia nigra and the locus ceruleus. Huntington's disease is characterized by degeneration of the intrastriatal and cortical chohnergic. neural cells and GABA-ergic neural cells. Parkinson's and Huntmgton's diseases are usually associated with movement disorders, but often show cognitive impairment (memory loss) as well.

Age- Associated Memory Impairment (AAMI) is another age-associated disorder that is characterized by memory loss in healthy, elderly individuals in the later decades of life. Presently, the neural basis for AAMI has not been precisely defined. However, neural death with aging has been reported to occur in many species in brain regions implicated in memory, including cortex, hippocampus, amygdala, basal ganglia, cholinergic basal forebrain, locus ceruleus, raphe nuclei, and cerebellum. Aging rodent brains do not develop senile plaques and neurofibrillary tangles. Most recent studies suggest, however, that loss or shrinkage of neurons, dendrites, and/or synapses is more closely correlated with either dementia or aging than are plaques and tangles. Aging rats exhibit neural cell loss in the pyramidal cells of the hippocampus, especially in field CAl, as well as cell loss or dendritic/synaptic changes in some other brain regions. Moreover, aging rodents show extensive hippocampal astrocyte hypertrophy just as do aging humans. In addition, loss of neural cells in field CAl of the hippocampus is a consistent correlate of aging across species, and is also prominent in human neurodegenerative diseases, such as Alzheimer's disease. For these reasons, the study of neural loss in aging rats, for example, is predictive of general mechanisms of brain aging and associated neural loss in humans.

Because of the great difficulty associated with measuring brain neural loss in living humans or even in autopsy material, which is highly variable and often shows massive changes due to the postmortem interval prior to fixation, treatment methods have been based on in vitro neural tissue culture systems from embryonic rodent pups (See e.g. U.S. Pat. No. 5,179,109-fetal rat tissue culture), or other mammalian (See e.g. U.S. Pat. No. 5,089,517-fetal mouse tissue culture) or non-mammalian animal models. These prior art treatment methods have been for protection of peripheral, as well as central nervous system neural cells, in animal or tissue culture models of ischemia, stroke, trauma, nerve crush, AD, and PD, etc, Neural deterioration in these model systems i&often proved by experimental trauma or intervention (e.g. application of toxins, nerve crush, interruption of oxygen supply, etc.).

However, other animal models, such as the models described in U.S. Pat. No.5,939,407 and Haughey et al. (2002), J Neurochem. 83:1509-1524, represent improvement in models for age-associated disease and decline because they relate to an intact animal, which is generally preferred over tissue culture models. Further, the animal model described in U.S. Pat. No.5,939,407 employs a strain of rat that was developed by the National Institute on Aging as a premier model of mammalian aging. The particular rat strain (Brown Norway/Fischer 344 FI cross rats) was selected as such a model due to its normal pattern of aging, with few indications of abnormal pathology. This strain also loses neural cells in field CAl of the hippocampus with aging and exhibits memory loss. This system represents one of the most natural animal models of neural degeneration and/or deterioration because it reflects a gradual loss of neural cells. Furthermore, the neural loss is not provoked by experimental intervention or abnormal pathology. Its brain aging pattern is also highly analogous to human and other mammalian species' brain aging patterns.

The therapeutic, neuro-enhancing agents and compositions provided in the present invention include progesterone and progesterone variants, including precursors of progesterone, progesterone metabolites and progesterone derivatives in its metabolic pathway, as well as the salts or hydrates of these progesterone variants. In a preferred embodiment, the present invention includes therapeutic agents and compositions comprising a naturally occurring metabolite of progesterone, namely 3α-hydroxy-5α- pregnan-20-one, also known as tetrahydroprogesterone (THP), as well as the pharmaceutically acceptable salts and hydrates of THP. Other preferred embodiments include 3α variant molecules of 3α-hydroxy-5α-pregnan-20-one or substituted derivatives of 3α-hydroxy-5α-pregnan-20-one, such as 3α-oxy derivatives, 3α-alkyl derivatives, 3 α- alkenyl derivatives, 3α-ester derivatives, 3α-ether derivatives, and the like, as well as the stereoisomers of 3 variant molecules of 3α-hydroxy-5α-pregnan-20- one or substituted derivatives of 3α-hydroxy-5α-pregnan-20-one derivatives.

Other preferred embodiments include substituted 3β-phenylethynyl derivatives of 3α -hydroxy-5α-pregnan-20-one, as well as pharmaceutically acceptable salts and hydrates of the 3β-phenylethynyl derivatives of 3 -hydroxy-5α-ρregnan-20-one, as described in Hawkinson, et al. (1998) J. Pharmacology & Experimental Therapeutics 287: 198-207. Other neuro-enhancing agents include steroids derivatives of the 5α - pregnan-20-one series as described in U.S. Pat. Nos. 5,925,630, 6,143,736 and 6,277,838. 3α-hydroxy-5 -pregnan-20-one (THP) is generally considered to be a neurosteriod as it is produced in the central nervous system and previously has been found to be an allosteric modulator of GABA receptors. See, for example, U.S. Pat. Nos. 5,925,630, 6,143,736 and 6,277,838.

Other preferred embodiments of the compositions and method of the present invention include variant molecules of 3α-hydroxy-5α-pregnan-20-one that exhibit substantially equivalent neuro-enhancing activity as 3α-hydroxy-5α-pregnan-20-one. Variant molecules of 3α-hydroxy-5α-ρregnan-20-one include progesterone-like molecules that are either natural precursors or metabolites of progesterone or synthetic variants of progesterone that exhibit substantially equivalent neurogenic activity as 3α- hydroxy-5α-pregnan-20-one. Substantially equivalent neuro-enhancing activity is defined as approximately 30% to approximately 300%> of the neuro-enhancing activity of 3 α-hydroxy-5 α-pregnan-20-one .

Neural loss through disease, age-related decline or physical insult leads to neurological disease and impairment. Embodiments of the present invention can counteract the deleterious effects of neural loss to an individual by providing therapeutic agents, compositions and methods that can lead to development of new neurons, new neurites and/or neural connections, result in the neuroprotection of existing neural cells, neurites and/or neural connection, or one or more these processes. Thus, the neuro- enhancing properties of the agents, compositions and methods of the present invention provide an eTfecϊive -strategy to generally reverse the neural loss associated with degenerative diseases, aging and physical injury or trauma.

Further, the administration of 3α -hydroxy-5 -pregnan-20-one, or a substantially equivalent variant molecule, to an individual who is undergoing or has undergone neural loss, as a result of a disease, defect or age-related decline, can generally provide an effective therapeutic strategy for the treatment of neurological conditions caused by neural loss. The defects and diseases that can benefit from administering the agents, compositions and methods of the present invention include, but are not limited to, spinal cord injury, stroke, head injury, epilepsy, Parkinson's disease and Alzheimer's disease. Moreover, given that 3α-hydroxy-5α-pregnan-20-one, and substantially equivalent variant molecules, possess neuro-enhancing activities, these agents and compositions may also be administered to improve age-related memory and learning impairments.

Particular embodiments of the present invention were discovered through the inventor's study of progestins] where the naturally occurring metabolite of progesterone, 3α -hydroxy-5 -pregnan-20-one or tetrahydroprogesterone (THP), was found to induce or stimulate the formation of new hippocampal neurons. Results of these analyses demonstrate that the number of mitotic neural cells was approximately doubled in the presence of tetrahydroprogesterone, as depicted in Figures 1 and 2.

Without being held to any mechanistic scheme or theory, the neuro-enhancing agents of the present invention are hypothesized to act through neural progenitor and/or stem cells (NP/SC). In certain regions of the brain of adult mammals, small populations of NP/SC are found that are capable of dividing and differentiating into neurons and glial cells. Moreover, it is the NP/SC populations of neural cells that respond to changing environmental demands, including brain injury and incipient neurological disease states, by increasing their proliferation and/or survival. Additionally, the rate of NP/SC proliferation is reduced in aged populations, leading to memory and learning impairments. Transplantation of NP/SC, however, has been shown to reverse age- associated memory impairment. Recently, amyloid beta protein, a protein implication in the onaetof Alzheimer's- disease, has been shown -to. alter the.proliferation and differentiation of NP/SC, which suggest a role for perturbed NP/SC behavior in the pathogenesis of Alzhiemer's disease.

The precise signals that influence neural progenitor cell fate are currently beginning to be identified. The data presented herein, however, are suggestive of a mechanism whereby the neuro-enhancing agents of the present invention signal neural progenitor and/or stem cells to divide. In this context, the neuro-enhancing agents of the present invention belong to a class of neural expansion signals. Neural expansion signals can provide for the growth of new neurons by causing a neural progenitor and/or stem cells to divide without exhausting the existing population of NP/SC. Thus, the neuro- enhancing agents of the present invention may be critically important to combat the effects of neurodegenerative disease and age-related mental decline and disability. Further, the neuro-enhaning agents of the present invention may induce or stimulate NP/SC to divide by modulating calcium and/or phosphate levels and/or homeostasis, and/or by restoring dysregulated calcium to normal levels. Changes in intracellular calcium and/or states of protein phosphorylation can also protect against neural loss, and thereby act to protect neurons from apotosis or other like processes leading to cell death. Finally, although certain progesterone metabolites, such as 3 α-hydroxy-5α-pregnan-20- one, are known to be neuroactive steroids that act as positive allosteric modulators of gamma-aminobutyric acid A (GABAA) receptor complexes (see U.S. Pat. No. 5,939,545), this may or may not be the mechanism of action from NP/SC.

Given this mechanistic theory, another therapeutic area where the agents, compositions and methods of the present invention may be used is in the treatment of neural damage caused by therapies aimed at combating certain cancers that affect the brain. For instance, cranial radiation therapy is crucial to the successful treatment of many primary brain tumors, cancers metastatic to the brain, CNS involvement of leukemia lymphoma, and head and neck cancers. Such irradiation that involves the cerebrum causes a debilitating cognitive decline in both children and adults. Experiments have shown that hippocampus-dependent learning and memory are strongly influenced by-the-activity of neural progenitor and/or stem cells and their proliferative prαgeny. Since the hippocampal granule cell layer undergoes continuous renewal and structuring by the addition of new neurons, radiation at much lower does than that needed to injure the more resistant post-mitotic neurons and glia of the brain, has been found to affect these highly proliferative progenitors and/or stem cells severely. The progenitor and/or stem cell, therefore, is considered to be so sensitive to radiation that a single low dose to the cranium of a mature rat is sufficient to ablate hippocampal neurogenesis. Recent experiments have further found that progressive learning and memory deficits following irradiation may be caused by the accumulating hippocampal dysfunction that results from a long-term absence of normal progenitor and/or stem cell activity. See. Monje and Palmer (2003) Current Opinions in Neurology 16(2):129-134. Thus, given the neurogenic effect of the agents and compositions of the present invention on hippocampal cell cultures, as depicted in Figures 1 and 2, therapeutic methods utilizing these agents and compositions would benefit individuals who are undergoing or have undergone radiation therapy for brain-related cancers.

The agents and compositions provided in the present invention are administered through a route which allows the neuro-enhancing agents to perform their intended function of stimulating or inducing new neurons and/or protecting against neural loss in an individual. Examples of routes of administration which may be used in this method include parenteral (subcutaneous, intravenous, intramuscular, intraarterial, fntraperitoneal, intrathecal, intracardiac, intrasternal and the like), enteral administration (i.e. administration via the digestive tract), mucosal administration, percutaneous administration and the like. Depending on the route of administration, the compositions may be coated with or in a material to protect it from the natural conditions which may detrimentally affect its ability to perform its intended function. A particularly convenient method of administering compositions of the present invention is via oral administration.

The administration of the compositions of the present invention is performed at dosages and for periods of time effective to stimulate or induce neural proliferation and/or to protect against the neural loss in an individual. Dosage regimes may be adjusted for purposes of improving the therapeutic response to the particular composition administered. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. Dosages of compositions comprising the neuro-enhancing agents of the present invention that are to be administered to an individual for in-vivo distribution are generally in the range of about 0.1 mg to about 1000 mg for human subjects, more preferably in the range of about 1 mg to about 500 mg. However, the particular dose depends on the particular neurological disease or defect being targeted.

Further, in the case of particular treatment regimens aimed at improving a particular neurological disease, neurological defect or age-related neurological decline, other agents and steroids can also be administered along with the neuro-enhancing agents or compositions of the present invention. In particular, biologically active forms of vitamin D₃ and D₂ material, as described in U.S. Pat. Nos. 4,897,388 and 5,939,407, may be co-administered to further aid in neurogenic stimulation or induction and/or prevention of neural loss, particularly for treatments of Alzheimer's disease. Estrogen and estrogen related molecules also may be co-administered with the neuro-enhancing agents and compositions of the present invention to enhance neuroprotection as described in Brinton (2001) Learning and Memory 8(3): 121-133, and other related publications. Other neuroactive steroids, such as various forms of DHEA (dehydriepiandrosterone) as described in U.S. Pat. No. 6,552,010, can also be co-administered along with the neuro- enhancing agents or compositions of the present invention to further aid in neurogenic stimulation or induction and/or prevention of neural loss. Other factors that cause neural growth and outgrowth of neural networks, such as Nerve Growth Factor (NGF), Brain- derived Neurotrophic Factor (BDNF), or the like, can also be administered either along with or before or after the administration of 3α-hydroxy-5α-pregnan-20-one. Additionally, inhibitors of neural apoptosis, such as inhibitors of calpains and capases as described in Haughey et al. (2002) J. Neurochemistry 83:1509-1524, and other cell death mechanisms, such as necrosis, can be co-administered with the neuro-enhancing agents of the present invention to further prevent neural loss associated with certain neurological diseases and neurological defects.

An aspect of the present invention provides for methods of treating an individual with a neurological disease, a neurological injury or age-related neurological deficiency or impairment with one or more neuro-enhancing agents or compositions disclosed herein. As stated above, the individual can be an animal that has a neurological disease, defect or condition. Generally the individual is a mammal, and preferably the individual is a human with a neurological disease, a neurological injury or age-related neurological deficiency or impairment. Criteria for assessing improvement in a particular neurological disease, neurological injury or age-related neurological change include methods of evaluating cognitive skills, motor skills, memory capacity or the like, as well as methods for assessing physical changes in selected areas of the central nervous system, such as magnetic resonance imaging (MRI) and computed tomography scans (CT) or other imaging methods. Such methods of evaluation are well known in the fields of medicine, neurology, psychology and the like, and can be appropriately selected to diagnosis the status of a particular neurological impairment. To assess a change in a neurological disease, neurological injury or age-related neurological change, the selected assessment or evaluation test, or tests, are given prior to the start of administration of the neuro- enhancing agents or compositions of the present invention. Following this initial assessment, treatment methods for the administration of the neuro-enhancing agents of the present invention are initiated and continued for various time intervals. At a selected time interval subsequent to the initial assessment of the neurological defect impairment, the same assessment or evaluation test(s) is again used to reassess changes or improvements in selected neurological criteria.

As stated above, the neuro-enhancing agents or compositions of the present invention may be administered in a variety of ways, such as orally, parenterally, transcutaneously, transmucosally, subcutaneously, by inhalation, infusion, particularly via intracerebro ventricular infusion, although oral administration is generally preferred. Depending upon the manner of introduction, the neuro-enhancing agents of the invention may be formulated in a variety of ways. Formulations containing THP or other substantially equivalent variant molecules can be prepared in various pharmaceutical forms, such as granules, tablets, capsules, suppositories;, powders^ controlled release formulations, suspensions, emulsions, creams, ointments, salves, lotions, or aeresols and the like. Preferably, these formulations are employed in solid dosage forms suitable for simple, and preferably oral, administration of precise dosages. Solid dosage forms for oral administration are preferably tablets, capsules, or the like. However, liquid dosage forms can also be utilized.

Formulations comprising the neuro-enhancing agents of the present invention can include one or more pharmaceutical grade organic or inorganic carriers, excipients, and/or diluents, especially those suitable for oral or topical use. Such carriers include tocopherol, dimethyl sulfoxide, and the like. For oral administration, suitable excipients include lactose, mannitol, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, gelatin, sucrose, magnesium carbonate, and the like. To prepare orally deliverable tablets, one or more neuro-enhancing agents of the present invention is mixed with at least one pharmaceutical excipient, and the solid formulation is compressed to form a tablet according to known methods, for delivery to the gastrointestinal tract. The tablet composition is typically formulated with additives, e.g. a saccharide or cellulose carrier, a binder such as starch paste or methyl cellulose, a filler, a disintegrator, or other additives typically usually used in the manufacture of medical preparations. To prepare orally deliverable capsules, one or more neuro-enhancing agents of the present invention is mixed with at least one pharmaceutical excipient, and the solid formulation is placed in a capsular container suitable for delivery to the gastrointestinal tract. Diluents known in the art include, for example, vegetable and animal oils and fats. Stabilizing agents, wetting and emulsifying agents, salts for varying the osmotic pressure, buffers for securing an adequate pH value, and/or skin penetration enhancers can be used as auxiliary agents in the neuro- enhancing formulations. Methods for preparing various conventional dosage forms are known or will be apparent to those skilled in the art; for example, see Remington's Pharmaceutical Sciences 1995, 19th ed., Williams & Wilkins.

The proportion of pharmaceutically active neuro-enhancing agent to carrier and/or other substances may vary from about 0.5 to about 100 wt.% (weight percent). For oral use, the pharmaceutical formulation will generally contain from about. 5 to about 100% by weight of the active material. For other uses, the pharmaceutical formulation will generally have from about 0.5 to about 50 wt.% of the active material.

Formulations employed in embodiments of the invention provide an effective amount of one or more neuro-enhancing agents upon administration to an individual. As used in this context, an "effective amount" of one or more neuro-enhancing agents is an amount that is effective to improve or ameliorate one or more symptoms associated with the particular neurological disease, neurological defect or age-related neurological decline or impairment. Such a therapeutic effect is generally observed within about 4 to about 6 weeks of initiating administration of an effective amount of one or more neuro-enhancing agents or compositions of the present invention.

The subject formulations are preferably, though not necessarily administered daily, in an amount to provide about a 1%, to about 25% increase in the blood level of the one or more neuro-enhancing agents disclosed herein. Generally, the total daily dosage will be at least about 10 mg and more preferably at least about 50 mg, and preferably not more than 500 mg per day, administered orally. Capsules or tablets for oral delivery can conveniently contain up to a full daily oral dose, e.g., 100 mg or more. Where the administration is by other than an oral route, the neuro-enhancing agents or compositions of the present invention may be delivered over an extended period, e.g., 3-10 days, in an amount effective to produce at least an average daily dose of, e.g., 50 mg.

The treatment methods described herein are carried out for an extended period, typically at least about 10, at least about 30, or at least about 60 weeks, and preferably as long as the patient is receiving noticeable benefit from the treatment method. In preferred embodiments, one or more neuro-enhancing agents of the present invention is administered at a dose and for a period effective to produce an improvement in at least one criterion set forth as indicative of an improvement in the neurological disease, neurological defect or neurological age-related decline or impairment, such as an improvement in cognitive abilities, memory, motor skills, learning or the like, preferably an improvement is observed in at least two such criteria.

The invention also provides a pharmaceutical products for use in treating various neurological diseases, such as neurodegenerative diseases, neurological defects, such as spinal cord injuries, or age-related neurological decline or impairments, such as changes in memory and learning. The pharmaceutical product includes a plurality of doses of a pharmaceutically active form of one or more neuro-enhancing agents of the present invention, and instructions for performing the treatment methods of the invention. Specifically, the instructions direct that an effective amount of a pharmaceutically active form of the neuro-enhancing agents of the present invention be administered to an individual with a particular neurological disease, defect or impairment as indicated. The pharmaceutically active form of the neuro-enhancing agents of the present invention can be formulated as described above with reference to a particular treatment method of the invention and can be packaged in any convenient manner. Generally, the instructions direct the administration of a composition comprising one or more neuro-enhancing agents as described above with reference to the treatment method. Oral administration is preferred. The instructions can be affixed to the packaging material or can be included as a package insert. While the instructions typically comprise written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this invention. Such media include, but are not limited to, electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. As used herein, the term "instructions" can include the address of an internet site that provides the instructions. Embodiments of the present invention also include the use of the above-described pharmaceutical products for the treatment of a human patient with a neurological disease, neurological defect or age-related neurological decline or impairment.

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure of how to make, to use and to evaluate the therapeutic agents, compositions and methods of the present invention, and are not intended to limit the scope of what is regarded as the invention. Efforts have been made to ensure accuracy with respect to numbers presented (e.g_.. amounts, concentrations, etc.), but some experimental errors and deviations should be allowed for.

EXAMPLES Example 1 Hippocampal neural cells obtained from the embryonic day 18 rat hippocampus,~l 2,000 neurons / sample. Sample was 95% neuronal. No selection for neuronal subtypes was conducted. Hippocampal neurons were treated with 3 -hydroxy- 5 -pregnan-20-one (THP). 3 α-hydroxy-5α-pregnan-20-one (THP) was added to two samples containing hippocampal neural cells at either 10 nanomolar (nm) and 100 nanomolar (nm) for 24 hrs at 37°C. Neurons were grown in a defined medium, Neurobasal + B27 supplement in the absence (control) or presence of THP or other test molecule (experimental). The samples with added THP were compared with a control sample containing only hippocampal neural cells. Changes in the mitotic appearance of the neural cells were observed. A mitotic appearance of a particular neural cell is defined as a doublet form in the cell body of the neural cell. The doublet form is indicative of a neural cell undergoing mitosis. A graphic comparison among the three samples studied is shown in Figure 1. These data reveal that there is approximate 2 fold increase in the mitotic phenotype of the neural cells studied at either 10 nm THP or 100 nm THP, as compared with the control sample. Data are expressed as percent the total number of neurons exhibiting mitotic phenotype, mean + SEM, **p<.01, ***p 001.

Example 2 Hippocampal neural obtained from the embryonic day 18 rat hippocampus, -12,000 neurons / sample. Sample was 95% neuronal. No selection for neuronal subtypes was conducted. Hippocampal neurons were treated with 3α-hydroxy-5α - pregnan-20-one (THP). THP was added to three samples containing hippocampal neural cells at 100 nm, 250 nm and 500 nm The samples with added THP were compared with a control sample containing only hippocampal neural cells. Neurons were grown in a defined medium, Neurobasal + B27 supplement in the absence (control) or presence of THP or other test molecule (experimental) . Changes in the mitotic appearance of the neural cells were observed. A mitotic appearance of a particular neural cell is defined as a doublet form in the cell body of the neuron. The doublet form is indicative of a neural cell undergoing mitosis. A graphic comparison among the four samples studied is shown in Figure 2. These data reveal that there is an approximate 2-3 fold increase in the mitotic phenotype of the neural cells studied, as compared with the control sample. The greatest effect in induction of the mitotic phenotype was observed at 500 nm THP. Data are expressed as percent of mean + SEM, **p<.01, ***p<.001. Example 3 THP also was shown to increase the expression of cell proliferating markers. Expression of cell cycle proteins have been successfully used to evaluate cellular proliferation. One such protein is the nuclear proliferation protein, Ki-67, which is expressed during the G_ls S, G₂, and M phases of the cell cycle, but is not expressed during the Go (resting) phase. Because Ki-67 antigen has a short half-life, it can be used as a marker of actively proliferating cells. Another cell cycle protein is cell division control protein 2 (cdc2) which is a cyclin dependent kinase (also called CDK1) which plays a crucial role in the Gl/S and G2/M phase. If THP induces neuronal proliferation, cell proliferation markers should be elevated. This question was first addressed in hippocampal neurons in primary culture and the results are shown in Figure 3. Hippocampal neurons were treated with THP 250 nM for 72 hours and immunostained with antibodies for the nuclear proliferation marker, ki-67 antigen, which appears yellow. The results indicate that THP induced the expression of the nuclear proliferation marker Ki-67 (see Fig. 3; note that the cytoplasm of the donor and the daughter cells have not yet completely separated (orange arrow). (Bar=25 μM). The cell cycle protein cdc2 is also observed in a dose dependent fashion (see Fig. 4). As shown in the Figure 4, THP increases expression of cell division control protein 2 (cdc2) in hippocampal neurons. For this experiment, neurons were collected following 24hrs of THP exposure. Forty μg protein of the total cell lysate was loaded and separated by 12% SDS-gel using antibody (Abcom) specifically against cdc2 and analyzed using Un-Scan-It image software (Silk Scientific Corp.). This figure shows a representative Western blot from one of three different experiments which have the similar results.

Example 4 Having determined that THP increases the expression of the cell proliferating markers, a determination was sought as to whether the increase in cell proliferation markers translated into an increase in neuronal number. It was found that THP induces neuronal proliferation by increasing the total cell number and the dividing speed. As shown in Figure 5, THP increased the neuron number by approximately 30%. These results are highly consistent across different experiments and are also comparable to the results we obtained using the mouse hippocampal neuron cell line (HT-22) (Fig. 6 and tablel below). As shown in Figure 6 and Table 1, THP-increases neuron number as assessed in MuLV-GFP infected mouse neurons. The effect of THP on HT-22 cells proliferation was detected on MuLV infected cells. Left panel shows the FACS profile of vehicle. Right Panel shows the FACS profile of THP treated MuLV-GFP infected cells. The table summarizes the FACS results. V - vehicle; THP (250 nM). THP treatment increased the dividing cell number ~22% as determined by fluorescent associated cell sorting (FACS). Therefore, these data demonstrate that THP can increase the proliferation of neuronal cells either in primary cultured cells or continuous cell lines, from rat and mouse.

Example 5 Biochemical analyses of ³H-thymidine uptake, as a measure of DNA synthesis, were used as the experimental vehicle to confirm the morphological observations described in examples 1 and 2. These morphological observations were confirmed by ³H-thymidine uptake data. As shown in Figure 7, THP induced a 80% increase in ³H- thymidine uptake relative to control (F=12.31,df 3,19, p <. 0001) from about 0.1 nm THP to about 250 nm THP. Thus, the range for the neurogenic effect of THP on neural cells is quite is sensitive and quite broad. Furthermore, DNA synthesis is specifically induced in the presence of THP (F=9.15,df 6,27, p < 0001), as compared with other structurally and chemically similar steroids, as shown in Figure 8. For these experimental results, cultured hippocampal neural cells, derived from embryonic day 18 rat fetuses, were allowed to adhere to polylysine coated plastic coverslips for 40 min in serum containing medium. Following adhesion, neurons were exposed to 1 μCi/ml ³H- thymidine in the presence or absence of 100-500 nM THP and allowed to incubate at 37° for 24 hours in the absence or presence of the indicated steroids. Data are expressed as mean + SEM, *p<.05, **p<01, ***p 001. The results of this experiment demonstrated that THP induction of H-thymidine incorporation is highly specific. Progesterone induced a modest increase in ³H-thymidine incorporation, however, the stereoisomers of THP, i.e., 5α, 3β-THP and 5β, 3β -THP, as well as 5α,3 β -pregnen were without effect. Additionally, 5α, 3α -pregnan-diol, 5α, 3α -pregnan-triol and pregnenolone sulfate (PS), which are known to increase morphological differentiation, induceD a significant decresase in ³H-thymidine incorporation which is consistent with their differentiation effect. The steroid specificity analysis provides evidence for the specificity of THP- induced mitogenesis. Moreover, consistent with this evidence is the observation that differentiation factors have an effect opposite to that of THP in that these agents cause a decrease in H-thymidine incorporation.

Example 6 The time course of THP-induced H-thymidine incorporation in hippocampal neuronal cells is shown in Figure 9 where cultured hippocampal nerve cells, derived from embryonic day 18 rat fetuses, were allowed to adhere to polylysine coated plastic coverslips for 40 min in serum containing medium. Following adhesion, serum containing medium in the presence or absence of 10-250 nM THP plus 1 μCi/ml ³H~ thymidine and allowed to incubate at 37° for 1, 8 or 24 hours. Data are expressed as mean ± SEM, *p<.05, **p<.01, ***p<.001.

Example 7 Experiments to determine whether THP promotes neural stem cells growth were also performed. In Figure 10, neural spheres were generated from the periventricular areaand hippocampus of embryonic day 1 .5 rat embryos with THP alone or-w th using EGF and FGF-2 as mitogens. In this experiment approximately the third passage of neural spheres were collected and randomly disturbed evenly to each dish. Dishes were treated with reagents as labeled in the absence of progesterone for 36 hours. Cells were then collected and trypsinized in to single cells. The cell numbers were counted blinded using a hemacytometer and plotted in Excel. Another experiment was performed to assess the neurogenic effects of THP administration on human neural stem cells, and the results are shown in Figure 11. In this experiment, neural stem cells derived from human fetal cortex were treated with varying concentrations of APα [1- 1000 nM] or with bFGF [20 ng/ml] + heparin [ 5 μg/ml] as a THP positive control. The proliferation marker, BrdU [10 μM] was added simultaneously with test molecules and cells incubated at 37°C for 24 hours. Quantitative Elisa chemiluminescence of BrdU signal was conducted at 24 hrs following addition of substrate and chemiluminescence determined by LMax microplate luminometer (Molecular Devices) (Roche Diagnostics Corp., Cell Proliferation ELISA, BrdU (chemiluminescence). THP at 250 and 500 nM, significantly increased BrDU chemilunescence relative to vehicle control condition and was consistently greater than the positive control bFGF + heparin. Data are presented as mean ± SEM and are derived from three separate experiments.

Example 8 An animal model of Alzheimer's disease will be utilized to determine the effects of administration of 3α-hydroxy-5α-pregnan-20-one (THP), and substantially equivalent variant molecules, on the relative formation of age-related amyloid deposits in mice. The animal model to be utilized has been described by Borchelt et al. (1996) Neuron 17:1005- 1013 and Haughey et al. (2002) J. Neurochemistry 83:1509-1524. Male mice(12-14 months old) overexpressing a mutant form of amyloid precursor protein (APP) are maintained on a 12 hour light/ 12 hour dark cycle with free access to food and water. This line of mice exhibits increased levels of soluble amyloid beta protein and develops amyloid deposits in an age-dependent manner with diffuse deposits first appearing at about 12 months of age and plaque-like deposits developing later, typically by 18-22 months of age. To determine proliferation and survival of NP/SC in the dentate gyrus in the-absence and presence of 3 -hydroxy--5α -pregnan-20=one (THP), mice are given-five daily injections of 5-bromo-2'deoxyuridine (BruU; 50 mg/kg, i.p.). 12 experimental mice and six control mice are used to assess the effects of 3α-hydroxy-5α-pregnan-20- one (THP) on the formation of amyloid deposits. The 12 experimental mice are also given daily injections of 3 -hydroxy-5α-pregnan-20-one (THP) (6 mice are given 10 mg/kg THP and 6 mice are given lOOmg/kg THP) for one week, two weeks and 4 weeks prior to the injections of 5-bromo-2'deoxyuridine (BruU). The six control mice are given daily injections of a saline solution contained in the same volume as the volume given to the experimental mice. All mice are sacrificed 12 days following the injections of BruU. Brain sections from experimental and control mice are prepared and compared for incorporation of BruU. For the quantification of neuro-enhancement, immunpositive cells in the dentate gyrus from three sections in each of the 12 experimental and six control mice are counted and compared. Rate of cell division is usually determined using nucleotide (BrDU (bromodeoxy-uridine, or ³H-thymidine) incorporation into DNA. The ratio of the BrDU or ³H-thymindine labeled cells vs non-labeled cells will indicate the dividing speed. DNA incorporation into cells is not only present in dividing cells but also occurs during DNA repair thereby generating the potential of a false positive by counting the mismatch DNA repair cells which also incorporate a relative high amount BRDU or ³H thymidine. To circumvent this source of BrDU incorporation, we will use, the MuLV- enhanced green fluorescent protein (GFP) to label the dividing cells. The cell proliferation rate will be determined by measuring the ratio of GFP expression cell vs non-GFP expression cells by FACS. Murine leukemia virus (MuLV) has been demonstrated to only infect cells during mitosis but not to infect non-dividing cells (Lewis and Emerman 1994). Moreover, this strategy has been successfully used in the labeling of the dividing neurons in vitro and in vivo (van Praag, H., Schinder, A. F., Christie, B. R., Toni, N., Palmer, T. D., Gage, F. H, Functional neurogenesis in the adult hippocampus. Nature. 2002. 415, p 1030-1034.)The infected cells can express GFP stably which makes it possible to follow neurons that have divided in vivo as they differentiate and migrate to sites within the brain. In addition, MuLV infection-GFP strategy permits a more precise comparison of the dividing speed between the THP treated cells and the non-treated cells by FACS analysis. Furthermore, this use of the retroviral infection strategy mayalso provide data for future application for future gene therapy purposes. Here we use a strain of virus in which the GFP was inserted in frame with the authentic start codon of the internal ribosome entry site-transgene cassette which is positioned between the env gene and the 3' long terminal repeat. This virus vector exhibited replication kinetics similar to those of the wild-type MuLV and mediated efficient delivery of transgene (Logg, C. R. Tai, C. K. Logg, A. Anderson, W. F.Kasahara, N., A uniquely stable replication-competent refrovirus vector achieves efficient gene delivery in vitro and in solid tumors. Human Gene Therapy. 2001. 8:p 921-932). Example 9 An aged population of mice is assessed at various time intervals for the effects of the treatment methods described herein. The particular rat strain (Brown Norway/Fischer 344 FI cross rats) was selected as such a model due to its normal pattern of aging, with few indications of abnormal pathology. Experimental treatment methods include administering one or more of the neuro-enhancing agents of the present invention to a set of aged mice, as compared to mice from the same population who are not receiving treatment methods of the present invention. For example, 12 experimental mice are given daily injections of 3α-hydroxy-5α-pregnan-20-one (THP) ( 6 mice arelO mg/kg THP and 6 mice are given 50 mg/kg THP) for a selected time interval for treatment. The control mice are given daily injections of a saline solution contained in the same volume as the volume given to the experimental mice. Treatments regimens are continued for 2 week, 1 month, 3 months and 6 months time intervals. At the end of the selected time period for treatment, proliferation and survival of NP/SC in the dentate gyrus in the absence and presence of 3α-hydroxy-5α-pregnan-20-one (THP) is determined. To assess neurological differences between the experimental and control mice, all mice are given five daily injections of 5-bromo-2'deoxyuridine (BruU; 50 mg/kg, i.p.). All mice are sacrificed 12 days following the injections of BruU. Brain sections from experimental and control mice are prepared and compared for changes in generation and survival of neural cells. For the quantification of neuro-enhancement, immunpositive cells in the dentate gyrus -are from three sesiionsin each of the experimental and ccαifroLmice are counted and compared. DNA incorporation into cells is not only present in dividing cells but also occurs during DNA repair thereby generating the potential of a false positive by counting the mismatch DNA repair cells which also incorporate a relative high amount BRDU or ³H thymidine. To circumvent this source of BrDU incorporation, we will use, the MuLV- enhanced green fluorescent protein (GFP) to label the dividing cells. The cell proliferation rate will be determined by measuring the ratio of GFP expression cell vs non-GFP expression cells by FACS. Murine leukemia virus (MuLV) has been demonstrated to only infect cells during mitosis but not to infect non-dividing cells (Lewis and Emerman 1994). Moreover, this strategy has been successfully used in the labeling of the dividing neurons in vitro and in vivo (van Praag, H., Schinder, A. F., Christie, B. R., Toni, N., Palmer, T. D., Gage, F. H., Functional neurogenesis in the adult hippocampus. Nature. 2002. 415, p 1030-1034.)The infected cells can express GFP stably which makes it possible to follow neurons that have divided in vivo as they differentiate and migrate to sites within the brain. In addition, MuLV infection-GFP strategy permits a more precise comparison of the dividing speed between the THP treated cells and the non-treated cells by FACS analysis. Furthermore, this use of the retroviral infection strategy may also provide data for future application for future gene therapy purposes. Here we use a strain of virus in which the GFP was inserted in frame with the authentic start codon of the internal ribosome entry site-trans gene cassette which is positioned between the env gene and the 3 ' long terminal repeat. This virus vector exhibited replication kinetics similar to those of the wild-type MuLV and mediated efficient delivery of transgene (Logg, C. R. Tai, C. K. Logg, A. Anderson, W. F.Kasahara, N., A uniquely stable replication-competent refrovirus vector achieves efficient gene delivery in vitro and in solid tumors. Human Gene Therapy. 2001. 8:p 921-932).

Example 10 Neural progenitor and or stem cells (NP/SC) are prepared for ex-vivo expansion, i.e., to be contacted with one or more neuro-enhancing agents described in the present invention. Methods of obtaining and maintaining NP/SC are known in the art (Eriksson, P. S., E. Perfilieva, et al. (1998). "Neurogenesis in the adult human hippocampus." Nat Med 4(11): 1313-7; Song, H. J., C. F. Stevens, et al. (2002). "Neural stem cells from adult hippocampus develop essential properties of functional CNS neurons, [comment]." Nature Neuroscience 5(5): 438-45; van Praag, H, A. F. Schinder, et al. (2002). "Functional neurogenesis in the adult hippocampus." Nature 415(6875): 1030-4; Erlandsson, A., M. Enarsson, et al. (2001). "Immature neurons from CNS stem cells proliferate in response to platelet-derived growth factor." Journal of Neuroscience 21(10): 3483-91). To this culture of cells, about 100 nm to about 1000 nm THP or another substantially equivalent variant neuro-enhancing molecule is added. Appropriate growth conditions are maintained to maximize mitosis of the NP/SC with minimal differentiation of the NP/SC, thus leading to a culture containing more NP/SC than present prior to the addition of one or more neuro-enhancing agents of the present invention. The expanded population of neural cells is then infused into an individual suffering from neuronal loss, or other neurological defect or damage, via intracerebroventricular infusion. The infusion of expanded cells is monitored to assess further proliferation, differentiation and survival.

All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference.

Although the foregoing embodiments of the invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A method for enhancing neurological function in an individual, comprising contacting the neural cells of the individual with one or more neuro-enhancing agents, wherein the one or more neuro-enhancing agents comprise 3α-hydroxy-5α-pregnan-20- one or a substantially equivalent variant molecule.

2. A method for ex-vivo expansion of neural cells comprising contacting a population neural cells with one or more neuro-enhancing agents ex-vivo to yield an expanded population of neural cells.

3. The method of claim 2, wherein the population of neural cells comprise neural progenitor cells, neural stem cells or both neural progenitor cells and neural stem cells.

4. The method of claim 1, wherein the neural cells are contacted with the one or more neuro-enhancing agents via in-vivo administration of the agent to the individual.

5. The method of claim 2, wherein the expanded population of neurons are directly administered to one or more central nervous regions of an individual.

6. The method of claim 5, wherein the expanded population of neural cells are infused via intracerebroventricular infusion.

7. The method of claim 5, further comprising directly administering the one or more neuro-enhancing agents to the individual in-vivo before, after, or before and after the expanded population of neural cells is administered to the individual.

8. The method of claim 4, wherein the one or more neuro-enhancing agents is administered via injection, infusion, implantation, inhalation, orally or topically. O 2005/009359

9. A pharmaceutical composition for enhancing neurological function in an individual comprising: one or neuro-enhancing agents comprising 3α-hydroxy-5 -pregnan-20-one, or a substantially equivalent variant molecule, contained in a pharmaceutically acceptable carrier, diluent or stabilizer, wherein the carrier, diluent or stabilzer maintains the active form of the neuro-enhancing agents and is suitable for administration to the individual.

10. The pharmaceutical composition of claim 9, wherein the pharmaceutically acceptable carrier, diluent or stabilizer is suitable for oral administration to the individual.

11. The pharmaceutical composition of claim 9, wherein the composition includes at least about 10 mg of pharmaceutically active form of 3 -hydroxy-5α- pregnan-20-one or a substantially equivalent variant molecule.

12. The pharmaceutical composition of claim 9, wherein the composition includes about 0.1 to about 100 mg of pharmaceutically active foπn of 3α-hydroxy-5α- ρregnan-20-one or a substantially equivalent variant molecule.

13. The pharmaceutical composition of claim 9, wherein the composition includes about 1 mg to about 10 mg of pharmaceutically active form of 3α-hydroxy-5α- pfegnan-20-ohe or a substantially equivalent variant molecule.

14. The pharmaceutical composition of claim 9, wherein the composition includes about 10 mg to about 500 mg of pharmaceutically active form of 3α-hydroxy- 5α-pregnan-20-one or a substantially equivalent variant molecule.

15. A therapeutic method for enhancing neurological function in an individual with a neurological disease, neurological injury or age-related neural decline or impairment comprising: administering to the individual an effective amount of one or more neuro- enhancing agents comprising 3α -hydroxy- 5 α -pregnan-20-one or a substantially equivalent variant molecule over a period of time effective to stimulate neural mitosis, to stimulate neurite growth and organization, to prevent neural loss, or one or more of these processes, wherein administration of the effective amount the one or more agents to the individual over the effective period of time yields an improvement in one or more neurological criteria related to the individual's improvement in neurological function.

16. The therapeutic method of claim 15, wherein the period of administration is about one month or longer.

17. The therapeutic method of claim 15, wherein the period of administration is about six months or longer.

18. The therapeutic method of claim 15, wherein the period of administration is about one year or longer.

19. The therapeutic- method of claim 15 , wherein the amount of the one or more neuro-enhancing agents is about 10 mg or greater.

20. The therapeutic method of claim 15, wherein the amount of the one or more neuro-enhancing agents is about 50 mg or greater.

21. The therapeutic method of claim 15, wherein the amount of the one or more neuro-enhancing agents is less than about 500 mg.

22. The method of claim 15, wherein the neurological disease in Alzheimer's disease.

23. The method of claim 15, wherein the neurological injury is due to radiation injury.

24. The method of claim 15, wherein the improvement in neurological function is an improvement in age-related memory decline.

25. The method of claim 15, wherein the improvement in neurological function is an improvement in age-related learning impairment.

26. The method of claim 15, wherein the agent is administered via injection, infusion, implantation, inhalation, orally or topically.

27. A method of stimulating neurogenesis in a subject comprising: contacting neural cells of the subject with one or more neuro-enhancing agents, wherein the one or more neuro-enhancing agents comprise 3 -hydroxy-5α- pregnan-20-one or a substantially equivalent variant molecule.

28. The method of claim 27, wherein the one or more agents contact neural cells by administration to a subject via injection, infusion, implantation, inhalation, orally or topically.

29. The method of claim 28, wherein administration of the one or more agents is continued at a dose and for a period of time effective for ameliorating a neurological defect or disease.

30. The method of claim 29, wherein the neurological defect or disease includes Alzheimer's Disease, dementia or cognitive impairment.