CA2175603C - Estrogen compositions and methods for neuroprotection - Google Patents
Estrogen compositions and methods for neuroprotection Download PDFInfo
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- CA2175603C CA2175603C CA002175603A CA2175603A CA2175603C CA 2175603 C CA2175603 C CA 2175603C CA 002175603 A CA002175603 A CA 002175603A CA 2175603 A CA2175603 A CA 2175603A CA 2175603 C CA2175603 C CA 2175603C
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
- A61K31/565—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/02—Drugs for disorders of the nervous system for peripheral neuropathies
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/08—Antiepileptics; Anticonvulsants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
- A61P25/16—Anti-Parkinson drugs
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/18—Antipsychotics, i.e. neuroleptics; Drugs for mania or schizophrenia
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
Abstract
The use of an estrogen compound is described for protecting a population of nerve cells from death. The compound can be used in the treatment of a subject affected by a neurodegenerative disorder, in orde r to prevent the loss of neuronal activity.
Description
ESTROGEN COMPOSITIONS AND METBODS FOR NEUROPROTECTION
TECHNICAL FIELD
The present invention relates to methods for protecting cells in the central nervous system of subjects from cell death and for stimulating neuronal survival through enhanced growth factor production.
BACKGROIIND OF THE INVENTION
Neurodegenerative diseases have a major impact on society. For example, approximately 3 to 4 million Americans are afflicted with a chronic neurodegenerative disease known as Alzheimer's disease. Other examples of chronic neurodegenerative diseases include diabetic peripheral neuropathy, multiple sclerosis, amyotrophic lateral sclerosis, Huntingdon's disease and Parkinson's disease. Not all neurodegenerative diseases are chronic.
Some acute neurodegenerative diseases include stroke, schizophrenia, and epilepsy as well as hypoglycemia and trauma resulting in injury of the brain, peripheral nerves or spinal cord. There is a need for improved therapeutic agents and methods for reversing or retarding neuronal damage associated with each of these conditions.
Neurodegenerative diseases and aging are characterized by a wide range of symptoms which vary in severity and range from individual to individual. For example, Alzheimer's disease is characterized by symptoms such as depression, R'o 95/12402 2 1 7 5 6 0 3 PCTIUS94/12782 ~
aggression, impairment in short-term memory, impairment in intellectual ability, agitation, irritability and restlessness. Since estrogen becomes deficient in post-menopausal women, and since estrogen is believed to affect mood, some studies have been undertaken to assess the relief of behavioral symptoms associated with Alzheimer's disease.
Unfortunately, those clinical trials that have been performed to establish the beneficial effect of estrogen on Alzheimer's disease have concluded that no statistically significant improvements in the disease course or symptoms resulted from the treatment. Fillet et al. 1986, Psvchoneuroendocrinoloav 11:337-345; Honjo et al. 1989, Steroid Biochemistry 34:521-524. In one study where only 1 female and 1 male patient were studied and no statistics were available, a rapid reduction in symptoms of senile dementia was observed when estrogen was administered to the female patient in a coclctail of drugs together with chorionic gonadotrophin, a vasodilator and a non-steroidal anti-inflammatory agent after a period as short as one week (Aroonsakul 1990, US patent 4,897,389). There is a need for a better understanding of the underlying process of neurodegeneration such that improved treatment protocols and effective drugs may be designed that are effective at treating the disease itself so as to bring about a longterm meaningful reversal of symptoms.
A common feature of neurodegenerative disorders and the process of aging in animals is the progressive cell damage of neurons within the central nervous system (CNS) leading to loss of neuronal activity and cell death. This loss of activity has been correlated with adverse behavioral symptoms including memory loss and cognitive deficits.
Therapeutic agents that have been developed to retard loss of neuronal activity either have toxic side effects or are prevented from reaching their target site because of their inability to cross the blood-brain barrier. The blood-brain barrier is a complex of morphological and enzymatic components that retards the passage of both large and charged small molecules thereby limiting access to cells of the brain. There is a need for novel therapeutic agents that are readily transported across the blood-brain barrier as well as for novel methods of treatment of neurodegenerative disorders that directly target the damaged site and are non-toxic.
Traditional methods of treating neurological symptoms focus on modifying the electrical impulse itself as it moves between and along neurons; or modifying the release or degradation of neurotransmitters. It is now recognized that neuronal cell density has an important impact on function.
In various pathological conditions, loss of cell density has been observed resulting from accelerated neuronal cell death. The pattern of degeneration of neurons typically originates from the nerve terminals and progresses "backward" toward the cell body (retrograde degeneration).
In several systems, lesioning of certain brain regions results in compensatory sprouting of axons. This plasticity of neurons is attributed at least in part to the presence of trophic growth factors.
These findings have spurred efforts to identify therapeutic agents that compensate for cell loss by stimulating sprouting of dendrites and axons of remaining cells so as to improve the structural integrity of the damaged region. However, the optimal density of neurons and neuronal extensions is a delicate balance between deficiency and excess, a balance that varies with the'environment of the cells. This balance can be disrupted when therapeutic agents act on normal or inappropriate tissue. There is a neea therefore to target therapeutic agents at a therapeutic dose specifically to those regions where they are required, or, alternatively, to identify agents that have a natural specificity for the target site only.
To date, there are no safe and effective methods for treating loss of neuronal activity. However, considerable attention has recently been focused on naturally occurring proteins, collectively called neurotrophic factors (see ~
TECHNICAL FIELD
The present invention relates to methods for protecting cells in the central nervous system of subjects from cell death and for stimulating neuronal survival through enhanced growth factor production.
BACKGROIIND OF THE INVENTION
Neurodegenerative diseases have a major impact on society. For example, approximately 3 to 4 million Americans are afflicted with a chronic neurodegenerative disease known as Alzheimer's disease. Other examples of chronic neurodegenerative diseases include diabetic peripheral neuropathy, multiple sclerosis, amyotrophic lateral sclerosis, Huntingdon's disease and Parkinson's disease. Not all neurodegenerative diseases are chronic.
Some acute neurodegenerative diseases include stroke, schizophrenia, and epilepsy as well as hypoglycemia and trauma resulting in injury of the brain, peripheral nerves or spinal cord. There is a need for improved therapeutic agents and methods for reversing or retarding neuronal damage associated with each of these conditions.
Neurodegenerative diseases and aging are characterized by a wide range of symptoms which vary in severity and range from individual to individual. For example, Alzheimer's disease is characterized by symptoms such as depression, R'o 95/12402 2 1 7 5 6 0 3 PCTIUS94/12782 ~
aggression, impairment in short-term memory, impairment in intellectual ability, agitation, irritability and restlessness. Since estrogen becomes deficient in post-menopausal women, and since estrogen is believed to affect mood, some studies have been undertaken to assess the relief of behavioral symptoms associated with Alzheimer's disease.
Unfortunately, those clinical trials that have been performed to establish the beneficial effect of estrogen on Alzheimer's disease have concluded that no statistically significant improvements in the disease course or symptoms resulted from the treatment. Fillet et al. 1986, Psvchoneuroendocrinoloav 11:337-345; Honjo et al. 1989, Steroid Biochemistry 34:521-524. In one study where only 1 female and 1 male patient were studied and no statistics were available, a rapid reduction in symptoms of senile dementia was observed when estrogen was administered to the female patient in a coclctail of drugs together with chorionic gonadotrophin, a vasodilator and a non-steroidal anti-inflammatory agent after a period as short as one week (Aroonsakul 1990, US patent 4,897,389). There is a need for a better understanding of the underlying process of neurodegeneration such that improved treatment protocols and effective drugs may be designed that are effective at treating the disease itself so as to bring about a longterm meaningful reversal of symptoms.
A common feature of neurodegenerative disorders and the process of aging in animals is the progressive cell damage of neurons within the central nervous system (CNS) leading to loss of neuronal activity and cell death. This loss of activity has been correlated with adverse behavioral symptoms including memory loss and cognitive deficits.
Therapeutic agents that have been developed to retard loss of neuronal activity either have toxic side effects or are prevented from reaching their target site because of their inability to cross the blood-brain barrier. The blood-brain barrier is a complex of morphological and enzymatic components that retards the passage of both large and charged small molecules thereby limiting access to cells of the brain. There is a need for novel therapeutic agents that are readily transported across the blood-brain barrier as well as for novel methods of treatment of neurodegenerative disorders that directly target the damaged site and are non-toxic.
Traditional methods of treating neurological symptoms focus on modifying the electrical impulse itself as it moves between and along neurons; or modifying the release or degradation of neurotransmitters. It is now recognized that neuronal cell density has an important impact on function.
In various pathological conditions, loss of cell density has been observed resulting from accelerated neuronal cell death. The pattern of degeneration of neurons typically originates from the nerve terminals and progresses "backward" toward the cell body (retrograde degeneration).
In several systems, lesioning of certain brain regions results in compensatory sprouting of axons. This plasticity of neurons is attributed at least in part to the presence of trophic growth factors.
These findings have spurred efforts to identify therapeutic agents that compensate for cell loss by stimulating sprouting of dendrites and axons of remaining cells so as to improve the structural integrity of the damaged region. However, the optimal density of neurons and neuronal extensions is a delicate balance between deficiency and excess, a balance that varies with the'environment of the cells. This balance can be disrupted when therapeutic agents act on normal or inappropriate tissue. There is a neea therefore to target therapeutic agents at a therapeutic dose specifically to those regions where they are required, or, alternatively, to identify agents that have a natural specificity for the target site only.
To date, there are no safe and effective methods for treating loss of neuronal activity. However, considerable attention has recently been focused on naturally occurring proteins, collectively called neurotrophic factors (see ~
Table I), that promote growth and maintenance of cells of the central nervous system (CNS) and sympathetic and sensory neurons of the peripheral nervous system. In particular, the administration of nerve growth factor (NGF), a protein which is normally transported retrogradely in the intact brain from the hippocampus to the septal cholinergic cell bodies as well as from the cortex to the nucleus basalis, provides trophic support to cholinergic neurons and has been shown in animal models to have utility in reducing the effects of neurodegeneration due to trauma, disease or aging. The septum and the nucleus basalis are part of a region of the brain known as the basal forebrain. The effectiveness of administering NGF in response to damage is supported by experiments that demonstrate that cholinergic neurons in the medial septum can be protected from retrograde degeneration by chronic infusion of exogenous NGF
(Rosenberg et al. 1988, Science 242:1575-1578). Indeed, infusion of NGF has been shown to significantly attenuate retrograde degeneration of cholinergic neurons after transection of their connections in the fimbria (the septo-hippocampal pathway).
One of the major problems confronting the use of NGF as a therapeutic agent is finding an appropriate method of increasing the levels of NGF at the appropriate target site.
NGF is a large molecule and as such cannot normally pass across the blood-brain barrier and therefore has very limited access to the cells of the brain. Invasive methods are commonly used to place externally administered NGF
within the brain. These methods are not sufficient to target NGF specifically to those cells where it is required.
Non-localized targeting not only decreases the amount of protein available at the target site but also results in stimulation of growth of neurons at inappropriate sites resulting in potential harmful effects for the subject.
Another disadvantage of administering NGF as a therapeutic agent is that of induction of an immunological response to this protein. There is a need therefore for a ~
(Rosenberg et al. 1988, Science 242:1575-1578). Indeed, infusion of NGF has been shown to significantly attenuate retrograde degeneration of cholinergic neurons after transection of their connections in the fimbria (the septo-hippocampal pathway).
One of the major problems confronting the use of NGF as a therapeutic agent is finding an appropriate method of increasing the levels of NGF at the appropriate target site.
NGF is a large molecule and as such cannot normally pass across the blood-brain barrier and therefore has very limited access to the cells of the brain. Invasive methods are commonly used to place externally administered NGF
within the brain. These methods are not sufficient to target NGF specifically to those cells where it is required.
Non-localized targeting not only decreases the amount of protein available at the target site but also results in stimulation of growth of neurons at inappropriate sites resulting in potential harmful effects for the subject.
Another disadvantage of administering NGF as a therapeutic agent is that of induction of an immunological response to this protein. There is a need therefore for a ~
compound that does not in itself cause an immune response but could stimulate the production of endogenous NGF.
Current methods for administering nerve growth factor across the blood-brain barrier include: polymeric implants, osmotic minipumps, cell therapy using genetically engineered autologous or heterologous cells secreting NGF for implantation into the brain, and methods of increasing the permeability of the blood-brain barrier thereby allowing diffusion of these molecules to cells in the brain. Where exogenous NGF is used, a relatively large amount of relatively costly recombinant protein is required.
Rather than these aforestated solutions to delivery of proteins, it would be desirable to: avoid invasive techniques; to control the amount and the site of delivery of neurotrophic proteins to sites where they are most needed thereby minimizing toxic side effects; and to minimize the health care costs of treatment.
An additional approach to treating neurological symptoms has followed the observation that certain amino acids (glutamic acid and aspartic acid) act as excitatory neurotransmitters that bind the N-methyl D-aspartate (NMDA) receptor. Excess release of these amino acids (EAA) causes overstimulation of the neurons in neurodegenerative diseases as well as in conditions of hypoglycemia or trauma, resulting in neuronal loss and behavioral dysfunctions.
NMDA is a potent and toxic analogue of glutamate which has been shown in animal studies to mediate much of the neuronal death associated with head trauma, hypoglycemia, anoxia, hypoxia and other conditions, and compromises the flow of blood, oxygen or glucose to the central nervous system.
A number of synthetic compounds that act as antagonists of the receptor have been described and tested in animal models. The possibility that these compounds are toxic in humans remains unresolved. Despite many years of clinical research, these antagonists are not as yet available as therapeutic products for treating patients.
Current methods for administering nerve growth factor across the blood-brain barrier include: polymeric implants, osmotic minipumps, cell therapy using genetically engineered autologous or heterologous cells secreting NGF for implantation into the brain, and methods of increasing the permeability of the blood-brain barrier thereby allowing diffusion of these molecules to cells in the brain. Where exogenous NGF is used, a relatively large amount of relatively costly recombinant protein is required.
Rather than these aforestated solutions to delivery of proteins, it would be desirable to: avoid invasive techniques; to control the amount and the site of delivery of neurotrophic proteins to sites where they are most needed thereby minimizing toxic side effects; and to minimize the health care costs of treatment.
An additional approach to treating neurological symptoms has followed the observation that certain amino acids (glutamic acid and aspartic acid) act as excitatory neurotransmitters that bind the N-methyl D-aspartate (NMDA) receptor. Excess release of these amino acids (EAA) causes overstimulation of the neurons in neurodegenerative diseases as well as in conditions of hypoglycemia or trauma, resulting in neuronal loss and behavioral dysfunctions.
NMDA is a potent and toxic analogue of glutamate which has been shown in animal studies to mediate much of the neuronal death associated with head trauma, hypoglycemia, anoxia, hypoxia and other conditions, and compromises the flow of blood, oxygen or glucose to the central nervous system.
A number of synthetic compounds that act as antagonists of the receptor have been described and tested in animal models. The possibility that these compounds are toxic in humans remains unresolved. Despite many years of clinical research, these antagonists are not as yet available as therapeutic products for treating patients.
For the foregoing reasons there is a need for methods of protecting neurons from accelerated cell death caused by trauma or disease or by the aging process or by combinations of these factors. There is also a need for methods that stimulate the production of neurotrophic growth factors using small molecules that are capable of crossing the blood-brain barrier and that have minimal side effects.
SUMMARY
A preferred embodiment of the-invention is directed toward a method of protecting a population of nerve cells from death that includes administering to a nerve cell population in an animal subject an effective dosage of an estrogen compound sufficient to cause the nerve cell population to be protected from progressive cell damage leading to the death of the cells otherwise occurring without any intervention, wherein the estrogen has a general structure :
i~2 20. AP
N
a tautomer thereof, or a pharmaceutically acceptable salt thereof.
A further embodiment of the invention is directed toward a method of protecting a population of nerve cells from death, that includes administering an estrogen compound 3-0 in an ef-fe-c-ta.ve dosage sufficient to cause the nerve cell population to be protected from progressive cell damage leading to death of the cells otherwise occurring without any intervention.
A further embodiment of the invention is a method of treating a subject, undergoing loss of neuronal activity at a site within a subject, comprising administering an estrogen compound to the subject in an effective amount so as to allow the compound to decrease the loss of neuronal activity.
A further embodiment of the invention is a method of increasing the amount of a naturally occurring neurotrophic factor available to a neurodegenerating site in CNS tissue, comprising administering an estrogen compound to neurons in a subject at a physiologically relevant dose so as to achieve a plasma level of the compound sufficient to cause an increase in the amount of the neurotrophic factor available to the neurons.
A further embodiment of the invention is a method for preventing a neurodegenerative disorder induced by overstimulation of excitatory amino acid receptors comprising; administering to a subject suffering from such neurodegenerative disorder, an effective amount of an estrogen compound.
SRIEF DESCRIPTION OF THE DRAWINGS
These and other features will become better understood with regard to the following description, appended claims and accompanying drawings where:
Figure 1 shows a histogram of the effects of 179-estradiol (E2) on the age related release of lactate dehydrogenase (LD) in primary cortical neuronal cultures.
Figure 2 shows a histogram of the protective effects of EZ on the cytotoxicity induced by various levels of hypoglycemia in C6 cells.
Figure 3 shows a histogram of the protective effects of Ez on the cytotoxic effects of N-methyl D-aspartate (NMDA) in neuroblastoma cells.
Figure 4 shows active avoidance performance following 5 weeks of ovariectomy in intact, ovariectomized and Ez replaced animals.
Figure 5 shows active avoidance performance following 28 weeks of ovariectomy in intact, ovariectomized and EZ
replaced animals.
SUMMARY
A preferred embodiment of the-invention is directed toward a method of protecting a population of nerve cells from death that includes administering to a nerve cell population in an animal subject an effective dosage of an estrogen compound sufficient to cause the nerve cell population to be protected from progressive cell damage leading to the death of the cells otherwise occurring without any intervention, wherein the estrogen has a general structure :
i~2 20. AP
N
a tautomer thereof, or a pharmaceutically acceptable salt thereof.
A further embodiment of the invention is directed toward a method of protecting a population of nerve cells from death, that includes administering an estrogen compound 3-0 in an ef-fe-c-ta.ve dosage sufficient to cause the nerve cell population to be protected from progressive cell damage leading to death of the cells otherwise occurring without any intervention.
A further embodiment of the invention is a method of treating a subject, undergoing loss of neuronal activity at a site within a subject, comprising administering an estrogen compound to the subject in an effective amount so as to allow the compound to decrease the loss of neuronal activity.
A further embodiment of the invention is a method of increasing the amount of a naturally occurring neurotrophic factor available to a neurodegenerating site in CNS tissue, comprising administering an estrogen compound to neurons in a subject at a physiologically relevant dose so as to achieve a plasma level of the compound sufficient to cause an increase in the amount of the neurotrophic factor available to the neurons.
A further embodiment of the invention is a method for preventing a neurodegenerative disorder induced by overstimulation of excitatory amino acid receptors comprising; administering to a subject suffering from such neurodegenerative disorder, an effective amount of an estrogen compound.
SRIEF DESCRIPTION OF THE DRAWINGS
These and other features will become better understood with regard to the following description, appended claims and accompanying drawings where:
Figure 1 shows a histogram of the effects of 179-estradiol (E2) on the age related release of lactate dehydrogenase (LD) in primary cortical neuronal cultures.
Figure 2 shows a histogram of the protective effects of EZ on the cytotoxicity induced by various levels of hypoglycemia in C6 cells.
Figure 3 shows a histogram of the protective effects of Ez on the cytotoxic effects of N-methyl D-aspartate (NMDA) in neuroblastoma cells.
Figure 4 shows active avoidance performance following 5 weeks of ovariectomy in intact, ovariectomized and Ez replaced animals.
Figure 5 shows active avoidance performance following 28 weeks of ovariectomy in intact, ovariectomized and EZ
replaced animals.
Figure 6 shows the effect of 5 week ovariectomy and E2 replacement on high affinity choline uptake in the frontal cortex of behaviorally naive rats.
Figure 7 shows the effect of 5 week ovariectomy and E2 replacement on high affinity choline uptake in the hippocampus of behaviorally naive rats.
Figure 8 shows the protective effect of a-E2 on SK-N-SH
cell viability at 24 hrs. and 8(b) at 48 hrs.
DETAILED DESCRIPTION OF THE INVBNTION
The present invention is directed toward methods for protecting a population of nerve cells in a subject from death and toward stimulating neurotrophic factors for protecting cells from cell death in an animal subject.
An "estrogen compound". is defined here and in the claims as any of the structures described in the llth edition of "Steroids" from Steraloids Inc., Wilton N.H.
Included in this definition are non-steroidal estrogens described in the aforementioned reference. Other estrogen compounds included in this definition are estrogen derivatives, estrogen metabolites and estrogen precursors as well as those molecules capable of binding cell associated estrogen receptor as well as other molecules where the result of binding specifically triggers a characterized estrogen effect. Also included are mixtures of more than 1 estrogen, where examples of such mixtures are provided in Table II below. Examples of a estrogen structures having utility either alone or in combination with other agents are provided in Tables XIII and XIV.
p estrogen is the P isomer of estrogen compounds. a estrogen is the a isomer of estrogen components. The term "estradiol" is either a or P estradiol unless specifically identified.
Figure 7 shows the effect of 5 week ovariectomy and E2 replacement on high affinity choline uptake in the hippocampus of behaviorally naive rats.
Figure 8 shows the protective effect of a-E2 on SK-N-SH
cell viability at 24 hrs. and 8(b) at 48 hrs.
DETAILED DESCRIPTION OF THE INVBNTION
The present invention is directed toward methods for protecting a population of nerve cells in a subject from death and toward stimulating neurotrophic factors for protecting cells from cell death in an animal subject.
An "estrogen compound". is defined here and in the claims as any of the structures described in the llth edition of "Steroids" from Steraloids Inc., Wilton N.H.
Included in this definition are non-steroidal estrogens described in the aforementioned reference. Other estrogen compounds included in this definition are estrogen derivatives, estrogen metabolites and estrogen precursors as well as those molecules capable of binding cell associated estrogen receptor as well as other molecules where the result of binding specifically triggers a characterized estrogen effect. Also included are mixtures of more than 1 estrogen, where examples of such mixtures are provided in Table II below. Examples of a estrogen structures having utility either alone or in combination with other agents are provided in Tables XIII and XIV.
p estrogen is the P isomer of estrogen compounds. a estrogen is the a isomer of estrogen components. The term "estradiol" is either a or P estradiol unless specifically identified.
The term "Ez" is synonymous with 6-estradiol 17j9-estradiol and p-E2. aE2, a-E2, and a-estradiol is the a isomer of P-EZ estradiol.
An "animal subject" is defined here and in the claims as a higher organism including humans having neurons subjected to forces that result in progressive cell damage and cell death.
"Neurotrophic growth factors" are defined here and in the claims as endogenous soluble proteins regulating survival, growth, morphological plasticity or synthesis of proteins for differentiated function of neurons.
"Neurodegenerative disorder" is defined here and in the claims as a disorder in which progressive loss of neurons occurs either in the peripheral nervous system or in the central nervous system. Examples of neurodegenerative disorders include: chronic neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's chorea, diabetic peripheral neuropathy, multiple sclerosis, amyotrophic lateral sclerosis; aging; and acute neurodegenerative disorders including: stroke, traumatic brain injury, schizophrenia, peripheral nerve damage, hypoglycemia, spinal cord injury, epilepsy, and anoxia and hypoxia.
These examples are not meant to be comprehensive but serve merely as an illustration of the term "neurodegenerative disorder."
- .10 -TABLE I
List of Characterized Proteins Exhibiting Neurotrophic Activities Growth Factor References*
Proteins initially characterized as neurotrophic factors:
1,2,3 Nerve growth factor (NGF) Brain-derived neurotrophic factor (BDNF) 4,5 Neurotrophin-3 (NT-3) 6,7,8,9 Neurotrophin-4 (NT-4) 10 Neurotrophin-5 (NT-5) 11 Ciliary neurotrophic factor (CNTF) 12,13 Heparin-binding neurotrophic factor (HBNF) 14 Growth factors with neurotraphie activity:
Basic fibrobtast growth factor (bFGF) 15,16 Acidic fibroblast growth factor (aFGF) 17 Insulin-like growth factors (IGF*s), insulin 18,19 EPidenat growth factor (EGF) 20,21 Transforming growth factor e(iGFa) 22,23 Interleukin 1 24 Interleukin 3 interteukin 6 25 Protease ?? I and II 26 27,28,29 Cholinergic neuronat differentiation factor 30 * References given refer to recent reviews or recent key publications.
=
TABLE II
Commercial Estrogen Preparations 1. Estrone Aqueous Suspensions Usual dosage IN, 0.1 to 0.5 ag, 2 to 3 times weekly.
2. EstradioL Estrase: 1 to 2 mg P0, daily for three weeks; one week off.
3. Estradiot Conjugates in Oil.
a. Estradiot-cypionate I to 5 mg IM; every 3 to 4 weeks-more than 20 preparations.
b. Estradiol-vaterate 10 to 20 mg IM; every 4 weeks - more than 30 preparations.
4. OraL estrogen preparations 50 to 65 % estrone sutfate and 20 to 35 %
equilin sulfate.
a. Premarin 0.3 to 2.5 mg PO daily for 3 weeks; one week off.
b. Estrocon 0.625 to 2.5 mg PO daily for 3 weeks; one week off.
c. Progens 0.625 to 2.5 mg PO daily for 3 weeks; one week off.
d. Many others 0.625 to 2.5 ag P0 daily for 3 weeks; one week off.
5. Oral Esterified Estrogens 75 to 85 % estrone sulfate and 6 to 15 X equitin sulfate.
a. Estratab 0.3 to 2.5 mg PO daily for three weeks; one week off.
b. Menest 0.3 to 2.5 mg PO daily for three weeks; one week off.
6. Estropipate Piperazine Estrone Sulfate.
2 5 a. Ogen 0.625 to 5 mg PO daity for 3 weeks: one week off.
7. EthinyL Estradiol.
a. Estinyt 0.02 to 0.5 ag P0 daily for three weeks; one week off.
3 0 b. Feminone 0.05 ng PO daily for three weeks; one week off.
8. Quinestrot a fat stored, stow release form of ethinyl estradioL.
a. Estrovis 100 ug P0 daily for 7 days; 100,ug weekly thereafter.
35 9. Diethylstitbestrot (0.2 to 0.5 mg P0 daily for three weeks; one week off.
An "animal subject" is defined here and in the claims as a higher organism including humans having neurons subjected to forces that result in progressive cell damage and cell death.
"Neurotrophic growth factors" are defined here and in the claims as endogenous soluble proteins regulating survival, growth, morphological plasticity or synthesis of proteins for differentiated function of neurons.
"Neurodegenerative disorder" is defined here and in the claims as a disorder in which progressive loss of neurons occurs either in the peripheral nervous system or in the central nervous system. Examples of neurodegenerative disorders include: chronic neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's chorea, diabetic peripheral neuropathy, multiple sclerosis, amyotrophic lateral sclerosis; aging; and acute neurodegenerative disorders including: stroke, traumatic brain injury, schizophrenia, peripheral nerve damage, hypoglycemia, spinal cord injury, epilepsy, and anoxia and hypoxia.
These examples are not meant to be comprehensive but serve merely as an illustration of the term "neurodegenerative disorder."
- .10 -TABLE I
List of Characterized Proteins Exhibiting Neurotrophic Activities Growth Factor References*
Proteins initially characterized as neurotrophic factors:
1,2,3 Nerve growth factor (NGF) Brain-derived neurotrophic factor (BDNF) 4,5 Neurotrophin-3 (NT-3) 6,7,8,9 Neurotrophin-4 (NT-4) 10 Neurotrophin-5 (NT-5) 11 Ciliary neurotrophic factor (CNTF) 12,13 Heparin-binding neurotrophic factor (HBNF) 14 Growth factors with neurotraphie activity:
Basic fibrobtast growth factor (bFGF) 15,16 Acidic fibroblast growth factor (aFGF) 17 Insulin-like growth factors (IGF*s), insulin 18,19 EPidenat growth factor (EGF) 20,21 Transforming growth factor e(iGFa) 22,23 Interleukin 1 24 Interleukin 3 interteukin 6 25 Protease ?? I and II 26 27,28,29 Cholinergic neuronat differentiation factor 30 * References given refer to recent reviews or recent key publications.
=
TABLE II
Commercial Estrogen Preparations 1. Estrone Aqueous Suspensions Usual dosage IN, 0.1 to 0.5 ag, 2 to 3 times weekly.
2. EstradioL Estrase: 1 to 2 mg P0, daily for three weeks; one week off.
3. Estradiot Conjugates in Oil.
a. Estradiot-cypionate I to 5 mg IM; every 3 to 4 weeks-more than 20 preparations.
b. Estradiol-vaterate 10 to 20 mg IM; every 4 weeks - more than 30 preparations.
4. OraL estrogen preparations 50 to 65 % estrone sutfate and 20 to 35 %
equilin sulfate.
a. Premarin 0.3 to 2.5 mg PO daily for 3 weeks; one week off.
b. Estrocon 0.625 to 2.5 mg PO daily for 3 weeks; one week off.
c. Progens 0.625 to 2.5 mg PO daily for 3 weeks; one week off.
d. Many others 0.625 to 2.5 ag P0 daily for 3 weeks; one week off.
5. Oral Esterified Estrogens 75 to 85 % estrone sulfate and 6 to 15 X equitin sulfate.
a. Estratab 0.3 to 2.5 mg PO daily for three weeks; one week off.
b. Menest 0.3 to 2.5 mg PO daily for three weeks; one week off.
6. Estropipate Piperazine Estrone Sulfate.
2 5 a. Ogen 0.625 to 5 mg PO daity for 3 weeks: one week off.
7. EthinyL Estradiol.
a. Estinyt 0.02 to 0.5 ag P0 daily for three weeks; one week off.
3 0 b. Feminone 0.05 ng PO daily for three weeks; one week off.
8. Quinestrot a fat stored, stow release form of ethinyl estradioL.
a. Estrovis 100 ug P0 daily for 7 days; 100,ug weekly thereafter.
35 9. Diethylstitbestrot (0.2 to 0.5 mg P0 daily for three weeks; one week off.
10. Chlorotrianisene Taee; 12 to 25 mg PO daiLy for three weeks; on week off.
11. OraL Estrogen-Antianxiety Agent Coabinations Oral.
a. MiLprem-200 or -400 (Conjugated estrogens and meprobamate).
40 b. PM8 20 or 400 (Conjugated estrogens aid meprobamate).
C. Menrium 5-2, 5-4, or 10-4 esterified estrogens and chlordiazepoxide.
a. MiLprem-200 or -400 (Conjugated estrogens and meprobamate).
40 b. PM8 20 or 400 (Conjugated estrogens aid meprobamate).
C. Menrium 5-2, 5-4, or 10-4 esterified estrogens and chlordiazepoxide.
12. Estrogen and Androgen Coabinations IM in Oil.
a. 13 preparations with 2 mg estradiol cypionate and 50 mg testosterone cypionate.
4 5 b. 12 preparations with 4 mg estradioL valerate and 90 mg testosterone enanthate.
c. 4 preparations with 8 mg estradiol valerate and 180 sg testosterone enanthate.
d. 6 preparations with various combinations of other estrogens and androgens.
50 Properties of estrogen.
Estrogen occurs in at least two isomeric forms, including p estrogen and a estrogen. ~ estrogens are pleotrophic molecule with many biological activities.
Clinical uses include treatment of osteoporosis, symptoms of 55 menopause and fertility control. In embodiments of the invention, p estrogen has also been shown to protect a subject against neuronal loss.
In comparison to A estrogen, a estrogen is typically believed to be at least 100-1000 times less potent in WO 95"2402 217 5 6 0 3 PCT/US94/12782 =
estrogenic activity. Numerous examples have been reported in the literature that show that the biological effects of ~
estrogen are not shared by the a isomer. in fact, in the art, a estrogen is typically used as a negative control for /3 estradiol.
We have shown for the first time that a estrogen has a comparable activity to that of p estrogen for neuroprotection. The novel activity identified for a estrogen presents a number ofadvantages in the treatment of neurodegenerative diseases, trauma and aging. These advantages arise in situations which require treatment of males where the development of female traits is to be avoided and the treatment of females where the subject has increased susceptibility to endometrial, breast and cervical cancer.
In an embodiment of the invention, a novel use for estrogen has been identified, namely to cause arrest and/or reversal of progressive degeneration of neurons. The degenerative process ultimately leads to behavioral defects that accompany reduction in observed neuronal density.
According to this invention, improvements in cognition, memory and other behavioral symptoms occur in subjects suffering from neurodegenerative disorders caused individually by disease, trauma or aging or a combination of these factors following administration of estrogen. Also according to this invention is an arrest in progression of a neurodegenerative disease such as by intervention and/or prevention of neuronal loss the patient will not develop or manifest a further decline in disease course. Evidence for this effect is shown in the accompanying examples and is correlated with biochemical effects identified both in vivo and in vitro.
In Example 1, in vitro studies have demonstrated cytoprotective properties of estrogen where comparatively low concentrations of a-E2 and p-EZ enhance viability in neuronal cells. In a further embodiment of the invention, pretreatment of cells with p-EZ-has been shown to protect WO 95112402 217 5 6 0 3 PCTfUS94/12782 cells from death caused by glucose reduction or deprivation, a condition called hypoglycemia. Post-treatment with E2 can rescue cells from the effects of hypoglycemia in a manner t~z- dissipates with increasing length of hypoglycemia (Example 2).
These examples demonstrate that both a and p estradiol at physiologically relevant doses exert a cytoprotective effect on both glial and neuroblastoma cells lines in vitro and that this cytoprotective effect can be distinguished from a mitogenic action. While not wishing to be bound by theory, we hypothesize that estrogen exerts a direct protective effect on neuronal cells.
In vivo studies described in Examples 3-6 have shown that estrogen can reverse an impairment in non-spatial learning. This impairment is correlated with a time dependent decline in choline acetyl transferase (ChAT) in both the frontalcortex and the hippocampus which is attenuated in animals treated with estradiol. The ChAT-containing nerve terminals in these two brain regions have cell bodies located in the basal forebrain. In Example 3, rats have shown improvements in behavioral performances as determined by the active avoidance test following the addition of estrogen to estrogen deficient ovariectomized animals. Collectively, these data provide a method of treating subjects through the modulation of basal forebrain cholinergic function by means of treating with estrogen so as to reduce loss of learning and memory associated with neuronal damage.
Examples 1-6 demonstrate a number of different pathways through which estrogen compounds exert a cytoprotective effect on cells in the CNS. These include protection against hypoglycemia (Examples 2a, 2b, and 6) and protection from overstimulation of EAA (Examples 2c and 5), as well as by the stimulation of neurotrophic growth factor production (Examples 4a and 4b).
Example 4a describes experiments demonstrating the increase in brain derived neurotrophic factor (BDNF) ~
a. 13 preparations with 2 mg estradiol cypionate and 50 mg testosterone cypionate.
4 5 b. 12 preparations with 4 mg estradioL valerate and 90 mg testosterone enanthate.
c. 4 preparations with 8 mg estradiol valerate and 180 sg testosterone enanthate.
d. 6 preparations with various combinations of other estrogens and androgens.
50 Properties of estrogen.
Estrogen occurs in at least two isomeric forms, including p estrogen and a estrogen. ~ estrogens are pleotrophic molecule with many biological activities.
Clinical uses include treatment of osteoporosis, symptoms of 55 menopause and fertility control. In embodiments of the invention, p estrogen has also been shown to protect a subject against neuronal loss.
In comparison to A estrogen, a estrogen is typically believed to be at least 100-1000 times less potent in WO 95"2402 217 5 6 0 3 PCT/US94/12782 =
estrogenic activity. Numerous examples have been reported in the literature that show that the biological effects of ~
estrogen are not shared by the a isomer. in fact, in the art, a estrogen is typically used as a negative control for /3 estradiol.
We have shown for the first time that a estrogen has a comparable activity to that of p estrogen for neuroprotection. The novel activity identified for a estrogen presents a number ofadvantages in the treatment of neurodegenerative diseases, trauma and aging. These advantages arise in situations which require treatment of males where the development of female traits is to be avoided and the treatment of females where the subject has increased susceptibility to endometrial, breast and cervical cancer.
In an embodiment of the invention, a novel use for estrogen has been identified, namely to cause arrest and/or reversal of progressive degeneration of neurons. The degenerative process ultimately leads to behavioral defects that accompany reduction in observed neuronal density.
According to this invention, improvements in cognition, memory and other behavioral symptoms occur in subjects suffering from neurodegenerative disorders caused individually by disease, trauma or aging or a combination of these factors following administration of estrogen. Also according to this invention is an arrest in progression of a neurodegenerative disease such as by intervention and/or prevention of neuronal loss the patient will not develop or manifest a further decline in disease course. Evidence for this effect is shown in the accompanying examples and is correlated with biochemical effects identified both in vivo and in vitro.
In Example 1, in vitro studies have demonstrated cytoprotective properties of estrogen where comparatively low concentrations of a-E2 and p-EZ enhance viability in neuronal cells. In a further embodiment of the invention, pretreatment of cells with p-EZ-has been shown to protect WO 95112402 217 5 6 0 3 PCTfUS94/12782 cells from death caused by glucose reduction or deprivation, a condition called hypoglycemia. Post-treatment with E2 can rescue cells from the effects of hypoglycemia in a manner t~z- dissipates with increasing length of hypoglycemia (Example 2).
These examples demonstrate that both a and p estradiol at physiologically relevant doses exert a cytoprotective effect on both glial and neuroblastoma cells lines in vitro and that this cytoprotective effect can be distinguished from a mitogenic action. While not wishing to be bound by theory, we hypothesize that estrogen exerts a direct protective effect on neuronal cells.
In vivo studies described in Examples 3-6 have shown that estrogen can reverse an impairment in non-spatial learning. This impairment is correlated with a time dependent decline in choline acetyl transferase (ChAT) in both the frontalcortex and the hippocampus which is attenuated in animals treated with estradiol. The ChAT-containing nerve terminals in these two brain regions have cell bodies located in the basal forebrain. In Example 3, rats have shown improvements in behavioral performances as determined by the active avoidance test following the addition of estrogen to estrogen deficient ovariectomized animals. Collectively, these data provide a method of treating subjects through the modulation of basal forebrain cholinergic function by means of treating with estrogen so as to reduce loss of learning and memory associated with neuronal damage.
Examples 1-6 demonstrate a number of different pathways through which estrogen compounds exert a cytoprotective effect on cells in the CNS. These include protection against hypoglycemia (Examples 2a, 2b, and 6) and protection from overstimulation of EAA (Examples 2c and 5), as well as by the stimulation of neurotrophic growth factor production (Examples 4a and 4b).
Example 4a describes experiments demonstrating the increase in brain derived neurotrophic factor (BDNF) ~
determined by increased levels of BDNF mRNA while Example 4b describes the stimulation of nerve growth factor production determined by increased levels of NGF mRNA. A further cytoprotective effect of estrogen has been demonstrated by the amelioration of the toxic effects of overstimulation of excitatory amino acid (EAA) receptors (Examples 2c and 5).
Overstimulation of the EAA receptors has been identified as characteristic of a number of neurodegenerative disorders including epilepsy. In a further embodiment of the invention, the estrogen compounds ameliorate the toxic effects of hypoglycemia that is a further cause of progressive cell damage leading to cell death (Examples 2a, 2b, and 6).
While not wishing to be bound by theory, it is suggested that estrogen compounds act on a fundamental process that impacts cell viability and cell response to adverse conditions that result in damage and death and that this process underlies the observed phenomena. An example of such a mechanism includes the regulation of glucose to cells. -These observations contrast with observations of the prior art. Although estrogen has been identified as having utility in treating adverse behavioral symptoms that accompany fluctuations in hormones associated with menopause in aging women, the biochemical basis for these effects has never been determined. As such, the treatment of behavioral effects with estrogen in human subjects has been restricted to the treatment of menopause in women that demonstrate signs of deficiency in estrogen and use in prevention of the sequelae of menopause, namely osteoporosis, corrected by replacement therapy of estrogen.
The human clinical studies do however demonstrate that externally administered estrogen is non toxic. in addition, these studies demonstrate that estrogen administered intramuscularly subsequently reaches the brain as inferred by the behavioral effects of the treatment and as predicted from the structure of the molecule.
~
Overstimulation of the EAA receptors has been identified as characteristic of a number of neurodegenerative disorders including epilepsy. In a further embodiment of the invention, the estrogen compounds ameliorate the toxic effects of hypoglycemia that is a further cause of progressive cell damage leading to cell death (Examples 2a, 2b, and 6).
While not wishing to be bound by theory, it is suggested that estrogen compounds act on a fundamental process that impacts cell viability and cell response to adverse conditions that result in damage and death and that this process underlies the observed phenomena. An example of such a mechanism includes the regulation of glucose to cells. -These observations contrast with observations of the prior art. Although estrogen has been identified as having utility in treating adverse behavioral symptoms that accompany fluctuations in hormones associated with menopause in aging women, the biochemical basis for these effects has never been determined. As such, the treatment of behavioral effects with estrogen in human subjects has been restricted to the treatment of menopause in women that demonstrate signs of deficiency in estrogen and use in prevention of the sequelae of menopause, namely osteoporosis, corrected by replacement therapy of estrogen.
The human clinical studies do however demonstrate that externally administered estrogen is non toxic. in addition, these studies demonstrate that estrogen administered intramuscularly subsequently reaches the brain as inferred by the behavioral effects of the treatment and as predicted from the structure of the molecule.
~
Although clinical studies by Sherwin 1988, psvchoneuroendoarinoloav 13:345-357, and Sherwin and Phillips 1990, Annals of the New York Academy of Sciences 592:474-5, have shown a general mood enhancing effect in oophorectomized women following intramuscularly administration of estrogen at doses of 10 mg, the mechanism by which this effect occurred is unclear.
Clinical studies on women with Alzheimer's disease have shown acute benefit in some patients by improving mood and reducing depression in response to short-term duration of estrogen treatment (Fillit et al. 1986, Neuroscience Abst.
12:945; Fillit et al. 1986, Psychoneuroendocrinology 11:
337-345; Honjo et al, 1989, Steroid Biochem. 34:521-525).
The results from these studies were difficult to interpret because of the small number of patients used, because treatment only had limited effect on a few patients, and because treatment extended for a short period of time only.
Indeed neither study was of a duration long enough to evaluate or indeed to manifest any neuroprotective effects if any of estrogen. An additional study by Aroonsakul, 1990, US Patent 4,897,389 described the effect of anabolic hormones including estrogen on a single man and a single women where the relief of symptoms was proposed to result from stimulation of cell replication and human growth hormone production following a short-term administration of hormone mixtures.
Biochemical studies on the actibn of estrogen on cells of the CNS either in vivo or in vitro has resulted in conflicting reports. A number of studies have shown that estradiol has an effect on the plasticity of neurons. Morse et al. 1986, Experimental Neuroloav 94:649-658, reported that an estrogen derivative enhances sprouting of commissural-associational afferent fibers in the hippocampal dentate gyrus following entorhinal cortex lesions.
Additionally, cyclic changes in synaptic density in the CAl of the hippocampus were shown to be related to circulating E. levels (Woolley et al. 1992, Journal of Neuroscience =
Clinical studies on women with Alzheimer's disease have shown acute benefit in some patients by improving mood and reducing depression in response to short-term duration of estrogen treatment (Fillit et al. 1986, Neuroscience Abst.
12:945; Fillit et al. 1986, Psychoneuroendocrinology 11:
337-345; Honjo et al, 1989, Steroid Biochem. 34:521-525).
The results from these studies were difficult to interpret because of the small number of patients used, because treatment only had limited effect on a few patients, and because treatment extended for a short period of time only.
Indeed neither study was of a duration long enough to evaluate or indeed to manifest any neuroprotective effects if any of estrogen. An additional study by Aroonsakul, 1990, US Patent 4,897,389 described the effect of anabolic hormones including estrogen on a single man and a single women where the relief of symptoms was proposed to result from stimulation of cell replication and human growth hormone production following a short-term administration of hormone mixtures.
Biochemical studies on the actibn of estrogen on cells of the CNS either in vivo or in vitro has resulted in conflicting reports. A number of studies have shown that estradiol has an effect on the plasticity of neurons. Morse et al. 1986, Experimental Neuroloav 94:649-658, reported that an estrogen derivative enhances sprouting of commissural-associational afferent fibers in the hippocampal dentate gyrus following entorhinal cortex lesions.
Additionally, cyclic changes in synaptic density in the CAl of the hippocampus were shown to be related to circulating E. levels (Woolley et al. 1992, Journal of Neuroscience =
12:2549-2554) and these changes could be mimicked with exogenous E2 administration (Woolley et al. 1992). Indeed, it has further been shown that ovariectomy reduces and E2 replacement normalizes high affinity choline uptake (HACU) in the frontal cortex of rats.
Additionally, Gibbs et al. 1993 (Societv for Neuroscience Abstracts 19:5) have reported upregulation of choline acetyltransferase (ChAT) levels following estradiol treatment in the medial septum after 2 days and 2 weeks of treatment although no effect was observed after 1 week using in situ hybridization of ChAT mRNA. Luine et al. 1980, Brain Research 191:273-277, reported increased ChAT levels in the preoptic and hypothalamic regions of the rat brain in response to estradiol treatment.
In an embodiment of the invention, administration of estrogen in a physiological dose results in the reversal of impairment of non-spatial learning in female rats that have been ovariectomized (OVX). These behavioral effects of short-term OVX and E2-replacement are correlated with biochemical changes in the hippocampus and the frontal cortex of the brain; in particular, a reduction and increase in high affinity choline uptake (HACU) in OVX and E2-pellet treated rats, respectively. Short-term E2-replacement also had a positive effect on choline acetyltransferase activity (ChAT) in the hippocampus, but not in the frontal cortex.
Long-term E2 replacement appeared to prevent the time-dependent decline of ChAT in the frontal cortex and to attenuate ChAT activity decline in the hippocampus.
Collectively, these data show that estrogen has a cytoprotective effect on cells in the CNS and that the estrogen environment of adult female rats influences learning and the activity of basal forebrain cholinergic neurons and also demonstrate the importance of estrogens in the maintenance and proper function of basal forebrain cholinergic neurons in the female rat.
In 1977, Perez-Polo et al. (Life Sci. 21:1535-1543) published a paper entitled "Steroid Induction of Nerve WO 95112402 _217 C,LUQ3 PCT/US94112752 ~ !J
Additionally, Gibbs et al. 1993 (Societv for Neuroscience Abstracts 19:5) have reported upregulation of choline acetyltransferase (ChAT) levels following estradiol treatment in the medial septum after 2 days and 2 weeks of treatment although no effect was observed after 1 week using in situ hybridization of ChAT mRNA. Luine et al. 1980, Brain Research 191:273-277, reported increased ChAT levels in the preoptic and hypothalamic regions of the rat brain in response to estradiol treatment.
In an embodiment of the invention, administration of estrogen in a physiological dose results in the reversal of impairment of non-spatial learning in female rats that have been ovariectomized (OVX). These behavioral effects of short-term OVX and E2-replacement are correlated with biochemical changes in the hippocampus and the frontal cortex of the brain; in particular, a reduction and increase in high affinity choline uptake (HACU) in OVX and E2-pellet treated rats, respectively. Short-term E2-replacement also had a positive effect on choline acetyltransferase activity (ChAT) in the hippocampus, but not in the frontal cortex.
Long-term E2 replacement appeared to prevent the time-dependent decline of ChAT in the frontal cortex and to attenuate ChAT activity decline in the hippocampus.
Collectively, these data show that estrogen has a cytoprotective effect on cells in the CNS and that the estrogen environment of adult female rats influences learning and the activity of basal forebrain cholinergic neurons and also demonstrate the importance of estrogens in the maintenance and proper function of basal forebrain cholinergic neurons in the female rat.
In 1977, Perez-Polo et al. (Life Sci. 21:1535-1543) published a paper entitled "Steroid Induction of Nerve WO 95112402 _217 C,LUQ3 PCT/US94112752 ~ !J
Growth Factor Synthesis in Cell Culture". Although the title indicates a positive effect of steroids on the production of NGF by glial cells in vitro, closer inspection of =the paper revealed the opposite. By using non-specific polyclonal antibodies, increased levels of a high molecular weight molecule was identified in response to high levels of estrogen whereas there was no increase in the amount of a low molecular weight (MW) fraction now known to contain p NGF and similarly recognized by the antibody preparation.
The doses of estrogen used in the experiment were 5,500 times higher then physiological levels. The levels of the low MW fraction attributed to NGF and secreted into the media were found to be 1000-fold higher than predicted for NGF production using current methods. No further work was carried out by these investigators or others to determine in vivo effects of estrogen on NGF production. Subsequent in vivo studies by Gibbs et al. 1993 reported that steroids (17-Q estradiol) caused decreased levels of NGF mRNA in the hippocampus followed by decreased levels in the medial septum and the diagonal band of Broca.
Contrary to the above report, an embodiment of the invention describes how estradiol stimulates the production of neurotrophic growth factor mRNA. For the first time, an estrogen compound has been described that not only has ready access to brain tissue across the blood-brain barrier but can stimulate the availability of growth factor where it is most needed so as to reverse and offset the effects of neurodegeneration.
In a preferred embodiment of the invention, novel properties have been identified for estrogen compounds that provide for the first time a method of using estrogen to arrest and/or reverse progression of neurodegenerative disorders rather than merely treating the symptoms of the disease.
In a preferred embodiment, both stereoisomers of estradiol, 17-p-estradiol and 17-a-estradiol have been found effective in reversing neurodegeneration.
The doses of estrogen used in the experiment were 5,500 times higher then physiological levels. The levels of the low MW fraction attributed to NGF and secreted into the media were found to be 1000-fold higher than predicted for NGF production using current methods. No further work was carried out by these investigators or others to determine in vivo effects of estrogen on NGF production. Subsequent in vivo studies by Gibbs et al. 1993 reported that steroids (17-Q estradiol) caused decreased levels of NGF mRNA in the hippocampus followed by decreased levels in the medial septum and the diagonal band of Broca.
Contrary to the above report, an embodiment of the invention describes how estradiol stimulates the production of neurotrophic growth factor mRNA. For the first time, an estrogen compound has been described that not only has ready access to brain tissue across the blood-brain barrier but can stimulate the availability of growth factor where it is most needed so as to reverse and offset the effects of neurodegeneration.
In a preferred embodiment of the invention, novel properties have been identified for estrogen compounds that provide for the first time a method of using estrogen to arrest and/or reverse progression of neurodegenerative disorders rather than merely treating the symptoms of the disease.
In a preferred embodiment, both stereoisomers of estradiol, 17-p-estradiol and 17-a-estradiol have been found effective in reversing neurodegeneration.
In a preferred embodiment, estradiol is administered to rats and also to humans at concentrations sufficient to exert neuroprotective effects in the CNS. These doses vary according to interperson variability, the route of administration and the estrogen formulation used. For example, in rats, estradiol is administered subcutaneously by means of a silastic tube to achieve plasma levels of about 50 pg/ml. In humans, 0.2-10 mg or more specifically 1-2 mg of orally administered estrase (estradiol) given daily is commonly administered to patients suffering from post menopausal syndrome. These levels are also expected to be effective in treating neurodegenerative disorders in human subjects.
The recommended route of administration of the estrogen compound includes oral, intramuscular, transdermal, buccal, intravenous and subcutaneous. Methods of administering estrogen may be by dose or by controlled release vehicles.
Administration of estrogen may include the use of a single estrogen compound or a mixture of estrogens.
The protection of cholinergic neurons from severe degeneration is an important aspect of treatment for patients with acute or chronic neurodegenerative disorders, an example of chronic disease being Alzheimer's disease.
For Alzheimer's patients, estrogen replacement or supplementation may be of significant therapeutic use.
Other diseases for which estrogen treatment may be effective include Parkinson's disease, Huntington's disease, AIDS
Dementia, Wernicke-Korsakoff's related dementia (alcohol induced dementia), age related dementia, age associated memory impairment, brain cell loss due to any of the following; head trauma, stroke, hypoglycemia, ischemia, anoxia, hypoxia, cerebral edema, arteriosclerosis, hematoma and epilepsy; spinal cord cell loss due to any of the conditions listed under brain cell loss; and peripheral neuropathy. Because of its cytoprotective properties, it is suggested that one pathway of action for estrogen is the inhibition of apoptosis.
The recommended route of administration of the estrogen compound includes oral, intramuscular, transdermal, buccal, intravenous and subcutaneous. Methods of administering estrogen may be by dose or by controlled release vehicles.
Administration of estrogen may include the use of a single estrogen compound or a mixture of estrogens.
The protection of cholinergic neurons from severe degeneration is an important aspect of treatment for patients with acute or chronic neurodegenerative disorders, an example of chronic disease being Alzheimer's disease.
For Alzheimer's patients, estrogen replacement or supplementation may be of significant therapeutic use.
Other diseases for which estrogen treatment may be effective include Parkinson's disease, Huntington's disease, AIDS
Dementia, Wernicke-Korsakoff's related dementia (alcohol induced dementia), age related dementia, age associated memory impairment, brain cell loss due to any of the following; head trauma, stroke, hypoglycemia, ischemia, anoxia, hypoxia, cerebral edema, arteriosclerosis, hematoma and epilepsy; spinal cord cell loss due to any of the conditions listed under brain cell loss; and peripheral neuropathy. Because of its cytoprotective properties, it is suggested that one pathway of action for estrogen is the inhibition of apoptosis.
ExamDles Example la: In vitro studies demonstrate increased viability of nerve cells in the presence of an estrogen compound where estrogen exerts a cell protective effect.
Experimental design. A neuroblastoma (SK-N-SH) cell line was obtained from American Type Culture Collection (ATCC) (Rockville, MD). Culture conditions have been described previously (Keller et al. 1976, Kolbe et al.
1976). All experiments were accomplished in cells that were in passage number 3-6. Each experiment was divided into three treatment groups: Group 1 was RPMI media supplemented with 10% fetal bovine serum (FBS), group 2 was RPMI media without the FBS (serum free group) and group 3 was serum free RPMI media supplemented with 544 pg E2/ml (E2 provided by Steraloids, Inc., Wilton, NH), greatly in excess of the 16pg/ml of estrogen normally found in FBS. E2 was initially dissolved in l00 1 absolute ethanol and then diluted with media. Media for the other two groups were similarly prepared with the addition 100 l absolute ethanol with the media. Media was changed at 48 h into the experiment to maintain the normal schedule of nutrient replenishment.
The rate of growth of the population of cells in each experimental group was measured.
Quantification of cell viability. Cell viability was assessed using the trypan blue dye exclusion method of Black et al. 1964, Exn. Cell Research 35:9-13. Fcr each aliquot, two separate counts of total cells and dead cells were made.
Live cell number was determined from the difference between total and dead cell number. An important limitation of the trypan blue method of staining is its time dependence.
Therefore, care was taken to standardize time between resuspension of the cells, addition of the dye and actual counting on the hemacytometer.
Analysis of data. All data are presented as mean SEM
after correcting for the dilution factor allowing the data WO 95/12402 2 1 7 5[1 t13 PCT/US94/12782 to be expressed as the number of cells per ml. Data were analyzed by an analysis of variance followed by Scheffe's F
test. The criterion for significance was p < 0.05.
The mitosis ratio was calculated by dividing dead cells 5(number cells/ml) over total-cells (number cells/ml). This index provides a means of distinguishing between the mitotic and the cytoprotective effects of the treatments used in the experimental design. The ratio of dead/total cells was used to differentiate mitotic from cytoprotective effects of EZ
in culture.
Experimental results. The ratio of dead to total cells increased by 2 to 3 - fold with the removal of FBS from the culture media (Table III). The addition of E2 prevented this serum removal effect and produced ratios similar to those observed in the FBS group at each sample time.
Through 48 h, the ratio of dead to live cells remained constant for E2-treated cells, but nearly doubled for serum free cells. By 96 h, the ratio increased in all three groups, indicating the diminishing cytoprotective effect with time.
Although the total SK-N-SH cell number in the FBS group was approximately double that seen with E. treated cells at both 24 and 48 hours, the fraction of live cells was similar. However, E2-treated SK-N-SH cells did not show the exponential growth pattern seen for cells grown in FBS. The ratio of dead to total SK-N-SH treated cells increased in all three groups with time (Table III). However, at each sampling time, this ratio was similar and markedly lower in both E2 and FBS groups than under serum free conditions.
Experimental design. A neuroblastoma (SK-N-SH) cell line was obtained from American Type Culture Collection (ATCC) (Rockville, MD). Culture conditions have been described previously (Keller et al. 1976, Kolbe et al.
1976). All experiments were accomplished in cells that were in passage number 3-6. Each experiment was divided into three treatment groups: Group 1 was RPMI media supplemented with 10% fetal bovine serum (FBS), group 2 was RPMI media without the FBS (serum free group) and group 3 was serum free RPMI media supplemented with 544 pg E2/ml (E2 provided by Steraloids, Inc., Wilton, NH), greatly in excess of the 16pg/ml of estrogen normally found in FBS. E2 was initially dissolved in l00 1 absolute ethanol and then diluted with media. Media for the other two groups were similarly prepared with the addition 100 l absolute ethanol with the media. Media was changed at 48 h into the experiment to maintain the normal schedule of nutrient replenishment.
The rate of growth of the population of cells in each experimental group was measured.
Quantification of cell viability. Cell viability was assessed using the trypan blue dye exclusion method of Black et al. 1964, Exn. Cell Research 35:9-13. Fcr each aliquot, two separate counts of total cells and dead cells were made.
Live cell number was determined from the difference between total and dead cell number. An important limitation of the trypan blue method of staining is its time dependence.
Therefore, care was taken to standardize time between resuspension of the cells, addition of the dye and actual counting on the hemacytometer.
Analysis of data. All data are presented as mean SEM
after correcting for the dilution factor allowing the data WO 95/12402 2 1 7 5[1 t13 PCT/US94/12782 to be expressed as the number of cells per ml. Data were analyzed by an analysis of variance followed by Scheffe's F
test. The criterion for significance was p < 0.05.
The mitosis ratio was calculated by dividing dead cells 5(number cells/ml) over total-cells (number cells/ml). This index provides a means of distinguishing between the mitotic and the cytoprotective effects of the treatments used in the experimental design. The ratio of dead/total cells was used to differentiate mitotic from cytoprotective effects of EZ
in culture.
Experimental results. The ratio of dead to total cells increased by 2 to 3 - fold with the removal of FBS from the culture media (Table III). The addition of E2 prevented this serum removal effect and produced ratios similar to those observed in the FBS group at each sample time.
Through 48 h, the ratio of dead to live cells remained constant for E2-treated cells, but nearly doubled for serum free cells. By 96 h, the ratio increased in all three groups, indicating the diminishing cytoprotective effect with time.
Although the total SK-N-SH cell number in the FBS group was approximately double that seen with E. treated cells at both 24 and 48 hours, the fraction of live cells was similar. However, E2-treated SK-N-SH cells did not show the exponential growth pattern seen for cells grown in FBS. The ratio of dead to total SK-N-SH treated cells increased in all three groups with time (Table III). However, at each sampling time, this ratio was similar and markedly lower in both E2 and FBS groups than under serum free conditions.
- 21 - 2 1 7 5 6 0 3 PCT/US94,12782 TABLE III
Dead/Total Cell Ratio for SB-N-SH Cultures Sasple Size Ratio TotaL ~ells 9iabLe Cells Dead f4ells Tine Treatmrnt (i Flasks) (seao + sess) (x 10 /ol) (x 104/ml) (x 10 /sl) 24 hrs. Serua Free 6 0.30 + 0.03') 19 2F 13 4# 6 1 24 hrs. E2 (544 pg/ml) 6 0.16 + 0.04 31 + 5 26 + 5 4 1 24 hrs. FBS 6 0.15 + 0.03 34 + 3 29 3 5 1 48 hrs. Serus Free 6 0.53 0.07/ 21 3R 11 + 2'F 11 + 2 48 hrs. E2 (544 pg/ml) 6 0.21 0.05 41 9 34 9 7 2 48 hrs. FBS 6 0.14 + 0.06 52 i 7 46 t 8 6 2 96 hrs. Serun Free 5 0.62 0.12# 8 2# 4 2'1 4 1 96 hrs. E2 (544 pg/ml) 5 0.38 0.06* 22 4* 14 2* 8 t 2 96 hrs. FBS 5 0.22 + 0.04 50 + 8 40 + 8 10 1 # p< 0.05 vs both E2 and FBS 9roups.
* p< 0.05 vs both serua free and FBS groups.
The growth pattern of neuroblastoma cells in FBS-supplemented media revealed a doubling time of 6 to 9 days. A decrease in total number of cells with a corresponding increase in the fraction of dead cells was observed under serum free conditions across the five day evaluation.
Collectively, these data demonstrate that the enhanced total and live cell number observed results from a cytoprotective, rather than mitotic, effect of E2 under specific in vitro conditions employed.
Example lb. In vitro studies demonstrate increased viability of nerve cells in the presence of an estrogen compound where a-estrogen exerts a cell protective effect.
Experimental design. SK-iY-SH cells were backcultured with 2 ml 0.02% EDTA (Sigma Chemical Corporation, St. Louis, MO), incubated for thirty minutes at 37 C and resuspended at a density of 1 x 105 cells per ml as follows. Each experiment had three treatment groups: Group 1 was RPMI
media supplemented with 10% FBS (FBS group), group 2 was wo 95/12402 2175603 PCT/US94112782 RPMI media without the FBS (serum free group) and group 3 was serum free RPMI media supplemented with 544 pg/ml 17-a-E2 pg/ml (a-E2 group) (Steraloids, Inc., Wilton, NH).
Treatment media were made the day of the experiment. a-E2 was initially dissolved in 100 gl absolute ethanol and then diluted with media. Media for the other two groups was similarly prepared with the addition and dilution of 100 ml absolute ethanol. The FBS has been assayed for /3-EZ
determining concentrations of about 16 pg/ml. The RPMI 1640 media had undetectable P-EZ levels. However, neither FBS
nor media has been assayed for a-Ez.
Cell viability was assessed as described in Example la. After the cells had been incubated for 24 or 48 hours in the respective treatment media, cell suspensions were made by decanting media, topically washing each flask with 2 ml 0.02% EDTA, then incubating with 2 ml 0.02% EDTA for 30 minutes. Cells were subsequently resuspended in the appropriate media. 2 ml aliquots of five to six different flasks from each treatment were then treated with 500 l of 0.4% trypan blue stain (Sigma Chemical Corporation, St.
Louis, MO). An important limitation of the trypan blue staining method for differentiating dead and live cells is its time dependence. Therefore, care was taken to standardize time between resuspension of the cells, addition of the dye, and actual counting on the hemacytometer.
Experimental results. As in Example la, the ratio of dead to total cells increased 2-3 fold with the removal of FBS from the culture media (Figure 8). The addition of a-estrogen prevented this serum removal effect and produced ratios similar to those observed in the FBS group at each sample time.
Example ic. Estrogen delays or prevents time-dependent death of cells in primary cortical neuron cultures.
Experimental design. Primary cortical neurons were produced from rat brains that were 0-1 day old using a variation of methods described by Monyer et al. 1989, Brain Research 483:347-354. Dispersed brain tissue was grown in DMEM/10% PDT'.S (pregnant donor horse serum) for three days and then treated with cytosine arabinoside (ARC) for two days to remove contaminating glial cells. On day 5, the ARC
media was removed and replaced with DMEM/10% PDHS. The neuronal cells were cultured for a further 4-7 days before use.
Control primary neuronal cultures show progressive cell death between days 12 and 18 in culture. Twelve cultures were evaluated on days 12 and 16 for levels of the enzyme lactate dehydrogenase (LD) after adding estrogen to 6 cultures maintained in DMEM and 10% PDHS on day 9 and maintaining the remaining cultures as controls. LD was assayed using a variation of the method by Wroblewski et al.
1955, Proc. Soc. Exp. Biol. Med. 90:210-213. LD is a cytosolic enzyme which is commonly used in both clinical and basic research to determine tissue viability. An increase in media LD is directly related to cell death.
Results. A single treatment on day 9 with estrogen significantly reduced (p < 0.05) the increase in LD observed in all 6 replicates on days 12 and 16. These data suggest that in primary neurons, estrogen exposure delays or prevents time-dependent death in culture for at least 7 days (Figure 1), an observation that is further supported by examination of cultures by light microscopy. Here it was observed that estradiol prevented the retrograde degeneration (regression of neuronal extensions) and reduced the appearance of cytosolic inclusions (clusters of material) in cell bodies; both of which are normally observed with aging in primary neuronal cultures in vitro and with degenerative disorders in vivo.
Example 2a. In vitro studies show that estrogen compounds protect cells against cytotoxicity induced by hypoglycemia.
WO 95,12402 2175603 PCT/US9a,12782 i Experimental approach. C6 glioma cells obtained from ATCC were plated in RPMI media with FBS at a concentration of 1 x 106 cells/mi in Falcon 25 cm2 tissue culture flasks.
Four hours prior to the onset of hypoglycemia, the maintenance media was discarded, monolayers were washed twice in the appropriate media and then incubated for four hours at 37 C in either serum free or serum free plus 544 pg/mi EZ. Kreb's Ringer Phosphate buffer was used to wash the monolayers twice before the addition of appropriate glucose treatment. RPMI medium contains 2 mg glucose/ml;
flasks were divided into groups of 6 each receiving 100%
glucose (2 mg/ml), 80% glucose (1.6 mg/ml), 60% glucose (1.2 mg/ml) or 0% glucose (buffer) with no steroid addition or supplemented with 544 pg/ml EZ. All flasks were incubated for 20 hours and then evaluated for total, live, and dead cell number utilizing the trypan blue method previously described.
Results. Figure 2 shows that hypoglycemia caused a marked and dose-dependent reduction in both total and live C6 cell numbers in control flasks, which did not receive E2 treatment. By contrast, at each of the levels of hypoglycemia tested, E2 exposure prevented the loss in total and live cells associated with hypoglycemia. Trypan blue stains dying cells that have become permeable to the dye.
In adverse conditions, the number of cells in the total cell population that is measured is diminished as a result of disintegration of dead cells. Hence, the total cell numbers of cells in samples maintained for 20 hours in suboptimal levels of glucose show a reduction in total cell number in Figure 2. However, a large percentage of this diminishing population are live cells. The addition of estradiol to cultures, maintained in suboptimal levels of glucose, protects the population from cell death and results in an overall greater number of live cells. The asterisk marks those samples having a statistically significant reduction in total cells and live cells in the absence of estrogen when compared to samples in the presence of estrogen.
Dead/Total Cell Ratio for SB-N-SH Cultures Sasple Size Ratio TotaL ~ells 9iabLe Cells Dead f4ells Tine Treatmrnt (i Flasks) (seao + sess) (x 10 /ol) (x 104/ml) (x 10 /sl) 24 hrs. Serua Free 6 0.30 + 0.03') 19 2F 13 4# 6 1 24 hrs. E2 (544 pg/ml) 6 0.16 + 0.04 31 + 5 26 + 5 4 1 24 hrs. FBS 6 0.15 + 0.03 34 + 3 29 3 5 1 48 hrs. Serus Free 6 0.53 0.07/ 21 3R 11 + 2'F 11 + 2 48 hrs. E2 (544 pg/ml) 6 0.21 0.05 41 9 34 9 7 2 48 hrs. FBS 6 0.14 + 0.06 52 i 7 46 t 8 6 2 96 hrs. Serun Free 5 0.62 0.12# 8 2# 4 2'1 4 1 96 hrs. E2 (544 pg/ml) 5 0.38 0.06* 22 4* 14 2* 8 t 2 96 hrs. FBS 5 0.22 + 0.04 50 + 8 40 + 8 10 1 # p< 0.05 vs both E2 and FBS 9roups.
* p< 0.05 vs both serua free and FBS groups.
The growth pattern of neuroblastoma cells in FBS-supplemented media revealed a doubling time of 6 to 9 days. A decrease in total number of cells with a corresponding increase in the fraction of dead cells was observed under serum free conditions across the five day evaluation.
Collectively, these data demonstrate that the enhanced total and live cell number observed results from a cytoprotective, rather than mitotic, effect of E2 under specific in vitro conditions employed.
Example lb. In vitro studies demonstrate increased viability of nerve cells in the presence of an estrogen compound where a-estrogen exerts a cell protective effect.
Experimental design. SK-iY-SH cells were backcultured with 2 ml 0.02% EDTA (Sigma Chemical Corporation, St. Louis, MO), incubated for thirty minutes at 37 C and resuspended at a density of 1 x 105 cells per ml as follows. Each experiment had three treatment groups: Group 1 was RPMI
media supplemented with 10% FBS (FBS group), group 2 was wo 95/12402 2175603 PCT/US94112782 RPMI media without the FBS (serum free group) and group 3 was serum free RPMI media supplemented with 544 pg/ml 17-a-E2 pg/ml (a-E2 group) (Steraloids, Inc., Wilton, NH).
Treatment media were made the day of the experiment. a-E2 was initially dissolved in 100 gl absolute ethanol and then diluted with media. Media for the other two groups was similarly prepared with the addition and dilution of 100 ml absolute ethanol. The FBS has been assayed for /3-EZ
determining concentrations of about 16 pg/ml. The RPMI 1640 media had undetectable P-EZ levels. However, neither FBS
nor media has been assayed for a-Ez.
Cell viability was assessed as described in Example la. After the cells had been incubated for 24 or 48 hours in the respective treatment media, cell suspensions were made by decanting media, topically washing each flask with 2 ml 0.02% EDTA, then incubating with 2 ml 0.02% EDTA for 30 minutes. Cells were subsequently resuspended in the appropriate media. 2 ml aliquots of five to six different flasks from each treatment were then treated with 500 l of 0.4% trypan blue stain (Sigma Chemical Corporation, St.
Louis, MO). An important limitation of the trypan blue staining method for differentiating dead and live cells is its time dependence. Therefore, care was taken to standardize time between resuspension of the cells, addition of the dye, and actual counting on the hemacytometer.
Experimental results. As in Example la, the ratio of dead to total cells increased 2-3 fold with the removal of FBS from the culture media (Figure 8). The addition of a-estrogen prevented this serum removal effect and produced ratios similar to those observed in the FBS group at each sample time.
Example ic. Estrogen delays or prevents time-dependent death of cells in primary cortical neuron cultures.
Experimental design. Primary cortical neurons were produced from rat brains that were 0-1 day old using a variation of methods described by Monyer et al. 1989, Brain Research 483:347-354. Dispersed brain tissue was grown in DMEM/10% PDT'.S (pregnant donor horse serum) for three days and then treated with cytosine arabinoside (ARC) for two days to remove contaminating glial cells. On day 5, the ARC
media was removed and replaced with DMEM/10% PDHS. The neuronal cells were cultured for a further 4-7 days before use.
Control primary neuronal cultures show progressive cell death between days 12 and 18 in culture. Twelve cultures were evaluated on days 12 and 16 for levels of the enzyme lactate dehydrogenase (LD) after adding estrogen to 6 cultures maintained in DMEM and 10% PDHS on day 9 and maintaining the remaining cultures as controls. LD was assayed using a variation of the method by Wroblewski et al.
1955, Proc. Soc. Exp. Biol. Med. 90:210-213. LD is a cytosolic enzyme which is commonly used in both clinical and basic research to determine tissue viability. An increase in media LD is directly related to cell death.
Results. A single treatment on day 9 with estrogen significantly reduced (p < 0.05) the increase in LD observed in all 6 replicates on days 12 and 16. These data suggest that in primary neurons, estrogen exposure delays or prevents time-dependent death in culture for at least 7 days (Figure 1), an observation that is further supported by examination of cultures by light microscopy. Here it was observed that estradiol prevented the retrograde degeneration (regression of neuronal extensions) and reduced the appearance of cytosolic inclusions (clusters of material) in cell bodies; both of which are normally observed with aging in primary neuronal cultures in vitro and with degenerative disorders in vivo.
Example 2a. In vitro studies show that estrogen compounds protect cells against cytotoxicity induced by hypoglycemia.
WO 95,12402 2175603 PCT/US9a,12782 i Experimental approach. C6 glioma cells obtained from ATCC were plated in RPMI media with FBS at a concentration of 1 x 106 cells/mi in Falcon 25 cm2 tissue culture flasks.
Four hours prior to the onset of hypoglycemia, the maintenance media was discarded, monolayers were washed twice in the appropriate media and then incubated for four hours at 37 C in either serum free or serum free plus 544 pg/mi EZ. Kreb's Ringer Phosphate buffer was used to wash the monolayers twice before the addition of appropriate glucose treatment. RPMI medium contains 2 mg glucose/ml;
flasks were divided into groups of 6 each receiving 100%
glucose (2 mg/ml), 80% glucose (1.6 mg/ml), 60% glucose (1.2 mg/ml) or 0% glucose (buffer) with no steroid addition or supplemented with 544 pg/ml EZ. All flasks were incubated for 20 hours and then evaluated for total, live, and dead cell number utilizing the trypan blue method previously described.
Results. Figure 2 shows that hypoglycemia caused a marked and dose-dependent reduction in both total and live C6 cell numbers in control flasks, which did not receive E2 treatment. By contrast, at each of the levels of hypoglycemia tested, E2 exposure prevented the loss in total and live cells associated with hypoglycemia. Trypan blue stains dying cells that have become permeable to the dye.
In adverse conditions, the number of cells in the total cell population that is measured is diminished as a result of disintegration of dead cells. Hence, the total cell numbers of cells in samples maintained for 20 hours in suboptimal levels of glucose show a reduction in total cell number in Figure 2. However, a large percentage of this diminishing population are live cells. The addition of estradiol to cultures, maintained in suboptimal levels of glucose, protects the population from cell death and results in an overall greater number of live cells. The asterisk marks those samples having a statistically significant reduction in total cells and live cells in the absence of estrogen when compared to samples in the presence of estrogen.
Example 2b: Estrogen compounds protect cells from cytotoxicity induced by preexisting hypoglycomia.
Experimental design. C6 cells were cultured as described in Example 2a. On the day of the experiment, flasks (5 to 6 per treatment group) were divided into 5 groups. One group was maintained in normal glucose media (euglycemia: 2 mg/ml glucose in RPMI) and the remaining 4 groups were placed in RPMI with 80% of normal glucose concentration (hypoglycemia). At one hour or at 4 hours after the initiation of the hypoglycemia state, flasks were treated with either RPMI (controls) or with E2 (544 pg/ml RPMI). At 24 hours after the initiation of hypoglycemia, total, live and dead cells were counted as described above.
This study design permitted the determination of the time-course of C6 cell rescued from the cytotoxic effects of hypoglycemia.
Results. The results are shown in Table IV and demonstrate the ability of EZ to rescue C6 cells from the cytotoxic effects of a preexisting hypoglycemia state. The number of live cells was increased by more than 2-fold and the number of dead cells was reduced by half after treatment with E2 at one hour after the initiation of hypoglycemia.
2:_ In contrast, treatment with EZ at 4 hours after the initiation of hypoglycemia had no effect on live cell number and only slightly reduced dead cell number.
=
Experimental design. C6 cells were cultured as described in Example 2a. On the day of the experiment, flasks (5 to 6 per treatment group) were divided into 5 groups. One group was maintained in normal glucose media (euglycemia: 2 mg/ml glucose in RPMI) and the remaining 4 groups were placed in RPMI with 80% of normal glucose concentration (hypoglycemia). At one hour or at 4 hours after the initiation of the hypoglycemia state, flasks were treated with either RPMI (controls) or with E2 (544 pg/ml RPMI). At 24 hours after the initiation of hypoglycemia, total, live and dead cells were counted as described above.
This study design permitted the determination of the time-course of C6 cell rescued from the cytotoxic effects of hypoglycemia.
Results. The results are shown in Table IV and demonstrate the ability of EZ to rescue C6 cells from the cytotoxic effects of a preexisting hypoglycemia state. The number of live cells was increased by more than 2-fold and the number of dead cells was reduced by half after treatment with E2 at one hour after the initiation of hypoglycemia.
2:_ In contrast, treatment with EZ at 4 hours after the initiation of hypoglycemia had no effect on live cell number and only slightly reduced dead cell number.
=
TABLE IV
Demonstration of the Ability of E to Rescue C6 Cells from the Cytotoxic Effects oi Hypoglycemia.
GLucose C6 Fell Nueber Pretreataent N Treatment Tim ( x104/al) TotaL Live Dead Euglycemie 6 -- 243 16 235 16 9 2 Rypoglycemia 6 SF - E2 1 hr. 108+7 19+3 89+6 5 SF + E2 1 hr. 92+7 44+5* 49+4*
6 SF - E2 4 hr. 124+10 17+2 107+8 6 SF + E2 4 hr. 93 9 19+4 74 7 Depicted are mean + SEM.
SF = Serun Free RPMI media.
* P< 0.05 from Serum Free group at 1 hour.
Example 2a: Estrogen compounds can protect cells against cytotoxicity induced by excitotoxic amino acids.
Experimental design. Five culture dishes containing SK-N-SH neuroblastoma cells were treated with E2 (544 pg/ml) and 5 culture dishes were treated with RPMI media as described above. Four hours later, all cell were treated with NMDA (500 pM) for 5 minutes. Total live cells and dead cells were then determined.
Results. The protective effect of estradiol on the viability of the cell population is shown in Figure 3.
Pretreatment with estradiol increased the number of live cells and reduced the number of dead cells in these neuroblastoma cultures following treatment with NMDA. These data demonstrate that E2 pretreatment protects cells from the neurotoxicity associated with excitotoxic amino acids.
W095,12402 217 5 6 0 3 PCT/US94/12782 =
Demonstration of the Ability of E to Rescue C6 Cells from the Cytotoxic Effects oi Hypoglycemia.
GLucose C6 Fell Nueber Pretreataent N Treatment Tim ( x104/al) TotaL Live Dead Euglycemie 6 -- 243 16 235 16 9 2 Rypoglycemia 6 SF - E2 1 hr. 108+7 19+3 89+6 5 SF + E2 1 hr. 92+7 44+5* 49+4*
6 SF - E2 4 hr. 124+10 17+2 107+8 6 SF + E2 4 hr. 93 9 19+4 74 7 Depicted are mean + SEM.
SF = Serun Free RPMI media.
* P< 0.05 from Serum Free group at 1 hour.
Example 2a: Estrogen compounds can protect cells against cytotoxicity induced by excitotoxic amino acids.
Experimental design. Five culture dishes containing SK-N-SH neuroblastoma cells were treated with E2 (544 pg/ml) and 5 culture dishes were treated with RPMI media as described above. Four hours later, all cell were treated with NMDA (500 pM) for 5 minutes. Total live cells and dead cells were then determined.
Results. The protective effect of estradiol on the viability of the cell population is shown in Figure 3.
Pretreatment with estradiol increased the number of live cells and reduced the number of dead cells in these neuroblastoma cultures following treatment with NMDA. These data demonstrate that E2 pretreatment protects cells from the neurotoxicity associated with excitotoxic amino acids.
W095,12402 217 5 6 0 3 PCT/US94/12782 =
Example 3: In vivo studies show behavioral improvements caused by the neuroprotective effect of estrogen compounds.
The role of estradiol in protecting cholinergic function in the CNS and the association of this effect with learning and memory has been demonstrated as described below.
Esample 3a: Learning and memory improve in estrogen treated ovariectomized rats.
Experimental design. Three groups of animals were analyzed using a standard 2-way active avoidance paradigm.
The three groups of animals were: ovary-intact, ovariectomized, and estradiol-replaced ovariectomized animals.
Animals. Young adult female (3-4 months old) CD-Sprague-Dawley rats (Charles River Breeding Laboratories, Wilmington, MA) were maintained in standard breeding conditions.
Animal surgical procedure: Animals were anesthetized with methoxyflurane (Metofane, Pitman-Moore, Washington Crossing, NJ). Two-thirds of the rats underwent bilateral ovariectomy using a dorsal approach. Three weeks following ovariectomy, a subset of the ovariectomized animals (the E2 replaced group) received a 5 mm Silastic (Dow Corning, Midland, MI) pellet containing a 1:1 mixture of cholesterol (Steraloids, Inc., Wilton, NH) and 17-B estradiol that was implanted subcutaneously. Estradiol delivery through Silastic tubing results from diffusion down a large concentration gradient and the fibrosis, which occurs over time around the Silastic pellet, reduces diffusion. The EZ
replacement regimen was maintained for 2 or 25 weeks following the 3 week rest period after ovariectomy. At 5 weeks and 28 weeks post ovariectomy, animals were behaviorally tested. In the long term treatment regimen, we removed and repositioned the Silastic pellets every 2 to 3 ''VU 95/12402 - 2 1 f 5 6 0 3 PCT/US94/12782 r weeks to maintain EZ diffusion from the Silastic. The ovariectomized group received sham pellets that were similarly re-positioned every 2 to 3 weeks. Both E. and sham pellets were washed twice with 100% ethanol and were then incubated in PBS at room temperature for 48 hours prior to implantation. The resulting experimental groups were:
ovary intact (INTACT), ovariectomized (OVX) (5 or 28 weeks), and estradiol-replaced (E2 pellet).
Behavioral testing; active avoidance. To assess learning, the 2-way active avoidance paradigm was employed following the procedures of Mouton et al. 1988, Brain Research 444:104-108. All three groups of animals were tested for 14 consecutive days, each day consisting of 15 trials. Each trial lasted for 1 minute; and consisted of the simultaneous presentation of conditional stimuli (a light and sound cue) for the first 5 seconds and a 7 second interval followed by an electrical foot-shock of 1.4 mA for a 2 second duration. Successful learning was determined by the number of correct responses or "avoidances" and was defined as transferring from one side of the shuttle-box to the other within the first 12 seconds of each trial, before the onset of the footshock. In order to assess potential motivational differences between animals in different treatment groups, the number of "no transfers" were also recorded. This parameter describes the number of trials in which the animal did not transfer from one side of the shuttle box to the other upon stimulation with the electrical shock.
Statistical analyses. Behavioral data were analyzed non-parametrically using the Kruskal-Wallis one-way analysis of variance and the Mann Whitney U test for assessment of group differences.
Results. INTACT and E2-pellet animals performed better on the 2-way active avoidance paradigm relative to OVX
animals. Figures 4 and 5 show the average of the total number of avoidances made by each animal over the 14 day testing period starting at the end of the 5 week and 28 week wo 95112402 217 5 6 0 3 PCT/US94/12782 ~
The role of estradiol in protecting cholinergic function in the CNS and the association of this effect with learning and memory has been demonstrated as described below.
Esample 3a: Learning and memory improve in estrogen treated ovariectomized rats.
Experimental design. Three groups of animals were analyzed using a standard 2-way active avoidance paradigm.
The three groups of animals were: ovary-intact, ovariectomized, and estradiol-replaced ovariectomized animals.
Animals. Young adult female (3-4 months old) CD-Sprague-Dawley rats (Charles River Breeding Laboratories, Wilmington, MA) were maintained in standard breeding conditions.
Animal surgical procedure: Animals were anesthetized with methoxyflurane (Metofane, Pitman-Moore, Washington Crossing, NJ). Two-thirds of the rats underwent bilateral ovariectomy using a dorsal approach. Three weeks following ovariectomy, a subset of the ovariectomized animals (the E2 replaced group) received a 5 mm Silastic (Dow Corning, Midland, MI) pellet containing a 1:1 mixture of cholesterol (Steraloids, Inc., Wilton, NH) and 17-B estradiol that was implanted subcutaneously. Estradiol delivery through Silastic tubing results from diffusion down a large concentration gradient and the fibrosis, which occurs over time around the Silastic pellet, reduces diffusion. The EZ
replacement regimen was maintained for 2 or 25 weeks following the 3 week rest period after ovariectomy. At 5 weeks and 28 weeks post ovariectomy, animals were behaviorally tested. In the long term treatment regimen, we removed and repositioned the Silastic pellets every 2 to 3 ''VU 95/12402 - 2 1 f 5 6 0 3 PCT/US94/12782 r weeks to maintain EZ diffusion from the Silastic. The ovariectomized group received sham pellets that were similarly re-positioned every 2 to 3 weeks. Both E. and sham pellets were washed twice with 100% ethanol and were then incubated in PBS at room temperature for 48 hours prior to implantation. The resulting experimental groups were:
ovary intact (INTACT), ovariectomized (OVX) (5 or 28 weeks), and estradiol-replaced (E2 pellet).
Behavioral testing; active avoidance. To assess learning, the 2-way active avoidance paradigm was employed following the procedures of Mouton et al. 1988, Brain Research 444:104-108. All three groups of animals were tested for 14 consecutive days, each day consisting of 15 trials. Each trial lasted for 1 minute; and consisted of the simultaneous presentation of conditional stimuli (a light and sound cue) for the first 5 seconds and a 7 second interval followed by an electrical foot-shock of 1.4 mA for a 2 second duration. Successful learning was determined by the number of correct responses or "avoidances" and was defined as transferring from one side of the shuttle-box to the other within the first 12 seconds of each trial, before the onset of the footshock. In order to assess potential motivational differences between animals in different treatment groups, the number of "no transfers" were also recorded. This parameter describes the number of trials in which the animal did not transfer from one side of the shuttle box to the other upon stimulation with the electrical shock.
Statistical analyses. Behavioral data were analyzed non-parametrically using the Kruskal-Wallis one-way analysis of variance and the Mann Whitney U test for assessment of group differences.
Results. INTACT and E2-pellet animals performed better on the 2-way active avoidance paradigm relative to OVX
animals. Figures 4 and 5 show the average of the total number of avoidances made by each animal over the 14 day testing period starting at the end of the 5 week and 28 week wo 95112402 217 5 6 0 3 PCT/US94/12782 ~
period in the three groups of animals. OVX rats showed a 59% decrease in the number of avoidances achieved relative to the intact group, but this difference was not significant. Ez replacement of OVX rats caused a 8.5 - fold increase in the number of avoidances relative to the OVX
group. When these same animals were maintained on their respective treatments and were behaviorally tested at 28 weeks, the number of total avoidances was increased in all groups relative to the 5 week testing period (Figures 4 and 5.) However, at this 28 week testing point, OVX
significantly reduced the total number of avoidances by 61%
and E2 replacement continued to increase avoidances by 4.5-fold versus OVX rats. Furthermore, Ez-pellet rats showed a marked acceleration in their rate of learning at 28 weeks, achieving the criteria of performing correctly 11 out of a possible 15 times in a given day by 1.3 0.3 days of testing (Table V). INTACT rats did not show this acceleration in learning requiring 9 2.8 days to reach criteria. The OVX animals maintained their inability to learn the task in the allotted 14 days and were therefore assigned a value of 15 days.
At both 5 and 28 weeks, rats in each group maintained their relative order of proficiency in active avoidance and all animals performed better in the second trial than first (Figures 4 and 5). This enhanced performance during the second exposure to the paradigm likely reflects recall of the behavior learned during the first test. This long-term memory is particularly apparent in the E2-pellet group, which at the 28 week time-point, reached the performance criteria in 1.3 0.3 days(Table V). It appears that chronic exposure to low doses of E2 may enhance long-term memory in addition to its stimulation of learning of this active avoidance paradigm.
i TABLE V
Effect of Short-Term and Long-Term Ovariectomy and Estradiol Replacement on Learning and Retention Treatment Group Days to Reach Criteria 5 weeks 28 weeks (acute) (chronic) INTACT 14.0 9.0 9.0 2.8 OVARIECTOMIZED 15.0 0.0 15.0 0.0 E2 PELLET 9.5 2.1* 1.3 0.3*
'i p< 0.05 vs Ovariectomized arxl Intact animels.
* p< 0.05 vs Ovariectamized animels using the Mann-Yhitney U nonparametrie statistic.
Example 3b: Neurochemical Assays demonstrate the neuroprotective effect of estrogen.
Biochemical tests were performed on enzymes normally produced by healthy cholinergic neurons to establish whether a cytoprotective effect could be detected in vivo and correlated with behavioral improvements.
Experimental design. Two assays were used to measure the viability of neurons. These assays were high affinity choline uptake (HACU) and choline acetyl transferase (ChAT) activity. HACU was conducted on tissues from both the frontal cortex and hippocampus of the rats. The effect of different lengths of time following ovariectomy or Ez replacement on behavior (active avoidance behavior) and on the activity of cholinergic neurons (HACU and choline acetyl transferase (ChAT) activity) was measured.
Female Sprague-Dawley rats were either ovariectomized (OVX) only, or ovariectomized for 3 weeks followed by subcutaneous implantation of a silastic pellet containing 17-p-estradiol (E2 pellet) resulting in a replacement of EZ
to physiological levels. Ovary intact animals served as a positive control. Active avoidance behavior and choline acetyltransferase (ChAT) activity in the frontal cortex and hippocampus were assessed at 5 weeks and 28 weeks post ovariectomy while high affinity choline uptake (HACU) was measured only at the 5 week time point.
Active Avoidance Test. This test was carried out as described in Example 3a.
Biochemical Analyses.
(a) Plasma est=-adiol assay. Following behavioral testing, or in the case of the untested animals following the period of treatment, animals were decapitated and trunk blood was obtained. The blood was centrifuged at 13,500 x g for 1.5 minutes and resulting plasma was aliquoted into a separate tube for estradiol level determination at a later date. Plasma concentrations of Ez were assayed by radioimmunoassay (RIA) using commercial kits supplied by Diagnostic Product Corp. (Los Angeles, CA). The range of assay detectability was 20-3600 pg/mi. All samples were quantified in a single assay.
(b) High Affinity Choline Uptake (HACII). HACU was assessed in behaviorally naive animals. Following decapitation, brains were removed from the skull and placed on an ice-cooled surface. The hippocampus and frontal cortex were then dissected and immediately placed into ice cold 0.32 M sucrose buffer (0.32 M sucrose, 1.0 mM EDTA, 100 M TRIS-HC1, pH = 7.4 at 4 C). Average wet weights for the tissue regions dissected were 0 mg and 110 mg for the frontal cortex and hippocampus, respectively. Tissue samples were then homogenized with a dounce homogenizer at 400 rpm. Homogenized samples were subsequently centrifuged at 1000 x g for 8 minutes at 4"C. The supernatant (S1 fraction) was centrifuged at 30,000 x g for 15 minutes at 4"C. Following this spin, the supernatant was discarded and the resulting pellet (P2 fraction) was resuspended in 2 ml of cold, oxygenated Krebs buffer (139 mM NaCl, 5 mM KC1, 13 mM NaHCO3, 1 mM MgClZ1 1 mM NaHZPO41 10 mM glucose, 1 mM
CaC12; and was oxygenated for 15 minutes with 95% 02/5% COZ).
High affinity choline uptake was determined in triplicate in the presence of 1 M [3H]-choline (final specific activity:
4.5 Ci/mmol, New England Nuclear, Cambridge, MA).
Non-specific uptake was estimated by adding 5 M
hemicholinium-3 (Sigma Chemical Corp., St. Louis, MO); these hemicholinium-3 values were subtracted from total counts to obtain high affinity values. Each reaction tube contained 200 l of P2 suspension. Unused tissue was stored at -30 C
for subsequent determination of ChAT activity and protein levels. Analysis of protein in the P2 preparation was conducted according to the method of Bradford using Coomassie Blue Dye (5).
HACU differences were analyzed using a t-test since INTACT versus OVX and OVX versus E2-treated were evaluated only once each in two separate studies. Analysis of variance (ANOVA) was employed for other neurochemical analyses. Multiple comparisons among groups were performed using Scheffe's post-hoc test.
(c) ChAT assay. ChAT activity was determined in both behaviorally naive animals (5 week group) as well as the behaviorally tested group (28 week group). ChAT was assayed following a modified version of Fonnum 1975, J. Neurochem.
24:407-409. P2 samples were thawed and sonicated in the presence of 1% 1-butanol and centrifuged at 13000 x g for 5 min. 20 l of the resulting supernatant was used in each reaction tube. The reaction mixture contained 0.28 mM
[3H]-ACoA (specific activity: 45 Ci/mol, New England Nuclear, Cambridge, MA), 7.8 mM choline chloride and 0.2 mM
physostigmine (Sigma Chemical Corp., St. Louis, MO).
Incubation with the [3H]-ACoA was carried out for 30 min.
The reaction was terminated by the addition of ice cold glycyl-glycine buffer (GLY-GLY) at pH 8.6. Following a 10 minute incubation at 4 C, tetraphenyl boron dissolved in butyronitrile (10 mg/ml) was added to the reaction tube allowing for liquid cation exchange extraction of acetylcholine (ACh). Samples were vortexed and centrifuged in a bucket centrifuge at low speed (185 x g) for 5 minutes to allow settling and separation of the organic and aqueous =
group. When these same animals were maintained on their respective treatments and were behaviorally tested at 28 weeks, the number of total avoidances was increased in all groups relative to the 5 week testing period (Figures 4 and 5.) However, at this 28 week testing point, OVX
significantly reduced the total number of avoidances by 61%
and E2 replacement continued to increase avoidances by 4.5-fold versus OVX rats. Furthermore, Ez-pellet rats showed a marked acceleration in their rate of learning at 28 weeks, achieving the criteria of performing correctly 11 out of a possible 15 times in a given day by 1.3 0.3 days of testing (Table V). INTACT rats did not show this acceleration in learning requiring 9 2.8 days to reach criteria. The OVX animals maintained their inability to learn the task in the allotted 14 days and were therefore assigned a value of 15 days.
At both 5 and 28 weeks, rats in each group maintained their relative order of proficiency in active avoidance and all animals performed better in the second trial than first (Figures 4 and 5). This enhanced performance during the second exposure to the paradigm likely reflects recall of the behavior learned during the first test. This long-term memory is particularly apparent in the E2-pellet group, which at the 28 week time-point, reached the performance criteria in 1.3 0.3 days(Table V). It appears that chronic exposure to low doses of E2 may enhance long-term memory in addition to its stimulation of learning of this active avoidance paradigm.
i TABLE V
Effect of Short-Term and Long-Term Ovariectomy and Estradiol Replacement on Learning and Retention Treatment Group Days to Reach Criteria 5 weeks 28 weeks (acute) (chronic) INTACT 14.0 9.0 9.0 2.8 OVARIECTOMIZED 15.0 0.0 15.0 0.0 E2 PELLET 9.5 2.1* 1.3 0.3*
'i p< 0.05 vs Ovariectomized arxl Intact animels.
* p< 0.05 vs Ovariectamized animels using the Mann-Yhitney U nonparametrie statistic.
Example 3b: Neurochemical Assays demonstrate the neuroprotective effect of estrogen.
Biochemical tests were performed on enzymes normally produced by healthy cholinergic neurons to establish whether a cytoprotective effect could be detected in vivo and correlated with behavioral improvements.
Experimental design. Two assays were used to measure the viability of neurons. These assays were high affinity choline uptake (HACU) and choline acetyl transferase (ChAT) activity. HACU was conducted on tissues from both the frontal cortex and hippocampus of the rats. The effect of different lengths of time following ovariectomy or Ez replacement on behavior (active avoidance behavior) and on the activity of cholinergic neurons (HACU and choline acetyl transferase (ChAT) activity) was measured.
Female Sprague-Dawley rats were either ovariectomized (OVX) only, or ovariectomized for 3 weeks followed by subcutaneous implantation of a silastic pellet containing 17-p-estradiol (E2 pellet) resulting in a replacement of EZ
to physiological levels. Ovary intact animals served as a positive control. Active avoidance behavior and choline acetyltransferase (ChAT) activity in the frontal cortex and hippocampus were assessed at 5 weeks and 28 weeks post ovariectomy while high affinity choline uptake (HACU) was measured only at the 5 week time point.
Active Avoidance Test. This test was carried out as described in Example 3a.
Biochemical Analyses.
(a) Plasma est=-adiol assay. Following behavioral testing, or in the case of the untested animals following the period of treatment, animals were decapitated and trunk blood was obtained. The blood was centrifuged at 13,500 x g for 1.5 minutes and resulting plasma was aliquoted into a separate tube for estradiol level determination at a later date. Plasma concentrations of Ez were assayed by radioimmunoassay (RIA) using commercial kits supplied by Diagnostic Product Corp. (Los Angeles, CA). The range of assay detectability was 20-3600 pg/mi. All samples were quantified in a single assay.
(b) High Affinity Choline Uptake (HACII). HACU was assessed in behaviorally naive animals. Following decapitation, brains were removed from the skull and placed on an ice-cooled surface. The hippocampus and frontal cortex were then dissected and immediately placed into ice cold 0.32 M sucrose buffer (0.32 M sucrose, 1.0 mM EDTA, 100 M TRIS-HC1, pH = 7.4 at 4 C). Average wet weights for the tissue regions dissected were 0 mg and 110 mg for the frontal cortex and hippocampus, respectively. Tissue samples were then homogenized with a dounce homogenizer at 400 rpm. Homogenized samples were subsequently centrifuged at 1000 x g for 8 minutes at 4"C. The supernatant (S1 fraction) was centrifuged at 30,000 x g for 15 minutes at 4"C. Following this spin, the supernatant was discarded and the resulting pellet (P2 fraction) was resuspended in 2 ml of cold, oxygenated Krebs buffer (139 mM NaCl, 5 mM KC1, 13 mM NaHCO3, 1 mM MgClZ1 1 mM NaHZPO41 10 mM glucose, 1 mM
CaC12; and was oxygenated for 15 minutes with 95% 02/5% COZ).
High affinity choline uptake was determined in triplicate in the presence of 1 M [3H]-choline (final specific activity:
4.5 Ci/mmol, New England Nuclear, Cambridge, MA).
Non-specific uptake was estimated by adding 5 M
hemicholinium-3 (Sigma Chemical Corp., St. Louis, MO); these hemicholinium-3 values were subtracted from total counts to obtain high affinity values. Each reaction tube contained 200 l of P2 suspension. Unused tissue was stored at -30 C
for subsequent determination of ChAT activity and protein levels. Analysis of protein in the P2 preparation was conducted according to the method of Bradford using Coomassie Blue Dye (5).
HACU differences were analyzed using a t-test since INTACT versus OVX and OVX versus E2-treated were evaluated only once each in two separate studies. Analysis of variance (ANOVA) was employed for other neurochemical analyses. Multiple comparisons among groups were performed using Scheffe's post-hoc test.
(c) ChAT assay. ChAT activity was determined in both behaviorally naive animals (5 week group) as well as the behaviorally tested group (28 week group). ChAT was assayed following a modified version of Fonnum 1975, J. Neurochem.
24:407-409. P2 samples were thawed and sonicated in the presence of 1% 1-butanol and centrifuged at 13000 x g for 5 min. 20 l of the resulting supernatant was used in each reaction tube. The reaction mixture contained 0.28 mM
[3H]-ACoA (specific activity: 45 Ci/mol, New England Nuclear, Cambridge, MA), 7.8 mM choline chloride and 0.2 mM
physostigmine (Sigma Chemical Corp., St. Louis, MO).
Incubation with the [3H]-ACoA was carried out for 30 min.
The reaction was terminated by the addition of ice cold glycyl-glycine buffer (GLY-GLY) at pH 8.6. Following a 10 minute incubation at 4 C, tetraphenyl boron dissolved in butyronitrile (10 mg/ml) was added to the reaction tube allowing for liquid cation exchange extraction of acetylcholine (ACh). Samples were vortexed and centrifuged in a bucket centrifuge at low speed (185 x g) for 5 minutes to allow settling and separation of the organic and aqueous =
phases. 100 l of the organic phase was then aliquoted into 7 ml-scintillation vials and 4 ml of scintillation fluid (Liquiscint , National Diagnostics, Atlanta, GA) were added.
Vials were then counted in a Hewlett-Packard scintillation counter for 5 minutes each, the dpm were converted to pmoles and values were normalized for protein content.
Results.
(a) Estradiol concentrations. Serum E2 concentrations were 43 10 and 36 5 pg/ml for the INTACT and EZ pellet groups, respectively. Ovariectomy reduced serum E2 concentrations to below the sensitivity of the radioimmunoassay employed (20 pg/ml) in all but 5 animals sampled. These 5 animals, however, had serum levels that were very close to the sensitivity limit of the assay.
(b) High Affinity Choline Uptake. Ovariectomy significantly reduced HACU by 24% in the frontal cortex (Figure 6) and by 34% in the hippocampus (Figure 7).
E2-replacement resulted in a reversal of this effect of ovariectomy, increasing HACU by 82% in the frontal cortex (Figure 6) and by 46% in the hippocampus (Figure 7).
Because HACU is a measure of cholinergic activity, it is concluded from these results that estradiol reverses the decline of cholinergic activity in the frontal cortex and hippocampus and further stimulates activity of cholinergic projections to these regions.
(a) Choline Acetyl Transferase activity. In the frontal cortex, no significant differences in ChAT activity were detected within 5 weeks of ovariectomy (Table VI). At the 28 week time point, ChAT levels in the frontal cortex were reduced in both the INTACT and OVX groups by 61% and 56%, respectively. In the EZ-pellet group, however, this reduction was only 16%. In the hippocampus, five weeks of ovariectomy was a sufficient time period to induce a significant reduction in ChAT activity, and 3 weeks of E2-replacement reversed this effect (Table VII). The reductions in hippocampal ChAT activities in INTACT and OVX
animals between 5 and 28 weeks were comparable with those WO 95I12402 2 1 7 5 6 0 3 PCT1Us94/12782 seen in the frontal cortex. In E2-pellet animals, loss of hippocampal ChAT activity was larger than that seen in the frontal cortex but was less than that seen in OVX or INTACT
animals.
TABLE VI
Effect of Short Term and Long Term Ovariectomy and Estradiol Replacement on Choline Acetyltransferase Activity in the Frontal Cortex Treatnent Grap ChAT activity (nmt/30 miNs6 protein) 5 weeks 28 weeks INTACT 10.2 + 0.5 4.0 + 0.1 OVARIECTOMIZED 9.2 + 0.6 4.0 + 0.2 E2 PELLET 9.8 + 0.6 8.2 + 0.8*
2 5 n = 6 for ovarlectomized and E2 pellet groups for the 5 week time period.
n = 5 S for intact group for the 5 week time period.
n= 6 for all treatment groups for the 28 week time period.
* p< 0.05 vs intact group and OVX group.
TABLE VII
Effect of Short Term and Long Term Ovariectomy and Estradiol Replacement on Choline Acetyltransferase Activity in the Hippocampus.
Treatwent Group ChAT activity (root/30 miNag protein) 5 reeks 28 weeks INTACT 13.2 + 0.8 5.7 + 0.3 OVARIECTOMIZED 10.3 + 0.3* 6.2 + 1.1 E2 PELLET 12.7 + 0.5 8.0 + 1.1 n= 6 for ovariectomized and E2 pellet groups for the 5 week time period. =
n= 5 for intact group for the 5 week time period.
n = 6 for aLL treatment groups for the 28 week time period.
* p< 0.05 vs. intact group and Ep pellet group.
~
Vials were then counted in a Hewlett-Packard scintillation counter for 5 minutes each, the dpm were converted to pmoles and values were normalized for protein content.
Results.
(a) Estradiol concentrations. Serum E2 concentrations were 43 10 and 36 5 pg/ml for the INTACT and EZ pellet groups, respectively. Ovariectomy reduced serum E2 concentrations to below the sensitivity of the radioimmunoassay employed (20 pg/ml) in all but 5 animals sampled. These 5 animals, however, had serum levels that were very close to the sensitivity limit of the assay.
(b) High Affinity Choline Uptake. Ovariectomy significantly reduced HACU by 24% in the frontal cortex (Figure 6) and by 34% in the hippocampus (Figure 7).
E2-replacement resulted in a reversal of this effect of ovariectomy, increasing HACU by 82% in the frontal cortex (Figure 6) and by 46% in the hippocampus (Figure 7).
Because HACU is a measure of cholinergic activity, it is concluded from these results that estradiol reverses the decline of cholinergic activity in the frontal cortex and hippocampus and further stimulates activity of cholinergic projections to these regions.
(a) Choline Acetyl Transferase activity. In the frontal cortex, no significant differences in ChAT activity were detected within 5 weeks of ovariectomy (Table VI). At the 28 week time point, ChAT levels in the frontal cortex were reduced in both the INTACT and OVX groups by 61% and 56%, respectively. In the EZ-pellet group, however, this reduction was only 16%. In the hippocampus, five weeks of ovariectomy was a sufficient time period to induce a significant reduction in ChAT activity, and 3 weeks of E2-replacement reversed this effect (Table VII). The reductions in hippocampal ChAT activities in INTACT and OVX
animals between 5 and 28 weeks were comparable with those WO 95I12402 2 1 7 5 6 0 3 PCT1Us94/12782 seen in the frontal cortex. In E2-pellet animals, loss of hippocampal ChAT activity was larger than that seen in the frontal cortex but was less than that seen in OVX or INTACT
animals.
TABLE VI
Effect of Short Term and Long Term Ovariectomy and Estradiol Replacement on Choline Acetyltransferase Activity in the Frontal Cortex Treatnent Grap ChAT activity (nmt/30 miNs6 protein) 5 weeks 28 weeks INTACT 10.2 + 0.5 4.0 + 0.1 OVARIECTOMIZED 9.2 + 0.6 4.0 + 0.2 E2 PELLET 9.8 + 0.6 8.2 + 0.8*
2 5 n = 6 for ovarlectomized and E2 pellet groups for the 5 week time period.
n = 5 S for intact group for the 5 week time period.
n= 6 for all treatment groups for the 28 week time period.
* p< 0.05 vs intact group and OVX group.
TABLE VII
Effect of Short Term and Long Term Ovariectomy and Estradiol Replacement on Choline Acetyltransferase Activity in the Hippocampus.
Treatwent Group ChAT activity (root/30 miNag protein) 5 reeks 28 weeks INTACT 13.2 + 0.8 5.7 + 0.3 OVARIECTOMIZED 10.3 + 0.3* 6.2 + 1.1 E2 PELLET 12.7 + 0.5 8.0 + 1.1 n= 6 for ovariectomized and E2 pellet groups for the 5 week time period. =
n= 5 for intact group for the 5 week time period.
n = 6 for aLL treatment groups for the 28 week time period.
* p< 0.05 vs. intact group and Ep pellet group.
~
Example 4a: In situ hybridization demonstrates increased levels of brain derived neurotrophic factor (BDNF) mRNA is stimulated by estradiol.
This example demonstrates that estrogen can stimulate the production of neurotrophic growth factors such as BDNF.
Experimental design. Animals were ovariectomized for 12 weeks, while E2 treated animals were ovariectomized for 3 weeks followed by 9 weeks of E2 treatment. A second set of animals were ovariectomized for 28 weeks and the E. treated animals were similarly 'ovariectomized for 3 weeks followed by 25 weeks of E2 replacement. Intact control animals were run in parallel with each set of animals. The rats were deeply sedated with sodium pentobarbital. Immediately following, the brains of these animals were transcardially perfused with 4% paraformaldehyde in 0.1M phosphate buffer.
On the following day, the brains were removed from the skull and immersed in a series of solutions (cold 4%
paraformaldehyde solution for 2 days at 4 C followed by a 4 % paraformaldehyde solution containing 20% (wt/vol) sucrose). After removal from the sucrose solution, the brains were blocked (removed olfactory bulbs, brainstem and cerebellum), frozen on dry ice and immediately stored at -80=C until time of use. 25 m slices of the brain were made using a microtome and placed in a 4% paraformaldehyde solution. Within 3 weeks, the slices were hybridized according to the method described by Gall et al. 1989, Science 245:758-761. The BDNF probe was 750 b.p. of rat BDNF cDNA, corresponding to the entire amino acid coding region. The probes were labeled with 35S using the T3 polymerase kit. The hybridized slices were mounted onto Vectabond treated slides and exposed to autoradiographic film for 4 days. The varying intensities of radioactive signal on each slice (the hybridization signal) was translated into differing optical densities on film and was analyzed using the BRS 2 Imaging System (Imaging Research Inc.). The relative optical densities and the background levels were recorded. Signal was defined as the optical density of the region divided by the average background level as assessed by parts of the film adjacent to the brain slice. In the imaging system employed, the higher the O.D., the lower the signal. As such, the data were transformed to the inverse of the signal to noise ratio in order to facilitate the presentation of the data.
Results. Ovariectomy resulted in a significant reduction in the BDNF signal in cortical sections relative to INTACT controls. Estradiol replacement of ovariectomized rats increased the BDNF signal to that normally observed in INTACT controls. The data in Table VIII derived multiple slices of the cerebral cortex of an animal in each treatment group demonstrate the stimulatory effect of an estrogen on BDNF synthesis.
TABLE VIII
Relative Level of BDNF mRNA in the Cortex of Intact, Ovariectomized and E2 Pellet Animals Treatment Group BDNF Level INTACT 1.242 0.035 OVARIECTOMIZED 1.159 + 0.023*
EZ PELLET 1.224 0.026 * p< 0.05 vs Intact and E2 Pellet animaLs.
Example 4b: In vivo studies demonstrate increased levels of NGF mRNA stimulated by estradiol.
This example demonstrates that estrogen can stimulate the production of neurotrophic growth factors such as NGF.
Similarly treated animals as in example 4a were used to measure levels of NGF mRNA using the dot blot technique.
Animals were ovariectomized for 12 weeks, while E2 treated animals were ovariectomized for 3 weeks followed by 9 weeks of Ez treatment. RNA was isolated from the frontal cortex ~ 2175603 and hippocampus using acid guanidinium isothiocyanate followed by phenol/chloroform extraction. The use of the dot blot technique was first validated by performing Northern blots to ensure that hybridization with our NGF
probe of 771 b.p. recognizing the entire pre-pro sequence of NGF, resulting in a single band corresponding to the length of NGF mRNA. The NGF mRNA signal was normalized to the amount of RNA loaded as estimated by the amount of signal produced by subsequent actin hybridization.
Following 3 months of ovariectomy, NGF mRNA levels were significantly reduced (45%) in the frontal cortex (Table IX). E. treatment resulted in a partial recovery, albeit non-statistically significant from the OVX group.
Hippocampal NGF mRNA was not found to differ from controls using this method of detection. E2 treatment did, however, result in a significant increase in NGF mRNA levels (Table IX).
TABLE IX
The Effect of Three-Month Ovariectomy and Estrogen Replacement on NGF mRNA Levels in the Frontal Cortex and Hippocampus Treatsent NGF rlHA pnits per Ry actin) FrontaL Cortex Hippocaapus 3 0 INTACT 0.622 + 0.067 0.590 + 0.052 (n = 10) (n = 6) OVARIECTOMIZED 0.342 + 0.0500 0.616 + 0.051 (n = 12) (n = 12) E2 REPLACED 0.453 + 0.070 0.803 0.082*
(n ; 8) (n = 12) p p < 0.05 vs Intact.
4 0 = p< 0.05 vs Ovariectomized.
This example demonstrates that estrogen can stimulate the production of neurotrophic growth factors such as BDNF.
Experimental design. Animals were ovariectomized for 12 weeks, while E2 treated animals were ovariectomized for 3 weeks followed by 9 weeks of E2 treatment. A second set of animals were ovariectomized for 28 weeks and the E. treated animals were similarly 'ovariectomized for 3 weeks followed by 25 weeks of E2 replacement. Intact control animals were run in parallel with each set of animals. The rats were deeply sedated with sodium pentobarbital. Immediately following, the brains of these animals were transcardially perfused with 4% paraformaldehyde in 0.1M phosphate buffer.
On the following day, the brains were removed from the skull and immersed in a series of solutions (cold 4%
paraformaldehyde solution for 2 days at 4 C followed by a 4 % paraformaldehyde solution containing 20% (wt/vol) sucrose). After removal from the sucrose solution, the brains were blocked (removed olfactory bulbs, brainstem and cerebellum), frozen on dry ice and immediately stored at -80=C until time of use. 25 m slices of the brain were made using a microtome and placed in a 4% paraformaldehyde solution. Within 3 weeks, the slices were hybridized according to the method described by Gall et al. 1989, Science 245:758-761. The BDNF probe was 750 b.p. of rat BDNF cDNA, corresponding to the entire amino acid coding region. The probes were labeled with 35S using the T3 polymerase kit. The hybridized slices were mounted onto Vectabond treated slides and exposed to autoradiographic film for 4 days. The varying intensities of radioactive signal on each slice (the hybridization signal) was translated into differing optical densities on film and was analyzed using the BRS 2 Imaging System (Imaging Research Inc.). The relative optical densities and the background levels were recorded. Signal was defined as the optical density of the region divided by the average background level as assessed by parts of the film adjacent to the brain slice. In the imaging system employed, the higher the O.D., the lower the signal. As such, the data were transformed to the inverse of the signal to noise ratio in order to facilitate the presentation of the data.
Results. Ovariectomy resulted in a significant reduction in the BDNF signal in cortical sections relative to INTACT controls. Estradiol replacement of ovariectomized rats increased the BDNF signal to that normally observed in INTACT controls. The data in Table VIII derived multiple slices of the cerebral cortex of an animal in each treatment group demonstrate the stimulatory effect of an estrogen on BDNF synthesis.
TABLE VIII
Relative Level of BDNF mRNA in the Cortex of Intact, Ovariectomized and E2 Pellet Animals Treatment Group BDNF Level INTACT 1.242 0.035 OVARIECTOMIZED 1.159 + 0.023*
EZ PELLET 1.224 0.026 * p< 0.05 vs Intact and E2 Pellet animaLs.
Example 4b: In vivo studies demonstrate increased levels of NGF mRNA stimulated by estradiol.
This example demonstrates that estrogen can stimulate the production of neurotrophic growth factors such as NGF.
Similarly treated animals as in example 4a were used to measure levels of NGF mRNA using the dot blot technique.
Animals were ovariectomized for 12 weeks, while E2 treated animals were ovariectomized for 3 weeks followed by 9 weeks of Ez treatment. RNA was isolated from the frontal cortex ~ 2175603 and hippocampus using acid guanidinium isothiocyanate followed by phenol/chloroform extraction. The use of the dot blot technique was first validated by performing Northern blots to ensure that hybridization with our NGF
probe of 771 b.p. recognizing the entire pre-pro sequence of NGF, resulting in a single band corresponding to the length of NGF mRNA. The NGF mRNA signal was normalized to the amount of RNA loaded as estimated by the amount of signal produced by subsequent actin hybridization.
Following 3 months of ovariectomy, NGF mRNA levels were significantly reduced (45%) in the frontal cortex (Table IX). E. treatment resulted in a partial recovery, albeit non-statistically significant from the OVX group.
Hippocampal NGF mRNA was not found to differ from controls using this method of detection. E2 treatment did, however, result in a significant increase in NGF mRNA levels (Table IX).
TABLE IX
The Effect of Three-Month Ovariectomy and Estrogen Replacement on NGF mRNA Levels in the Frontal Cortex and Hippocampus Treatsent NGF rlHA pnits per Ry actin) FrontaL Cortex Hippocaapus 3 0 INTACT 0.622 + 0.067 0.590 + 0.052 (n = 10) (n = 6) OVARIECTOMIZED 0.342 + 0.0500 0.616 + 0.051 (n = 12) (n = 12) E2 REPLACED 0.453 + 0.070 0.803 0.082*
(n ; 8) (n = 12) p p < 0.05 vs Intact.
4 0 = p< 0.05 vs Ovariectomized.
Example 5: Proposed evaluation for the in vivo effects of estrogens in excitatory amino acid neurotoxicity.
Adult female rats are ovariectomized and two weeks later are treated with a Silastic" pellet containing cholesterol (controls) or estradiol in amounts sufficient to elevate plasma estradiol levels in the physiologic range.
After 1 to 2 weeks of such estrogen-replacement therapy, rats receive an intracerebral injection of N-methyl-D-aspartate (NMDA) or artificial cerebrospinal fluid in amounts to induce extensive toxicity of brain neurons.
As an endpoint of the study, the number of neurons in the hippocampal CAI region and the number of neurons in the cerebral cortex are determined. In addition, nerve terminals in both regions would be stained for detection choline acetyltransferase, the marker enzyme for cholinergic neurons.
It is expected that estrogen-replacement therapy will reduce or eliminate the loss of nerve cells in both the hippocampus and in the cerebral cortex. Such a result teaches that physiological estrogen-replacement can protect brain cells from the neurotoxic effects of excitatory amino acids.
Example 6: Estrogen enhances cerebral glucose uptake at sites in the brain.
The effect of estradiol benzoate (EZB) on cerebral glucose uptake in various brain regions which contain variable numbers of E2B receptors is described and the determination concerning increases in the amount of glucose transported into the brain in the presence of E2B is also described for a selected animal model.
Female rats were bilaterally ovariectomized to eliminate endogenous estrogens and two to three weeks later were implanted with an atrial cannula for the i.v.
administratioA of C14-2-deoxyglucose (C14-2-DG) to unanesthetized rats. Animals were allowed four to five days w0 95112402 2 i 7 5 6 0 3 PCT/US94/12782 =
Adult female rats are ovariectomized and two weeks later are treated with a Silastic" pellet containing cholesterol (controls) or estradiol in amounts sufficient to elevate plasma estradiol levels in the physiologic range.
After 1 to 2 weeks of such estrogen-replacement therapy, rats receive an intracerebral injection of N-methyl-D-aspartate (NMDA) or artificial cerebrospinal fluid in amounts to induce extensive toxicity of brain neurons.
As an endpoint of the study, the number of neurons in the hippocampal CAI region and the number of neurons in the cerebral cortex are determined. In addition, nerve terminals in both regions would be stained for detection choline acetyltransferase, the marker enzyme for cholinergic neurons.
It is expected that estrogen-replacement therapy will reduce or eliminate the loss of nerve cells in both the hippocampus and in the cerebral cortex. Such a result teaches that physiological estrogen-replacement can protect brain cells from the neurotoxic effects of excitatory amino acids.
Example 6: Estrogen enhances cerebral glucose uptake at sites in the brain.
The effect of estradiol benzoate (EZB) on cerebral glucose uptake in various brain regions which contain variable numbers of E2B receptors is described and the determination concerning increases in the amount of glucose transported into the brain in the presence of E2B is also described for a selected animal model.
Female rats were bilaterally ovariectomized to eliminate endogenous estrogens and two to three weeks later were implanted with an atrial cannula for the i.v.
administratioA of C14-2-deoxyglucose (C14-2-DG) to unanesthetized rats. Animals were allowed four to five days w0 95112402 2 i 7 5 6 0 3 PCT/US94/12782 =
to recover from the cannulation before the study was initiated.
On the day of the experiment, animals were randomized and assigned-to groups which received either E2B in oil or oil alone (controls) administered subcutaneously at the dose and times described below. In our initial study, animals were treated with oil or E2B (10 g/kg body weight) and were sacrificed at 2, 4, 8, 12 or 24 h. In an additional study, rats were treated with oil or E2B at doses of 1, 10 or 100 g/kg body weight and were sacrificed 4 h later. Forty-five minutes prior to sacrifice, all rats received a single injection via the atrial. cannulae of C14-2-DG (25 Ci/ml saline/kg body weight; specific activity 49-53 mCi/mmol, New England Nuclear, Boston, MA).
To assess the effects of E2B on transport of glucose across the blood-brain barrier we used the technique of Oldendorf (Oldendorf, Brain Res. 24:37-46; 1970; and An. J.
Physiol. 221:1629-1638 (1971)). One Ci/ml of C14-2-DG
(specific activity 49-53mCi/mmol, New England Nuclear, Boston, MA) and approximately five Ci/mi of 3HZo (specific activity 1 mCi/ml, New England Nuclear, Boston, MA) were mixed with Krebs' Ringer Phosphate solution buffered to pH
7.4 with lOmM HEPES and injected into the carotid artery of female rats.
Fifteen seconds after injection, the animals were killed by decapitation, trunk blood was collected for later assay ofserum E2B concentrations and the brain was removed from the cranium for dissection of the following regions:
medial basal hypothalamus (MBH), preoptic area (POA), cortex, hippocampus, striatum, cerebellum and brainstem.
The anterior pituitary (AP) was also isolated from the cranium. The dissection of brain tissues followed the methods of Glowinski and Iversen (Glowinski et al., J.
Neurochem. 13:655-669 (1966)). Tissues were immediately weighed and placed in scintillation vials for processing.
The mg weights (mean SEM) of tissues used in these studies were: MBH = 13.4t1.3; POA = 10.5t0.5; cortex = 38.7 3;
On the day of the experiment, animals were randomized and assigned-to groups which received either E2B in oil or oil alone (controls) administered subcutaneously at the dose and times described below. In our initial study, animals were treated with oil or E2B (10 g/kg body weight) and were sacrificed at 2, 4, 8, 12 or 24 h. In an additional study, rats were treated with oil or E2B at doses of 1, 10 or 100 g/kg body weight and were sacrificed 4 h later. Forty-five minutes prior to sacrifice, all rats received a single injection via the atrial. cannulae of C14-2-DG (25 Ci/ml saline/kg body weight; specific activity 49-53 mCi/mmol, New England Nuclear, Boston, MA).
To assess the effects of E2B on transport of glucose across the blood-brain barrier we used the technique of Oldendorf (Oldendorf, Brain Res. 24:37-46; 1970; and An. J.
Physiol. 221:1629-1638 (1971)). One Ci/ml of C14-2-DG
(specific activity 49-53mCi/mmol, New England Nuclear, Boston, MA) and approximately five Ci/mi of 3HZo (specific activity 1 mCi/ml, New England Nuclear, Boston, MA) were mixed with Krebs' Ringer Phosphate solution buffered to pH
7.4 with lOmM HEPES and injected into the carotid artery of female rats.
Fifteen seconds after injection, the animals were killed by decapitation, trunk blood was collected for later assay ofserum E2B concentrations and the brain was removed from the cranium for dissection of the following regions:
medial basal hypothalamus (MBH), preoptic area (POA), cortex, hippocampus, striatum, cerebellum and brainstem.
The anterior pituitary (AP) was also isolated from the cranium. The dissection of brain tissues followed the methods of Glowinski and Iversen (Glowinski et al., J.
Neurochem. 13:655-669 (1966)). Tissues were immediately weighed and placed in scintillation vials for processing.
The mg weights (mean SEM) of tissues used in these studies were: MBH = 13.4t1.3; POA = 10.5t0.5; cortex = 38.7 3;
hippocampus = 27.7 1.7; striatum = 24.3t2; cerebellum =
32.5 1.9; brainstem = 32.9 1.9 and AP = 8.6 0.6.
During the procedure, peripheral plasma glucose values were in the normal range of 90-120 mgt.
Alternatively, half of the brain was dissected rostral to the midbrain and ipsilateral to the injection side, tissue passed through a 20 gauge needle and the sample subjected to routine digestion and then prepared for liquid scintillation counting as described above. An aliquot of original isotope mixture was obtained by recovering the residual mixture in the injection syringes. Both aliquot and tissue samples were then counted for H3 and C14 by routine liquid scintillation counting. Uptake by the Brain Uptake Index (BUI) was calculated after correcting for counting efficiency using the following equation for extraction (E):
E= C16 in brain tissue/gH in brain tissue X 100 Clb in mixture/3H in mixture Table XI provides a sample of data generated within one experiment. Evaluations of the time or dose effect of E2B
on glucose uptake were done using a one way ANOVA. Post hoc comparisons were done with Dunnett's tests. These statistical analyses were performed on raw data (dpms C74/mg tissue) by comparing the dpms for the E2B-treated group with its control (oil) group at each dose and at each time point evaluated. For clarity of presentation, the raw data were then expressed as percent of mean control. The magnitude of the response of each E2B-treated animal was determined by calculating the percentage increase of 2-DG uptake relative to the mean value of 2-DG uptake in the oil-treated control group. BUI data were analyzed by a one-tailed t-test for independent samples. Statistical difference was set at p <
0.05 for all tests.
The time course of the effects of a 10 g/kg body weight dose of E2B on glucose uptake is shown in Table X.
Overall, E2B increased glucose uptake significantly in ovariectomized rats by 20 to 120% in 7 of 8 regions =
32.5 1.9; brainstem = 32.9 1.9 and AP = 8.6 0.6.
During the procedure, peripheral plasma glucose values were in the normal range of 90-120 mgt.
Alternatively, half of the brain was dissected rostral to the midbrain and ipsilateral to the injection side, tissue passed through a 20 gauge needle and the sample subjected to routine digestion and then prepared for liquid scintillation counting as described above. An aliquot of original isotope mixture was obtained by recovering the residual mixture in the injection syringes. Both aliquot and tissue samples were then counted for H3 and C14 by routine liquid scintillation counting. Uptake by the Brain Uptake Index (BUI) was calculated after correcting for counting efficiency using the following equation for extraction (E):
E= C16 in brain tissue/gH in brain tissue X 100 Clb in mixture/3H in mixture Table XI provides a sample of data generated within one experiment. Evaluations of the time or dose effect of E2B
on glucose uptake were done using a one way ANOVA. Post hoc comparisons were done with Dunnett's tests. These statistical analyses were performed on raw data (dpms C74/mg tissue) by comparing the dpms for the E2B-treated group with its control (oil) group at each dose and at each time point evaluated. For clarity of presentation, the raw data were then expressed as percent of mean control. The magnitude of the response of each E2B-treated animal was determined by calculating the percentage increase of 2-DG uptake relative to the mean value of 2-DG uptake in the oil-treated control group. BUI data were analyzed by a one-tailed t-test for independent samples. Statistical difference was set at p <
0.05 for all tests.
The time course of the effects of a 10 g/kg body weight dose of E2B on glucose uptake is shown in Table X.
Overall, E2B increased glucose uptake significantly in ovariectomized rats by 20 to 120% in 7 of 8 regions =
examined. However, the time at which a significant increase was observed varied among regions. For 5 of 8 regions, the POA, hippocampus, striatum, cerebellum and AP, the peak E2B
effect was observed at 2 to 4 hours, while 2 regions showed peak effects at 12 hours (the MBH and cerebral cortex).
Five regions showed a significant decline in glucose uptake at some time-point after an EZB-induced increase. These regions were the POA, the hippocampus, the striatum, the cerebellum and the AP. No significant effect of E2B on brainstem glucose uptake was observed.
The dose-dependency of the E2B effect on brain glucose uptake at 4 hours post-E2B injection is shown in Table XI.
The 1 g/kg dose of E2B had no effect on brain glucose uptake. In contrast, the 10 g/kg dose of E2B increased glucose uptake in 6 of 8 regions examined at the 4 h time point. Furthermore, only two regions exhibited increased glucose uptake at the 100 pg/kg dose of E2B, the MBH and the POA. All other regions examined exhibited a reduced glucose uptake, with three regions, the striatum, the cerebellum and the brainstem, showing a significant reduction.
Serum Ez concentrations were observed to increase in a dose-dependent manner 4 h post EZB injection. The 1 g/kg E2B dose did not elevate serum E2B, levels above those observed in ovariectomized rats, the 10 g/kg EZB dose increased serum E2B, levels into the physiological range, and the 100 g/kg dose increased serum E2B levels, which were 12 to 30 times those seen during peak serum E2B
concentrations on proestrus. Peak serum E2B levels were observed at 4 hours after administration of a 10 g/kg dose of E2B.
Our evaluation of BUI using the Oldendorf method indicates that 4 h of exposure to E2B increased the transport of glucose across the blood-brain barrier by about 40%. The 40% increase in BUI was accomplished by an EzB-induced increase in C14-2-DG extraction with no change in 3 HZ0 extraction across the blood-brain barrier (Table XII).
WO 95/12402 2175603 pCT/US94/12782 It is expected that estrogen-replacement therapy will reduce or eliminate the loss of nerve cells in both the hippocampus and in the cerebral cortex. Such a result would indicate that physiological estrogen-replacement can protect brain cells from the neurotoxic effects of hypoglycemia.
Although certain preferred embodiments of the present invention have been described, the spirit and scope of the invention is by no means restricted to what is described above. For example, while the examples describe experiments performed in rats and cultured cells, it is believed that these are accurate models for_predicting activity of compounds in humans.
WO 95/12402 217 5 6 0 3 pCT/US94112782 =
effect was observed at 2 to 4 hours, while 2 regions showed peak effects at 12 hours (the MBH and cerebral cortex).
Five regions showed a significant decline in glucose uptake at some time-point after an EZB-induced increase. These regions were the POA, the hippocampus, the striatum, the cerebellum and the AP. No significant effect of E2B on brainstem glucose uptake was observed.
The dose-dependency of the E2B effect on brain glucose uptake at 4 hours post-E2B injection is shown in Table XI.
The 1 g/kg dose of E2B had no effect on brain glucose uptake. In contrast, the 10 g/kg dose of E2B increased glucose uptake in 6 of 8 regions examined at the 4 h time point. Furthermore, only two regions exhibited increased glucose uptake at the 100 pg/kg dose of E2B, the MBH and the POA. All other regions examined exhibited a reduced glucose uptake, with three regions, the striatum, the cerebellum and the brainstem, showing a significant reduction.
Serum Ez concentrations were observed to increase in a dose-dependent manner 4 h post EZB injection. The 1 g/kg E2B dose did not elevate serum E2B, levels above those observed in ovariectomized rats, the 10 g/kg EZB dose increased serum E2B, levels into the physiological range, and the 100 g/kg dose increased serum E2B levels, which were 12 to 30 times those seen during peak serum E2B
concentrations on proestrus. Peak serum E2B levels were observed at 4 hours after administration of a 10 g/kg dose of E2B.
Our evaluation of BUI using the Oldendorf method indicates that 4 h of exposure to E2B increased the transport of glucose across the blood-brain barrier by about 40%. The 40% increase in BUI was accomplished by an EzB-induced increase in C14-2-DG extraction with no change in 3 HZ0 extraction across the blood-brain barrier (Table XII).
WO 95/12402 2175603 pCT/US94/12782 It is expected that estrogen-replacement therapy will reduce or eliminate the loss of nerve cells in both the hippocampus and in the cerebral cortex. Such a result would indicate that physiological estrogen-replacement can protect brain cells from the neurotoxic effects of hypoglycemia.
Although certain preferred embodiments of the present invention have been described, the spirit and scope of the invention is by no means restricted to what is described above. For example, while the examples describe experiments performed in rats and cultured cells, it is believed that these are accurate models for_predicting activity of compounds in humans.
WO 95/12402 217 5 6 0 3 pCT/US94112782 =
Estrogen and Glucose Uptake in Various Brain Regions Time Course Effects of a l0 g Dose of e2b on 4 Hour Glucose Uptake in a Variety of Brain Regions and the Anterior Pituitary Gland TIME
REGIONS
Medial BasaL 116 t 15 154 t 24* 110 x 20 265 1 15* 108 1 7 Hypothalaaus Preoptic 132 z 22* 221 a 62* 80 t 11 67 a 4* 98 a 15 Area Cerebral 76 a 10* 98 m 9 120 m 17 215 3 28* 100 t 7 Cortex Hi ca s 132 a 18* 125 x 14* 71 t 10* 88 f 6 93 x 5 Striatum 94 x 30 140 t 17* 62 t 9* 88 t 6 103 t 7 CerebelLum 121 1 29 120 14* 73 m 9* 110 x 62 107 t 9 Brainstem 111 x 24 90 t 7 70 6 118 s 12 94 1 11 Anterior 95 a 10 220 3 39* 142 x 28 66 a 6* 125 s 7*
Pituitary * p 0.05 versus control vaLue within a brain region. EvaLuation of the time effect was sede with 3 0 a one-way ANOVA. After anaLysis and for cLarity of presentation data was transformed to percent of mean control.
WO 95/12402 2 1 7 5 6 0 3 PCT/US94'12782 TABLE XI
Estrogen and Glucose Uptake in Various Brain Region Dose-Dependent Effects of 4 Hour E2B Exposure on Glucose Uptake $i a Variety of Brain Regions and the Anterior Pituitary Gland EZB DOSE
BRAIN 1 g/Kg 10 g/Kg 100 g/Kg REGIONS
Medial Basal 110 t 23 154 24* 192 28*
Hypothalamus Preoptic - 98 45 221 62* 155 47*
Area Cerebral 95 t 27 98 9 87 11 Cortex Hippocampus 90 t 19 125 14* 87 12 Striatum 111 # 29 140 17* 74 8*
Cerebellum 112 t 20 120 t 14* 57 t 11*
Brainstem 116 t 22 90 7 47 16 Anterior 112 t 32 220 39* 64 25 Pituitary * p < 0.05 versus control value. EvaLuation of the dose effect was accamptished with a one-way ANOVA. Post hoc ccaparisons were done with a Dunnett's test. After analysis and for clarity of presentation data was 3 0 transformed to percent of mean control.
REGIONS
Medial BasaL 116 t 15 154 t 24* 110 x 20 265 1 15* 108 1 7 Hypothalaaus Preoptic 132 z 22* 221 a 62* 80 t 11 67 a 4* 98 a 15 Area Cerebral 76 a 10* 98 m 9 120 m 17 215 3 28* 100 t 7 Cortex Hi ca s 132 a 18* 125 x 14* 71 t 10* 88 f 6 93 x 5 Striatum 94 x 30 140 t 17* 62 t 9* 88 t 6 103 t 7 CerebelLum 121 1 29 120 14* 73 m 9* 110 x 62 107 t 9 Brainstem 111 x 24 90 t 7 70 6 118 s 12 94 1 11 Anterior 95 a 10 220 3 39* 142 x 28 66 a 6* 125 s 7*
Pituitary * p 0.05 versus control vaLue within a brain region. EvaLuation of the time effect was sede with 3 0 a one-way ANOVA. After anaLysis and for cLarity of presentation data was transformed to percent of mean control.
WO 95/12402 2 1 7 5 6 0 3 PCT/US94'12782 TABLE XI
Estrogen and Glucose Uptake in Various Brain Region Dose-Dependent Effects of 4 Hour E2B Exposure on Glucose Uptake $i a Variety of Brain Regions and the Anterior Pituitary Gland EZB DOSE
BRAIN 1 g/Kg 10 g/Kg 100 g/Kg REGIONS
Medial Basal 110 t 23 154 24* 192 28*
Hypothalamus Preoptic - 98 45 221 62* 155 47*
Area Cerebral 95 t 27 98 9 87 11 Cortex Hippocampus 90 t 19 125 14* 87 12 Striatum 111 # 29 140 17* 74 8*
Cerebellum 112 t 20 120 t 14* 57 t 11*
Brainstem 116 t 22 90 7 47 16 Anterior 112 t 32 220 39* 64 25 Pituitary * p < 0.05 versus control value. EvaLuation of the dose effect was accamptished with a one-way ANOVA. Post hoc ccaparisons were done with a Dunnett's test. After analysis and for clarity of presentation data was 3 0 transformed to percent of mean control.
TABLE SI7:
Estrogen and Glucose Uptake in various Brain Regions Effect of EZB on the Extraction of C14-2-Deoxyglucose and sSZO in the Brain GROUP E2DG E3x20 Oil 0.0803 t 0.011 0.1318 0.943 E2B 0.1208 0.013* 0.1486 0.313 2 0 * p < 0.05 when compared to oil control. Analyzed with a t-test for indeperdent samples; n = 7, mean t sem.
Estrogen and Glucose Uptake in various Brain Regions Effect of EZB on the Extraction of C14-2-Deoxyglucose and sSZO in the Brain GROUP E2DG E3x20 Oil 0.0803 t 0.011 0.1318 0.943 E2B 0.1208 0.013* 0.1486 0.313 2 0 * p < 0.05 when compared to oil control. Analyzed with a t-test for indeperdent samples; n = 7, mean t sem.
Table XIII
Rl AND/OR R2 SUBSTITUTIONS
NAME STRUCTURE
HYDROXYI. -OH
ACETATE O-C
3, 3, (OR 17, 17) OCH 3 DIMETHYL KETAL < OCH3 ETHYNYL- a < C = CH
ON
BENZOATE O-C mop0 b BENZYL ETHER oCH2---9 SULFATE, SODIUM SALT OSO3Na OXIDE =0 CYCLOPEHM.PROPIONATE -O-C-(CH2)2 -Q
PROPIONATE -O-C-(CH2)2 Table XIV
NAME STRUCTURE
SODIUM PHOSPHATE -0-P03Na2 GLUCURONIDE, SODIUM SALT -C6H806No STEARATE -C1 g H34 0 TRIETHYL AMMONIUM SALT -N-(C2H5)3 n OH
OH 4p:~) 1 OH
17a ESTRADIOL
OH
Rl AND/OR R2 SUBSTITUTIONS
NAME STRUCTURE
HYDROXYI. -OH
ACETATE O-C
3, 3, (OR 17, 17) OCH 3 DIMETHYL KETAL < OCH3 ETHYNYL- a < C = CH
ON
BENZOATE O-C mop0 b BENZYL ETHER oCH2---9 SULFATE, SODIUM SALT OSO3Na OXIDE =0 CYCLOPEHM.PROPIONATE -O-C-(CH2)2 -Q
PROPIONATE -O-C-(CH2)2 Table XIV
NAME STRUCTURE
SODIUM PHOSPHATE -0-P03Na2 GLUCURONIDE, SODIUM SALT -C6H806No STEARATE -C1 g H34 0 TRIETHYL AMMONIUM SALT -N-(C2H5)3 n OH
OH 4p:~) 1 OH
17a ESTRADIOL
OH
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Claims (44)
1. Use of an estrogen compound in the absence of testosterone, the compound having a general structure:
or a tautomer thereof, or a pharmaceutically acceptable salt thereof, for protecting a population of neuronal cells from progressive cell damage leading to the death of the cells, wherein R1 is selected from hydrogen, hydroxyl, oxo, methyl, methyl ester, acetate, ethyl ether, benzoate, benzyl ether, glucuronide, valerate, cyclopentylpropionate, propionate, hemisuccinate, palmitate, enanthate, stearate and cypionate; and R2 is selected from hydrogen, hydroxyl, oxo, methyl, methyl ester, acetate, ethyl ether, 17, 17-dimethyl ketal, ethynyl-a, benzoate, benzyl ether, glucuronide, valerate, cyclopentylpropionate, propionate, hemisuccinate, palmitate, enanthate, stearate and cypionate.
or a tautomer thereof, or a pharmaceutically acceptable salt thereof, for protecting a population of neuronal cells from progressive cell damage leading to the death of the cells, wherein R1 is selected from hydrogen, hydroxyl, oxo, methyl, methyl ester, acetate, ethyl ether, benzoate, benzyl ether, glucuronide, valerate, cyclopentylpropionate, propionate, hemisuccinate, palmitate, enanthate, stearate and cypionate; and R2 is selected from hydrogen, hydroxyl, oxo, methyl, methyl ester, acetate, ethyl ether, 17, 17-dimethyl ketal, ethynyl-a, benzoate, benzyl ether, glucuronide, valerate, cyclopentylpropionate, propionate, hemisuccinate, palmitate, enanthate, stearate and cypionate.
2. The use according to claim 1, wherein the R2-group on C17 is an hydroxyl in an .alpha.-isomeric position, and R1 on C3 is a hydroxyl in a .beta.-isomeric position.
3. The use according to claim 1, wherein the R2-group on the C17 is an hydroxyl in a .alpha.-isomeric position, and R1 on the C3 is an hydroxyl in the .beta.-position.
4. The use according to claim 1, wherein the compound is a pharmaceutically acceptable salt independently selected from the group consisting of sodium glucoronide, sulfite sodium salt, sodium phosphate, and trimethyl ammonium salt.
5. The use according to claim 1, wherein the cell population is a population of nerve cells.
6. The use according to claim 1, wherein the neuronal cells are cholinergic nerve cells.
7. The use according to claim 1, wherein the neuronal cells are hippocampal cells.
8. The use according to claim 1, wherein the neuronal cells are cortex cells.
9. The use according to claim 1, wherein the cell population is a population of glial cells.
10. The use according to claim 1, wherein the R2 group is in an a-isomeric position and the estrogen compound is used at a site within a subject so as to decrease loss of neuronal activity in the subject.
11. The use according to claim 10, wherein the loss of neuronal activity in the subject is at a site selected from the group consisting of hippocampus, cortex and basal forebrain.
12. The use according to claim 10, wherein the loss of neuronal activity is associated with a chronic degenerative disorder.
13. The use according to claim 10, wherein the loss of neuronal activity is associated with an acute degenerative disorder.
14. The use according to claim 10, wherein the loss of neuronal activity is associated with trauma at the site.
15. The use according to claim 10, wherein the loss of neuronal activity is associated with overstimulation of the excitatory amino acid receptors on neurons.
16. The use according to claim 10, wherein the estrogen compound is in a controlled release vehicle.
17. The use according to claim 10, wherein use of the estrogen compound is oral.
18. The use according to claim 10, wherein use of the estrogen compound is subcutaneous.
19. The use according to claim 10, wherein the loss of neuronal activity is associated with loss of memory and loss of learning function.
20. Use of an estrogen compound in the absence of testosterone for protecting a population of cells in a subject in need thereof, from progressive damage leading to the death of the cells.
21. The use according to claim 20, wherein the cause of the progressive cell damage leading to death of cells is selected from the group consisting of a neurodegenerative disorder, trauma, and aging.
22. The use according to any one of claims 20 or 21, wherein the progressive cell damage is associated with loss of neuronal activity, and the dosage of the estrogen compound is sufficient to allow the compound to decrease the loss of neuronal activity in the subject.
23. The use according to any one of claims 20 to 22, wherein the cause of progressive cell damage includes an overstimulation of excitatory amino acid receptors.
24. The use according to claim 23, wherein the estrogen compound is reacted with an excitatory amino acid receptor so as to block receptor activity.
25. The use according to any one of claims 20 to 24, wherein the cause of progressive cell damage is a chronic neurodegenerative disorder.
26. The use according to any one of claims 20 to 24, wherein the cause of progressive cell damage is an acute neurodegenerative disorder and is associated with the loss of neuronal activity.
27. The use according to any one of claims 20 to 22 and 25 to 26 wherein protecting the population of cells from cell damage comprises increasing the amount of neurotrophic growth factor available to the population of cells.
28. The use according to claim 27, wherein the neurotrophic factor is nerve growth factor.
29. The use according to claim 27, wherein the neurotrophic factor is brain derived neurotrophic factor.
30. The use according to any one of claims 1 to 29, wherein the cell population is nerve cells and the nerve cells are protected from axonal retrograde degeneration in the presence of the estrogen compound.
31. The use according to any one of claims 1 to 30, wherein the cell population is nerve cells and the nerve cells are protected from apoptosis in the presence of the estrogen compound.
32. The use according to any one of claims 1 to 10 or 12 to 31, wherein the population of cells is located at a site selected from the hippocampus, the cortex, and the basal forebrain.
33. The use according to claim 11 or 32, wherein the population of cells is a population of cholinergic nerve cells.
34. The use according to any one of claim 1 to 16 or 19 to 33, wherein the use of the estrogen compound is oral, buccal, intramuscular, transdermal, intravenous or subcutaneous.
35. The use according to any of claims 20 to 33, wherein the use of the estrogen compound is oral.
36. The use according to any of claim 20 to 33, wherein the use of the estrogen compound is subcutaneous.
37. The use according to any of claims 20 to 33, wherein the use of the estrogen compound is intramuscular.
38. The use according to any one of claims 1 to 37, wherein the cells are nerve cells and the nerve cells are located within a first site in a brain of a subject, and wherein use of the estrogen compound is at a second site distal to the neurons.
39. The use according to any one of claims 1, 2, 4 to 38, the estrogen compound being used in an amount sufficient to provide a plasma level in the range 20 pg/ml to 250 pg/ml of the compound, wherein the estrogen compound has at least 100 times less estrogenic activity than 17-.beta.-estradiol.
40. The use according to any one of claim 1, 3 to 9, or 20- 38, wherein the estrogen compound is 17-.beta.-estradiol, the 17-.beta.-estradiol being used in an amount sufficient to provide a plasma level in the range 20 pg/ml to 250 pg/ml of 17-.beta.-estradiol.
41. The use according to claim 39, wherein the estrogen compound is 17-.alpha.-estradiol.
42. The use according to any one of claims 1 to 41, wherein the population of cells is a population of nerve cells and a decrease in nerve cell death is measured by monitoring levels of high affinity choline uptake by cholinergic nerve cells.
43. The use according to claim 42, wherein an increase in the level of choline acetyltransferase is caused.
44. The use according to any one of claims 12 to 14, 25, or 26, wherein the disorder, trauma or aging is selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, AIDS dementia, Wernicke-Korsakoff's related dementia (alcohol induced dementia), age related dementia, age-associated memory impairment, peripheral neuropathy, multiple sclerosis, amyotrophic lateral sclerosis, schizophrenia, and brain or spinal cord cell loss due to head trauma, stroke, hypoglycemia, ischemia, anoxia, hypoxia, cerebral edema, arteriosclerosis, hematoma and epilepsy.
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| US08/318,042 | 1994-10-04 | ||
| US08/318,042 US5554601A (en) | 1993-11-05 | 1994-10-04 | Methods for neuroprotection |
| PCT/US1994/012782 WO1995012402A1 (en) | 1993-11-05 | 1994-11-07 | Estrogen compositions and method for neuroprotection |
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| US6319914B1 (en) * | 1993-11-05 | 2001-11-20 | Apollo Biopharmaceuticals, Inc. | Cytoprotective effect of polycyclic phenolic compounds |
| US6350739B1 (en) * | 1999-08-11 | 2002-02-26 | University Of Florida Resarch Foundation, Inc. | Methods of prevention and treatment of ischemic damage |
| US5877169A (en) * | 1993-11-05 | 1999-03-02 | University Of Florida Research Foundation, Inc. | Methods of treatment of ischemic damage |
| DE4338314C1 (en) | 1993-11-10 | 1995-03-30 | Jenapharm Gmbh | Pharmaceutical preparations for the prophylaxis and therapy of radical-mediated cell damage |
| US5550029A (en) * | 1994-09-08 | 1996-08-27 | University Of Florida | Method for diagnosing estrogen responsiveness |
| WO1996022091A1 (en) * | 1995-01-20 | 1996-07-25 | Novo Nordisk A/S | Use of 3,4-diphenyl chromans for the manufacture of a pharmaceutical composition for the treatment or prophylaxis of cerebral degenerative disorders |
| FR2754179B1 (en) * | 1996-10-08 | 1998-12-24 | Theramex | NOVEL HORMONONAL COMPOSITION AND ITS USE |
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| US5554601A (en) | 1996-09-10 |
| AU1090195A (en) | 1995-05-23 |
| EP0799041A1 (en) | 1997-10-08 |
| WO1995012402A1 (en) | 1995-05-11 |
| US5843934A (en) | 1998-12-01 |
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