Regulation of amyloid precursor protein (APP) expression by administration of an estrogenic compound

It has been discovered that lipophilic hormones that interact with cytosolic or nuclear receptors regulate APP expression and synthesis, through modification of APP mRNA stability and/or regulation of APP gene transcription and translation activities. These studies demonstrate that the treatment of brain cells with estrone or 17.beta.-estradiol results in a reduction in the level of APP holoprotein expression, without a concomitant change in the total level of cell protein. The reduction in the level of APP holoprotein caused by estrone or 17.beta.-estradiol is also expected to reduce the production of neurotoxic APP fragments. In as much as estrogen deficiency in postmenopausal women is associated with a higher incidence of Alzheimer's disease, this discovery opens the possibility that estrogen therapy may prevent some of the neurodegenerative and cognitive changes associated with Alzheimer's disease, aging and other disease conditions associated with such neurodegenerative and cognitive decline.

FIELD OF THE INVENTION
 The present invention relates to compositions and methods for the treatment
 of various neurological diseases and neurodegenerative disorders
 associated with aging, particularly those affected by an overabundance of
 Amyloid Precursor Protein (APP). In particular, this invention relates to
 the APP holoprotein synthesis and the effect of lipophilic compounds on
 regulating the expression of this protein. In particular, it has been
 discovered that certain neurodegenerative or cognitive changes are
 associated with developing an imbalance in the serum levels of endogenous,
 gynecologically relevant substances, including certain neurotransmitters,
 neurotransmitter substrates or hormones. It has also been found that the
 level of APP holoprotein is reduced by lipophilic estrogenic compounds in
 brain cells, and that this reduction is expected to reduce the
 neurotoxicity or neurodegeneration associated with APP over expression.
 BACKGROUND OF THE INVENTION
 Alzheimer's Disease (AD) is the most common neurodegenerative disorder of
 aging, and is characterized by progressive dementia and personality
 dysfunction. The abnormal accumulation of amyloid plaques in the vicinity
 of degenerating neurons and reactive astrocytes is a pathological
 characteristic of AD.
 Several lines of studies suggest that postmenopausal women with lower
 levels of endogenous estrogen may be predisposed to the development of AD.
 Studies in experimental animal models provide a convincing rational for
 the role of estrogen replacement therapy and prevention of dementia. See,
 for example, Birge, J. Am. Geriatric. Soc. 44, 865, (1996). These studies
 suggest that estrogen deficiency in postmenopausal women apparently
 increases their susceptibility to the neurodegenerative changes of aging
 and AD, and that this risk can be decreased by estrogenic replacement
 therapy. Peganini et al. Am. J. Epidemiol, 140, 256 (1994). Estrogen
 treatment of cell culture reportedly promotes non-amyloidogenic APP
 processing and soluble APP (APPs) secretion. Jaffe et al., J. Biol. Chem.
 269, 13065 (1994).
 APP processing is regulated by neurotransmitters and synaptic activity.
 Amyloid plaques in AD accumulate near dystrophic neurons and reactive
 astrocytes. Cordell, Annu. Rev. Pharmacol. Toxicol. 34, 69 (1994); Selkoe,
 Annu. Rev. Neurosci. 17, 489 (1994). The activation of neurotransmitter
 receptors, which are coupled to phosphotidylinositol (PI) hydrolysis or to
 protein kinase C (PKC) activation, can promote APP metabolism and decrease
 amyloid formation. Nitsch, et al., Science 258, 304 (1992); Wolf et al.,
 J. Biol. Chem. 270, 4916 (1995); Buxbaum et al., Proc. Natl. Acad. Sci.
 U.S.A. 91, 4489 (1994); Lee, et al., Ibid., 92, 8083 (1995); Ulus and
 Wurtman, J. Pharm. Exp. Ther., 281,149 (1997); Lee et al., Proc. Natl.
 Acad. Sci. U.S.A. 92, 8083 (1995). Activation of neurotransmitters coupled
 to cAMP production suppresses both constitutive and PKC/PI-stimulated APPs
 secretion in astroglioma cells and in primary astrocytes. Eftimiopoulos et
 al., J. Neurochem., 67, 872 (1996); Lee et al., J. Neurochem., 68,1830
 (1997). The drastic alterations in neurotransmitter levels and second
 messenger signaling created by neurodegeneration and synapse loss in AD
 may disrupt APP processing in ways that promote the accumulation of
 amyloidogenic or neurotoxic APP fragments.
 Additionally, the loss of various neurotransmitters in AD may increase
 cellular levels of APP holoprotein containing amyloidogenic or neurotoxic
 peptides due to a decrease in proper APP metabolism. Yankner et al.,
 Science, 245, 417 (1989); M. R. Kozlowski, A. Spanoyannnis, S. P. Manly,
 S. A. Fidel, R. L. Neve, J. Neurosci, 12, 1679 (1992).
 Increased APP production in Down's syndrome is associated with a high
 incidence of AD at an early age due to the extra copy of the APP gene.
 Over expression of APP in cell cultures and in transgenic mice is also
 associated with neurodegeneration and age-related cognitive deficits,
 suggesting that over expression of APP could contribute to the
 neuropathology of AD. Maruyama, et al., Nature, 347, 566 (1990); Hsiao et
 al., Neuron 15, 1203-1218 (1995); Moran, et al., Proc. Natl. Acad Sci.
 U.S.A. 92, 5341 (1995).
 Several APP isoforms, ranging in size from 695-770 amino acids, are derived
 by differential splicing of a primary transcript of the three major APP
 isoforms, APP-695 is predominantly expressed in neurons; APP-751 and
 APP-770, which harbor an additional Kunitz-type protease inhibitor (KPI)
 insert at the N-terminus, are predominantly expressed in astrocytes and
 appear to be increased in AD brain. Golde, S. et al., Neuron 4, 253
 (1990); Neve, et al.,Ibid., 1, 669 (1990); Anderson et al., EMBO J. 8,
 3627 (1989); Nordstedt et al., Proc. Natl. Acad. Sci. U.S.A. 88, 8910
 (1991). The decreased amounts of APP-695 in postmortem AD brains may be
 due to neuronal loss. The increase in KPI-containing APP isoforms in AD
 and in regions surrounding senile plaques raises the possibility that APP
 overexpression in astrocytes contributes to AD neuropathology.
 Several lines of studies have indicated an apparently low incidence of
 Alzheimer's Disease in rheumatoid arthritis patients. These studies
 propose the possibility that anti-inflammatory therapy with NSAIDs
 (non-steroidal anti-inflammatory drugs) confers some protection against
 AD. See, e.g., McGeer, et al., Lancet, 335, 1037 (1990); Andersen, et al.,
 Neurology, 45, 1441 (1995); Rich, et al. Neurology, 45, 51 (1995);
 Lindsay, et al., Neurology, 44, 2073 (1994); Rogers, et al., Neurology,
 43, 1609 (1993). These studies suggest that the administration of a
 non-steroidal anti-inflammatory drug may reduce the onset of dementia in
 Alzheimer's patients. However, none of these studies discloses or suggests
 that the administration of NSDAs prevents the overproduction of APP.
 Persistent and rapid elevations in APP immunoreactivity have been observed
 in GFAP-positive astrocytes following brain injury. Siman et al., J.
 Neurosci., 3, 275 (1989); Banati and Kreutzberg, J. Cereb. Blood Flow
 Metab., 12, 257 (1995). In the AD brain, the loss of synapses is
 associated with an increase in the number of GFAP-positive astrocytes
 (Brun et al., Neurodegeneration, 4, 171 (1995), and increases in KPI
 containing APP mRNA in the frontal cortex have also been attributed to the
 astrocytic response during neuronal damage (Golde et al., Neuron, 4, 253
 (1990). It seems that the loss of synapses and neurons in AD might
 initiate a pathological cascade that includes APP overexpression by
 reactive astrocytes.
 In U.S. Pat. No. 5,385,915, Buxbaum et al. describe methods and
 compositions for affecting APP processing by the administration of agents
 that regulate protein phosphorylation, namely agents that regulate kinases
 or phosphatases. The modulation of APP processing leads, in turn, to the
 regulation of the production of .beta./A.sub.4 peptide, a peptide that
 accumulates in amyloidogenic plaques. See, e.g., col. 6, lines 8-10.
 Hence, Buxbaum et al. teach that one's objective should focus on the
 search for agents that alter the metabolism of APP.
 However, Buxbaum et al. make no mention, teaching, or suggestion that the
 step preceding the processing of APP, that is, the expression, production,
 or formation of APP, itself, can be at all affected by select groups of
 substances. Indeed, as Buxbaum et al. state (at col. 21, lines 7-9), "the
 effects observed are attributable to changes in APP metabolism rather than
 APP transcription (emphasis added)." Consistent with this notion, the
 claims of Buxbaum et al. are drawn to a method of regulating
 phosphorylation of proteins that control the processing of APP.
 Similarly, in U.S. Pat. No. 5,242,932, Gandy et al. disclose and claim a
 method of modulating or affecting the intracellular trafficking and
 processing of APP in the mammalian cell.
 For additional background information on the processing of APP, release of
 APP derivatives, or the processing, degradation and secretion of
 .beta./A.sub.4 APP, the interested reader is referred to the following
 publications: Nitsch, et al. Science, 258, 304 (1992); Lee, et al., Proc.
 Nat'l. Acad. Sci. USA, 92, 8083 (1995); Caporaso, et al. Proc. Nat'l.
 Acad. Sci. U.S.A, 89, 3055 (1992); Caporaso, et al. Proc. Nat'l. Acad.
 Sci. U.S.A, 89, 225 (1992); and Buxbaum, et al. Proc. Nat'l. Acad. Sci.
 U.S.A, 89, 10075 (1992).
 In contrast to the above studies, the present invention, as disclosed
 herein, concerns the expression, formation, or synthesis of APP. The
 present inventors have previously discovered that cAMP elevations caused
 by activation of neurotransmitter receptors increased APP mRNA and
 holoprotein production in astrocytes. Lee et al.,et al. Proc. Nat'l. Acad.
 Sci. U.S.A, 94, 5422 (1997). The present inventors have also shown that
 activation of prostaglandin E.sub.2 (PG E.sub.2) receptors coupled to
 increased cAMP formation also stimulates the synthesis of APP mRNA and
 holoprotein and that this effect can be mediated by cAMP-dependent protein
 kinases, and can be inhibited by various substances, including
 immunosuppressants and ion-channel modulators. Lee et al., J. Neurochem.
 (supp) 69, S103B (1997). As described herein, the present inventors have
 now shown that APP expression can also be regulated by lipophilic hormones
 that interact with cytosolic or nuclear receptors.
 Estrogen receptors within the brain have a regional distribution strikingly
 similar to that characteristics of AD-type brain pathology, Thomlinson, B.
 E.(1992): In Greenfield's neuropathology (Adams, J. H. and Duchen L. W.
 eds)pp 1284-1410 Oxford University Press. In addition recent findings
 suggest that estrogen replacement therapy my be protective against AD,
 Henderson, et al., Arch neurol (1994). In order to test the hypothesis
 that gonadal esteroids might play a role in regulating APP metabolism,
 Jaffe, et al., J. Biol. chem.269, 13065 (1994) investigated the possible
 effect of estrogen on the metabolism of Alzheimer's amyloid precursor
 protein. Using a cell line that contains high level of estrogen receptors,
 these authors found that the treatment with 17.beta.-estradiol is
 associated with the accumulation of APPs in the medium, indicative of
 non-amyloidogenic processing. However, these authors found no changes in
 the levels of intracellular immature or mature APP holoproteins,
 suggesting that estrogen may increase the secretory metabolism of APP.
 Additionally, Hsiao et al., Neuron, 15, 1203 (1995), disclose that
 age-related central nervous system (CNS) disorder and early death in mice
 were due to over expression of APP, rather than amyloid, with increasing
 level of intracellular APP, indicating that some deleterious processes
 related to APP over expression are dissociated from formation of arnyloid.
 Previously, it has been shown that neurotrophic effect of estrogen may be
 through uncoupling signal transduction mechanism of certain receptors
 linked to G-proteins. Lagrange, et al., Soc. Neursci. Abstr. 22, 378,
 (1996). Additionally, the present inventors have discovered that G-protein
 coupled receptors which stimulate cAMP formation or PI hydrolysis can
 accelerate the synthesis and metabolism of APP respectively.
 This invention, as disclosed herein, demonstrates for the first time that
 lipophilic compounds such as estrogen affects the synthesis of the APP
 holoprotein. The present invention also contemplates and provides an assay
 for identifying or screening potential drugs that can inhibit the
 excessive production of neurotoxic APP. For example, such an assay may
 utilize a tissue or cell culture comprising brain cells, as described
 herein.
 These and other objects of the invention will be evident to those of
 ordinary skill from a consideration of the discussions and descriptions
 provided in this specification, including the detailed description of the
 preferred embodiments.
 SUMMARY OF THE INVENTION
 It has now been discovered that APP expression can be regulated by
 lipophilic hormones that interact with cytosolic or nuclear receptors. By
 contrast to cell-surface receptors that activate second messengers,
 receptor estrogen complexes typically accumulate in the nucleus where they
 modify mRNA stability or DNA transcription. Thus, these substances can be
 used to prevent APP overexpression in brain cells.
 What is more, it has been shown that lipophilic estrogenic compounds, such
 as estrone or 17.beta.-estradiol, inhibit an abnormal increase in the
 level of APP synthesis (mRNA and cell-associated protein). We have shown
 that prolonged activation of receptors that are coupled to increased cAMP
 formation in cortical astrocytes up regulates both APP mRNA levels and APP
 holoprotein bearing the KPI insert. It has further been discovered that
 such overproduction, which is shown can result from the body's response to
 brain injury or trauma, can be inhibited by certain substances, including
 lipophilic hormones that interact with cytosolic or nuclear receptors.
 An object of this invention is to provide compositions and methods for
 modulating expression, production, or formation of amyloid precursor
 protein (APP) in a subject comprising administering to the subject an
 effective amount of a lipophilic hormone, an analog of a lipophilic
 hormone, a substance that is a ligand, an agonist, or an antagonist of a
 receptor that is coupled to a lipophilic hormone, and a pharmaceutically
 acceptable carrier or diluent.
 Another object of this invention is to provide a method of determining the
 capacity of a drug to modulate expression, production, or formation of
 amyloid precursor protein (APP) in an eukaryotic cell comprising,
 contacting a drug with an eukaryotic cell containing estrogen receptors.
 Yet another object of this invention is to provide compositions and methods
 for alleviating the negative effects of a neurological disorder or
 neurodegenerative disease stemming from the aberrant expression,
 production, formation, or overexpression of amyloid precursor protein
 (APP) in a subject, comprising administering to a subject suffering from
 the above-noted disorder or disease an effective amount of a lipophilic
 hormone, and a pharmaceutically suitable carrier or diluent.
 Consistent with the objectives of this invention, a method of modulating
 APP is provided which comprises administering to a subject in need of
 treatment an effective amount of a substance that regulates APP promoter
 activity, and other activities related to APP gene transcription and
 translation. Other embodiments includes methods of treating and
 alleviating neurodegenerative or cognitive changes in postmenopausal
 women, comprising administering to a woman in need of treatment an
 effective amount of an estrogenic compound.
 BRIEF DESCRIPTION OF THE DRAWINGS
 FIG. 1 illustrates the effects of estrone and 17.beta.-estradiol on APP
 holoprotein synthesis in confluent primary rat cortical neurons.
 FIG. 2 illustrates the effects of estrone and 17.beta.-estradiol on APP
 holoprotein synthesis in confluent primary rat cortical astrocytes.
 FIG. 3 illustrates the effects of estrone and 17.beta.-estradiol on APP
 holoprotein synthesis in human glioma cells (HS683).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
 The inventors have shown that APP expression can be regulated by lipophilic
 hormones that interact with cytosolic or nuclear receptors.
 In this study, confluent primary rat cortical neurons, astrocytes, or human
 glioma cells (HS683), which were treated over a 24 hour period with 1, 10,
 or 100 .mu.M of a lipophilic estrogenic compound, such as estrone or
 17.beta.-estradiol, gave rise to decreased levels of cellular APP,
 compared to those levels that are observed in untreated control cells.
 See, e.g., FIGS. 1, 2, and 3. The levels of APP holoprotein associated
 with cultured brain cells (e.g. astrocytes or neurons) and HS683 cells
 were measured with an antisera R37 (gift of Dr. F. Kametani, Tokyo Inst.
 of Psychiatry) or with monoclonal antibody 22C11 (BoehringerMannheim)
 directed against the C-terminus or N-terminus of APP, respectively, using
 Western blot analyses. The total cell protein content as measured by the
 bicinchoninic acid assay was not changed by these estrogenic compounds in
 the range of concentrations tested.
 These data suggests that estrogenic compounds reduce APP holoprotein levels
 by decreasing APP synthesis. The decrease in the level of APP synthesis is
 specially pronounced with 10 .mu.M estrone in cultured neurons and HS683
 cells, and 10 .mu.M 17.beta.-estradiol in cultured HS638 cells. The
 reduction in APP holoprotein caused by 17.beta.-estradiol or estrone is
 also expected to reduce neurotoxicity or neurodegeneration associated with
 APP over expression.
 Additionally, these results suggests that the mechanisms regulating APP
 synthesis or metabolism may differ among different types of cells.
 LeBlanc, A. C. J. Neurochem.2300, (1996); and LeBlanc. A. C. J. Neurochem.
 1183 (1997) disclose that the level of amyloidogenic processing of APP
 differs among brain cells such as neurons, astrocytes and microglia. These
 authors concluded that human neurons and astrocytes generate higher levels
 of amyloidogenic fragments than microglia.
 Jaffe et al. J. Biolo. Chem.269, 13065 (1994) disclose the possible effect
 of estrogen on the metabolism of the Alzheimer's amyloid precursor protein
 (APP). Using the breast carcinoma cell line, these authors found that
 treatment with physiological concentrations of 17.beta.-estradiol is
 associated with extracellular accumulation of soluble APP (APPs). However,
 these authors found no changes in the levels of intracellular immature or
 mature APP holoproteins, suggesting that estrogen may increase the
 secretory metabolism of APP.
 In contrast to Jaffe et al., our data, using brain cells, demonstrate a
 decrease in the level of intracellular APP holoprotein with administration
 of estrogenic compounds. The lower concentration of estradiol and the use
 of breast carcinoma cells by Jaffe et al may account for Jaffe et al's
 inability to detect a decrease in the level of APP holoprotein. There are
 functional differences between breast carcinoma cells and brain cells. The
 mechanisms regulating APP synthesis and metabolism between
 carcinoma/cancer cells and bona fide brain cells (e.g., neurons,
 astrocytes) are likely to be different (see, Mills and Reiner, J.
 Neurochem. 67, 1511 (1996)).
 The decrease in APP holoprotein levels, as disclosed herein, is associated
 with decreased production of APP mRNA, or decreased translation of mRNA to
 produce APP holoprotein. Northern blots are used to determine the levels
 of APP mRNA in the absence or presence of estrone or 17.beta.-estradiol.
 The results suggests a decrease in the level of APP mRNA in the presence
 of these compounds. Also, Western blot analysis and inhibitors of
 transcription (e.g., actinomycin D) or translation (e.g., cycloheximide)
 are used to measure the levels of APP holoprotein in cells treated with
 estrogenic compounds. Again, a decrease in the level of APP holoprotein is
 found to be associated with the administration of the estrogenic
 compounds.
 Estrogenic compounds are known to uncouple g-proteins. Since cell-surface
 receptors activate g-proteins in order to regulate intracellular signaling
 pathways, estrogenic compounds may also regulate signaling pathways that
 regulate APP synthesis and metabolism. For instance, activation of
 cell-surface receptors coupled to cAMP stimulates increases in
 amyloidogenic APP holoprotein. Uncoupling g-proteins by estrogenic
 compounds may reduce cAMP production and thus, prevent the formation of
 amyloidogenic APP holoprotein.
 Previously, we have shown that stimulation of certain cell-surface
 receptors coupled to cAMP formation in astrocytes increases the production
 of APP mRNA and APP holoprotein. It is suggested that the up regulation or
 aberrant activation of certain receptors in brain regions that are
 vulnerable to damage can stimulate APP overexpression in brain cells and,
 thereby, contribute to amyloid production. Because APP over expression can
 cause neurodegeneration and cognitive dysfunction, the inventors have
 shown that such substances as neuroactive steroids and related products,
 e.g., estrone and 17.beta.-estradiol and the like, are promising drug
 candidates for the treatment of Alzheimer's, Parkinson's, Lou Gehrig's
 Disease (amylotrophic lateral sclerosis), multiple sclerosis, ischemia,
 traumatic brain injury, epileptic seizure, and the like, which may have
 their roots in the formation or presence of amyloid plaques.
 Accordingly, it is an object of the present invention to provide methods
 and compositions that modulate or regulate the production or formation of
 APP in patients, including the expression of APP gene products and the
 transcription or translation of the APP gene in brain cells. For example,
 the production of APP by mammalian cells, in particular, by cells in the
 brain, can be increased or reduced.
 In attaining this objective, it is also an objective of the invention to
 inhibit excessive APP holoprotein formation, prevent neural dystrophy and
 alleviate pathological symptoms, such as neurodegeneration or cognitive
 deficits that may arise from the negative effects of inappropriately
 expressed, produced, or formed amounts of APP.
 FURTHER ASPECTS OF THE PREFERRED EMBODIMENTS
 Thus, the present invention is directed to a method of modulating the
 expression, production, or formation of amyloid precursor protein (APP) in
 a subject comprising administering to the subject an effective amount of a
 lipophilic hormone, such as estrone or 17.beta.-pestradiol, an analog of a
 lipophilic hormone, a substance that is a ligand, an agonist, or an
 antagonist of a receptor that is coupled to a lipophilic hormone, or a
 compound that regulates the activity of cytosolic or nuclear receptors. In
 specific embodiments of the invention the lipophilic hormones and related
 compounds comprise; Allopregnanolone; Allotetrahydrodeoxycorticosterone;
 Alphaxalone; Androsterone, 4-Androstane-3,17-dione, Corticosterone;
 Corticosterone: HBC complex; Danazol; Dehydroepiandrosterone sulfate
 sodium; Dexamethasone; 17.beta.-Estradiol; 17.beta.-Estradiol: HBC
 complex; Estrone; Etiocholanolone; FGIN I-27; Fluoxymesterone;
 Hydrocortisone; Hydrocortisone: HBC complex; Methandrostenolone;
 Nandrolone decanoate; Oxandrolone; Oxymetholone; Prednisolone;
 Pregnenolone sulfate sodium; Progesterone; Progesterone: HBC complex;
 Spironolactone; Stanolone; Tamoxifen; 3-hydroxy; citrate; (E)-; Tamoxifen,
 4-hydroxy-, (E)-; Tamoxifen, 4-hydroxy-, (Z)-; Testosterone: HBC complex;
 Testosterone, 17.beta.-cypionate; Testosterone, 17.beta.-decanoate;
 Testosterone, 17.beta.-heptanoate; Testosterone, 17.beta.-isocapronate;
 Testosterone, 17.alpha.-methyl-; Testosterone, 17.beta.-propionate;
 Triamcinolope; U-73122; U-73343; Endosulfan; dieldrin; methoxychlor;
 thyroid hormones; nethimazole (antithyroid hormones); human growth
 hormones; gonadotropin; vasopressin; calcitonin; adrenal cortical
 hormones; insulin; anabolic steroids; and antineoplastics.
 By "APP overexpression" is meant any activity that is exerted in the
 nucleus of a eukaryotic cell that ultimately gives rise to expression,
 production, or formation of APP in a subject, including but not limited to
 the regulation of the promoter of the APP gene, the modulation of APP
 metabolism transcriptional factors that affect APP expression, or the
 stimulation of the activity of second messengers.
 Indeed, according to the methods of the present invention, a suitable
 antagonist can comprise a receptor antagonist of a neurotransmitter, an
 antagonist of a lipophilic hormone, and the like. Examples of additional
 substances, which have been found to decrease or inhibit the APP synthesis
 include, but are not limited to H8, H9, cyclosporin A, FK-506, Win55212,
 and propranolol.
 When it is desirable to effect a decrease of endogenous APP production,
 cellular levels of cAMP are caused to diminish. For example, the cellular
 levels of cAMP can be decreased by retarding the production of endogenous
 cAMP or by stimulating the breakdown of cAMP. Substances, such as H8, H9,
 propranolol, or Win55212 can be administered to achieve this end.
 An important aspect of the present invention concerns the treatment of a
 subject that has suffered an injury or trauma, especially to the head or
 brain, or of a subject that is, for some other or related reason, may be
 experiencing overstimulation of cAMP expression, production, formation
 (or, collectively, "synthesis").
 It is particularly advantageous to treat the subject in need by
 administering an effective amount of a lipophilic hormone such as estrone
 or 17.beta.-estradiol. It is noteworthy that the present invention is also
 directed to a method of determining the capacity of a drug to inhibit the
 expression, production, or formation of amyloid precursor protein (APP) in
 a cell comprising contacting an estrogenic drug, an analog of an
 estrogenic drug, a substance that is a ligand, an agonist, or an
 antagonist of an estrogen receptor, with a cell culture that has the
 capacity to synthesize APP holoprotein. The level of APP mRNA or
 holoprotein produced from the cell culture in the presence of the drug is
 then compared with the level of APP mRNA or holoprotein produced from the
 cell culture in the absence of the drug. The cell can be any type of
 microbial, plant, or animal cell, so long as the cell has the capacity to
 express, produce, or otherwise form APP. The cell is preferably an
 eukaryotic cell. More preferably, the eukaryotic cell can further be a
 yeast cell, insect cell, invertebrate, vertebrate, or mammalian, including
 animal or human.
 It should be apparent that the present invention is directed to a method of
 alleviating the negative effects of a neurological disorder or
 neurodegenerative disease stemming from the aberrant expression,
 production, or formation of amyloid precursor protein (APP) in a subject.
 In a particular embodiment, an effective amount of a lipophilic hormone
 such as estrone or 17.beta.-estradiol, or an analog or agonist thereof, is
 administered to the subject suffering from the disorder or disease. As
 described herein, the present method of modulating amyloid precursor
 protein (APP) expression in a subject may also comprise administering to
 the subject an effective amount of a substance that regulates APP promoter
 activity, either by stimulating APP promoter activity or retarding it.
 The inhibition of APP promoter activity can, in turn, regulate the
 expression of abnormal forms of tau. And, hence, the present invention
 also contemplates a method of regulating the expression of abnormal forms
 of tau in a subject comprising modulating amyloid precursor protein (APP)
 expression in the subject.
 Moreover, compositions for modulating the expression, production, or
 formation of amyloid precursor protein (APP) in a subject are intended
 which comprise neuroactive steroids, such as estrone or
 17.beta.-estradiol, analogs of neuroactive steroids, a substance that is a
 ligand, an agonist, or an antagonist of a receptor that is coupled to a
 neuroactive steroid, or a compound that regulates the activity of
 cytosolic or nuclear receptors.
 COMPOSITIONS OF THE PRESENT INVENTION
 As should be apparent, the present invention also contemplates compositions
 comprising the active substances disclosed herein. Preferably, these
 compositions include pharmaceutical compositions comprising a
 therapeutically effective amount of one or more of the active compounds or
 substances along with a pharmaceutically acceptable carrier.
 As used herein, the term "pharmaceutically acceptable" carrier means a
 non-toxic, inert solid, semi-solid liquid filler, diluent, encapsulating
 material, formulation auxiliary of any type, or simply a sterile aqueous
 medium, such as saline. Some examples of the materials that can serve as
 pharmaceutically acceptable carriers are sugars, such as lactose, glucose
 and sucrose, starches such as corn starch and potato starch, cellulose and
 its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose
 and cellulose acetate; malt, gelatin, talc; excipients such as cocoa
 butter and suppository waxes; oils such as peanut oil, cottonseed oil,
 safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols,
 such as propylene glycol, polyols such as glycerin, sorbitol, mannitol and
 polyethylene glycol; esters such as ethyl oleate and ethyl laureate, agar;
 buffering agents such as magnesium hydroxide and aluminum hydroxide;
 alginic acid; pyrogen-free water; isotonic saline, Ringer's solution;
 ethyl alcohol and phosphate buffer solutions, as well as other non-toxic
 compatible substances used in pharmaceutical formulations.
 Wetting agents, emulsifiers and lubricants such as sodium lauryl sulfate
 and magnesium stearate, as well as coloring agents, releasing agents,
 coating agents, sweetening, flavoring and perfuming agents, preservatives
 and antioxidants can also be present in the composition, according to the
 judgment of the formulator. Examples of pharmaceutically acceptable
 antioxidants include, but are not limited to, water soluble antioxidants
 such as ascorbic acid, cysteine hydrochloride, sodium bisulfite, sodium
 metabisulfite, sodium sulfite, and the like; oil soluble antioxidants,
 such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated
 hydroxytoluene (BHT), lecithin, propyl gallate, aloha-tocopherol and the
 like; and the metal chelating agents such as citric acid, ethylenediamine
 tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid and the
 like.
 By a "therapeutically effective amount" or simply "effective amount" of an
 active compound, such as an analog of estrogen, is meant a sufficient
 amount of the compound to treat or alleviate the negative effects of a
 neurological disorder or neurodegenerative disease stemming from an
 increase in the level of expression, production, or formation of amyloid
 precursor protein (APP) at a reasonable benefit/risk ratio applicable to
 any medical treatment. It will be understood, however, that the total
 daily usage of the active compounds and compositions of the present
 invention will be decided by the attending physician within the scope of
 sound medical judgment. The specific therapeutically effective dose level
 for any particular patient will depend upon a variety of factors including
 the disorder being treated and the severity of the disorder; activity of
 the specific compound employed; the specific composition employed; the
 age, body weight, general health, sex and diet of the patient; the time of
 administration, route of administration, and rate of excretion of the
 specific compound employed; the duration of the treatment; drugs used in
 combination or coinciding with the specific compound employed; and like
 factors well known in the medical arts.
 The total daily dose of the active compounds of the present invention
 administered to a subject in single or in divided doses can be in amounts,
 for example, from 0.01 to 25 mg/kg body weight or more usually from 0.1 to
 15 mg/kg body weight. Single dose compositions may contain such amounts
 thereof to make up the daily dose. In general, treatment regimens
 according to the present invention comprise administration to a human or
 other mammal in need of such treatment from about 1 mg to about 1000 mg of
 the active substances of this invention per day in multiple doses or in a
 single dose of from 1 mg, 5 mg, 10 mg, 100 mg, 500 mg or 1000 mg.
 In certain situations, it may be important to maintain a fairly high dose
 of the active agent in the blood stream of the patient, particularly early
 in the treatment. Hence, at least initially, it may be important to keep
 the dose relatively high and/or at a substantially constant level for a
 given period of time, preferably, at least about six or more hours, more
 preferably, at least about twelve or more hours and, most preferably, at
 least about twenty-four or more hours.
 The compounds of the present invention may be administered alone or, in
 combination or in concurrent therapy with other agents which affect the
 central or peripheral nervous system, particularly selected areas of the
 brain.
 Liquid dosage forms for oral administration may include pharmaceutically
 acceptable emulsions, microemulsions, solutions, suspensions, syrups and
 elixirs containing inert diluents commonly used in the art, such as water,
 isotonic solutions, or saline. Such compositions may also comprise
 adjuvants, such as wetting agents; emulsifying and suspending agents;
 sweetening, flavoring and perfuming agents.
 Injectable preparations, for example, sterile injectable aqueous or
 oleaginous suspensions may be formulated according to the known art using
 suitable dispersing or wetting agents and suspending agents. The sterile
 injectable preparation may also be a sterile injectable solution,
 suspension or emulsion in a nontoxic parenterally acceptable diluent or
 solvent, for example, as a solution in 1,3-butanediol. Among the
 acceptable vehicles and solvents that may be employed are water, Ringer's
 solution, U.S.P. and isotonic sodium chloride solution. In addition,
 sterile, fixed oils are conventionally employed as a solvent or suspending
 medium. For this purpose any bland fixed oil can be employed including
 synthetic mono- or diglycerides. In addition, fatty acids such as oleic
 acid are used in the preparation of injectables.
 The injectable formulation can be sterilized, for example, by filtration
 through a bacteria-retaining filter, or by incorporating sterilizing
 agents in the form of sterile solid compositions, which can be dissolved
 or dispersed in sterile water or other sterile injectable medium just
 prior to use.
 In order to prolong the effect of a drug, it is often desirable to slow the
 absorption of a drug from subcutaneous or intramuscular injection. The
 most common way to accomplish this is to inject a suspension of
 crystalline or amorphous material with poor water solubility. The rate of
 absorption of the drug becomes dependent on the rate of dissolution of the
 drug, which is, in turn, dependent on the physical state of the drug, for
 example, the crystal size and the crystalline form. Another approach to
 delaying absorption of a drug is to administer the drug as a solution or
 suspension in oil. Injectable depot forms can also be made by forming
 microcapsule matrixes of drugs and biodegradable polymers, such as
 polylactide-polyglycoside. Depending on the ratio of drug to polymer and
 the composition of the polymer, the rate of drug release can be
 controlled. Examples of other biodegradable polymers include
 polyorthoesters and polyanhydrides. The depot injectables can also be made
 by entrapping the drug in liposomes or microemulsions, which are
 compatible with body tissues.
 Suppositories for rectal administration of the drug can be prepared by
 mixing the drug with a suitable nonirritating excipient, such as cocoa
 butter and polyethylene glycol which are solid at ordinary temperature but
 liquid at the rectal temperature and will, therefore, melt in the rectum
 and release the drug.
 Solid dosage forms for oral administration may include capsules, tablets,
 pills, powders, gel caps and granules. In such solid dosage forms the
 active compound may be admixed with at least one inert diluent such as
 sucrose, lactose or starch. Such dosage forms may also comprise, as is
 normal practice, additional substances other than inert diluents, e.g.,
 tableting lubricants and other tableting aids such as magnesium stearate
 and microcrystalline cellulose. In the case of capsules, tablets and
 pills, the dosage forms may also comprise buffering agents. Tablets and
 pills can additionally be prepared with enteric coatings and other
 release-controlling coatings.
 Solid compositions of a similar type may also be employed as fillers in
 soft and hardfilled gelatin capsules using such excipients as lactose or
 milk sugar as well as high molecular weight polyethylene glycols and the
 like.
 The active compounds can also be in micro-encapsulated form with one or
 more excipients as noted above. The solid dosage forms of tablets,
 capsules, pills, and granules can be prepared with coatings and shells
 such as enteric coatings and other coatings well known in the
 pharmaceutical formulating art. They may optionally contain opacifying
 agents and can also be of a composition that release the active
 ingredient(s) only, or preferably, in a certain part of the intestinal
 tract, optionally in a delayed manner. Examples of embedding compositions
 which can be used include polymeric substances and waxes.
 Dosage forms for topical or transdermal administration of a compound of
 this invention further include ointments, pastes, creams, lotions, gels,
 powders, solutions, sprays, inhalants or patches. The active component is
 admixed under sterile conditions with a pharmaceutically acceptable
 carrier and any needed preservatives or buffers as may be required.
 Ophthalmic formulations, ear drops, eye ointments, powders and solutions
 are also contemplated as being within the scope of this invention.
 The ointments, pastes, creams and gels may contain, in addition to an
 active compound of this invention, excipients such as animal and vegetable
 fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives,
 polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc
 oxide, or mixtures thereof.
 Powders and sprays can contain, in addition to the active compounds of this
 invention, excipients such as lactose, talc, silicic acid, aluminum
 hydroxide, calcium silicates and polyamide powder, or mixtures of these
 substances. Sprays can additionally contain customary propellants, such as
 chlorofluorohydrocarbons.
 Transdermal patches have the added advantage of providing controlled
 delivery of active compound to the body. Such dosage forms can be made by
 dissolving or dispersing the compound in the proper medium. Absorption
 enhancers can also be used to increase the flux of the compound across the
 skin. The rate can be controlled by either providing a rate controlling
 membrane or by dispersing the compound in a polymer matrix or gel.
 Accordingly, the present invention is useful in the treatment or
 alleviation of disease, especially those disorders related to neurological
 diseases or neurodegenerative disorders, such as Alzheimer's disease,
 Parkinson's disease, Lou Gehrig's disease, multiple sclerosis, central or
 peripheral nervous system damage, dysfunction, or complications involving
 same stemming from edema, injury, or trauma, or neurodegenerative changes
 in postmenopausal women. Such damage, dysfunction, or complications may be
 characterized by an apparent neurological, neurodegenerative,
 physiological, psychological, or behavioral aberrations, the symptoms of
 which can be reduced by the administration of an effective amount of the
 active compounds or substances of the present invention.
 The following examples are provided for further illustration of the present
 invention, and do not limit the invention.
 EXAMPLES
 Experiments and exemplary procedures are described below which provide
 additional enabling support for the present invention. In particular, in
 vitro studies using primary cultures of rat cortical astrocytes and human
 glioma cells are disclosed.
 General Methods
 Neurons and astrocytes are isolated from cortices from embryonic and
 postnatal rats, respectively. See, K. D. McCarthy and J. de Vellis, J.
 Cell Biol. 85, 890 (1980). In brief, dissected cortices were dissociated
 by trypsinization and trituration through a flame-polished Pasteur
 pipette. Cells were plated onto poly-L-lysine coated 35- or 100 mm culture
 dishes at densities of about 10-25 cells/mm.sup.2. The initial, culture
 media, minimal essential medium (MEM, Gibco) containing 10% horse serum
 (BioWhittaker), were aspirated after 2-5 h after plating to remove
 unattached cells and debris, and replaced with MEM containing 7.5% fetal
 bovine serum (FBS, BioWhittaker). Half the media was replaced with
 MEM/7.5% FBS twice weekly. Cells are kept at 37.degree. C. in a humidified
 5% CO.sub.2 /95% air incubator. Media are changed twice weekly.
 Immunocytochemical staining with antibodies against GFAP and tau shows
 that &gt;90% of cultured cells are astrocytes and &lt;5% are neurons.
 Pharmacological manipulations are performed in serum-free media on 7-14
 DIV confluent astrocytes.
 Detection of Cell-Associated Protein
 To detect cell-associated protein (APP or GFAP) astrocytes from 35 mm
 dishes are scraped in lysis buffer (60 mM Tris/HC1, 4% SDS, 20% glycerol,
 1 mM dithiothreitol), ultrasonicated and boiled for 5 min. The total
 amount of cell protein per dish, estimated using the bicinchoninic acid
 assay, is not altered by pharmacological treatments. Bromphenol blue
 (0.1%) is added to each sample and equal amounts of protein (.about.75
 mg/lane) are loaded on 10% SDS-polyacrylamide gels.
 To detect secreted APP, culture media was collected after drug treatments
 and phenylmethylsulfonyl fluoride was added to a final concentration of 2
 mM. The media samples were then applied to Sephadex PD-10 desalting
 columns (Pharmacia) and eluted with distilled water. Column eluates were
 frozen and dried by vacuum centrifugation. The lyophilized proteins were
 reconstituted in 25 .mu.L water followed by 25 .mu.L of 2.times.Laemmli
 gel loading buffer, and boiled for 5 min.
 The amount of media or cell protein loaded for sodium dodecyl
 sulfate-polyacrylamide gel electrophoresis (10-20% SDS PAGE; Bio-Rad) was
 Formalized for the amount of protein per sample. Proteins (equivalent to
 .about.100 .mu.g cell protein/lane) were separated by electrophoresis,
 electroblotted onto polyvinylidene difluoride membranes (Immobilon-P,
 Millipore) and blocked in Tris-buffered saline with 0.15% Tween 20 (TBST)
 containing 5% powdered milk for 30 min. After 2.times.10 min rinses in
 TBST, the membranes were incubated in TBST containing an appropriate
 antibody. Monoclonal antibodies 22Cll and GFAP (both from
 Boehringer-Mannheim) were used to detect the N-terminus of A. P. and glial
 fibrillary acidic protein respectively; antisera R37 and R98 (gifts of Dr.
 F. Kametani, Tokyo Institute of Psychiatry) were used to detected the
 C-terminus and KPI motifs of APP, respectively; antiserum C8 (gift of Dr.
 D. Selkoe, Women's Hospital, Harvard Medical School, Cambridge, Mass.) was
 used to detect the C-terminus of APP.
 After an overnight incubation, membranes were rinsed in TBST before being
 treated for 1h with a peroxidase-linked secondary antibody. After several
 rinses in TBST, protein bands were visualized on Kodak X-AR films by an
 enhanced chemiluminescence method (Amersham). Optical densities of the
 protein bands were quantitated by laser scanning densitometry (LKB,
 Bromma, Sweden), and normalized to the densities of those bands generated
 under control conditions.
 cAMP Assay
 Levels of cyclic AMP were measured with [8-.sup.3 H]- cAMP assay kit
 (Amersham TRK 432) in astrocytes grown on 35 mm dishes. In brief, after
 aspirating the medium and rinsing twice with 1 ml ice cold PBS, the cells
 were scraped in 0.8 ml ice cold ethanol and sonicated. The cell suspension
 was incubated for 5 min at room temperature, centrifuged and the
 supernatant was dried in a rotary evaporator. After resuspension in 120
 .mu.l Tris/EDTA buffer, two duplicate samples of 50 .mu.l each were mixed
 with the binding protein, [8-.sup.3 H] adenosine 3', 5'-cyclic phosphate
 tracer and incubated at 2-4.degree. C. for 2 h. A charcoal suspension (100
 .mu.l) was added to the samples before centrifugation and 200 .mu.l of the
 supernatant were removed for scintillation counting. The amount of cyclic
 AMP (pmol/mg protein) was estimated by comparing to known standards, and
 normalized to the amounts of whole cell protein as determined by the
 bicinchoninic acid assay (Sigma).
 Data Analysis
 Measurements of cellular and secreted proteins, or of mRNA in treatment
 groups were normalized against those of control groups which were prepared
 in parallel and loaded onto the same blot. Analysis of variance (ANOVA)
 and t-tests were used to evaluate differences between groups (significance
 level, p=0.05), using drug treatments as the independent variable.
 Analysis of RNA
 Total RNA from astrocytes grown on 100 mm dishes is extracted by the acid
 guanidium thiocyanate-phenolchloroform method. See, P. Chomcznski and N.
 Saachi, Anal. Biochem. 162, 156 (1987). In brief, the medium was aspirated
 and the cells were scraped in 1 mL of TRI Reagent. After incubation for 15
 min at room temperature, 0.2 ml chloroform was added, mixed vigorously
 with TRI Reagent and the mixture was stored for another 15 min at room
 temperature. After centrifugation at 12,000 g for 15 min, 0.5 ml
 isopropanol was added to the aqueous phase of the mixture to precipitate
 RNA. The RNA pellet collected by centrifugation (12,000 g, 15 min at
 4.degree. C.) was washed with 70% ethanol once and solubilized in an
 appropriate amount of Formazol (Molecular Research Center, Cincinnati,
 Ohio). RNA samples (-20 .mu.g) were denatured by heating for 15 min at
 60.degree. C. prior to loading onto 1.2% agarose-formaldehyde gels for
 electrophoresis. RNA was blotted onto Hybond polyvinyl membranes by
 overnight capillary transfer and fixed onto the membranes by UV light
 illumination. Membranes were pre-hybridized with Amersham Rapid-hyb
 (Amersham Lab, Arlington Heights, Ill.) buffer for 2 h and labeled
 overnight with a .about.1.8 kb human APP cDNA (gift of Dr. Rachael Neve,
 McLean Hospital, Harvard Medical School, Belmont, Mass.) or human
 glyceraldehyde-3-phosphate dehydrogenase probe (G3PDH; Clontech) labeled
 with (.sup.32 P]DCTP using random primed extension (Amersham Megaprime DNA
 labeling kit). Membranes were dried and exposed to Kodak X-ray film for
 24-48 h with an Amersham enhancer sheet. The relative amounts of mRNA
 obtained by hybridization were estimated using densitometric analysis of
 autoradiographs. The levels of APP mRNA were normalized to the amounts of
 G3PDH mRNA and expressed as a ratio to the levels of untreated, control
 cells.
 Exposure of Astrocytes, Neurons, and Human Glioma Cells to Lipophilic
 Estrogenic Compounds
 Confluent monolayers primary rat cortical neurons, astrocytes- prepared
 according to Example 5.1- and human glioma cells (HS683) were treated over
 24 hours with 1, 10, or 100 .mu.M of a lipophilic estrogenic compounds,
 such as estrone or 17.beta.-estradiol. The result shows a decrease in the
 level of APP holoprotein synthesis in the brain cells treated with
 lipophilic estrogenic compounds, as compared to those levels that are
 observed in untreated, control cells. See, e.g., FIGS. 1, 2, and 3. The
 levels of APP holoprotein associated with cultured brain cells (e.g.
 astrocytes or neurons) and HS683 cells were measured with an antisera R37
 (gift of Dr. F. Kametani, Tokyo Inst. of Psychiatry) or with monoclonal
 antibody 22C11 (Boehringer-Mannheim) directed against the C-terminus or
 N-terminus of APP, respectively. Using Western blot analysis, it is found
 that the total cell protein content as measured by the bicinchoninic acid
 assay was not changed by these estrogenic compounds in the range of
 concentrations tested.
 In Vivo Studies
 The present studies indicate that lipophilic hormones that interact with
 cytosolic or nuclear receptors reduce the level of APP holoprotein
 synthesis in rats and human's brain cells. Reactive astrocytes (that is,
 astrocytes that have been activated or stimulated in some fashion, e.g.,
 those associated with brain or neuronal injury) in vivo also up regulate
 GFAP expression. Indeed, the examination of post-mortem brains in patients
 with Alzheimer's disease shows that reactive astrocytes are found in
 proximity to amyloid plaques and regions of neurodegeneration. The
 inventors believe that neuronal, brain, or head injury gives rise to the
 formation of reactive astrocytes, which over express APP and contribute to
 the formation of amyloid or neurotoxic APP derivatives.
 Thus, animal models of head injury and Alzheimer's disease exhibit
 increased amounts of APP in the brain. The in vivo administration of a
 lipophilic hormone, like estrone, is found to inhibit APP overexpression
 and the associated neurological disorders. Inasmuch as estrogen deficiency
 in postmenopausal women is associated with a higher incidence of
 Alzheimer's disease, this discovery opens the possibility that estrogen
 therapy may prevent some of the neurodegenerative and cognitive changes
 associated with postmenopausal women, Alzheimer's disease, aging and other
 disease conditions associated with such neurodegenerative and cognitive
 decline.
 CONCLUSION
 Accordingly, the invention provides compositions and methods for
 preventing, alleviating, or inhibiting abnormal APP synthesis, or reducing
 APP expression, by the administration of lipophilic hormones that interact
 with cytosolic or nuclear receptors. Particularly where the upregulation
 of such receptors and APP overexpression accompanies brain trauma,
 neurological disease, or neurodegenerative disorder.
 Hence, the invention provides relief from the neuropathological symptoms of
 diseases, such as Alzheimer's disease, or postmenopausal neurodegenerative
 or cognitive syndromes by inhibiting aberrant APP gene expression, and
 reducing the level of APP holoprotein synthesis. It is the overexpression
 of APP that is believed to cause or strongly contribute to
 neurodegeneration and cognitive dysfunction in animals and humans.
 It should be apparent to those of ordinary skill that the discussion
 presented herein adequately supports the hypothesis that a reduction in
 APP synthesis (as evidenced by decrease in APP DNA transcription and
 translation) can be achieved by regulating cytosolic or nuclear receptors.
 It should also be apparent that other embodiments of the invention can be
 readily contemplated by those of ordinary skill in the art after reviewing
 the present specification and teachings. The present invention is not
 limited, however, to the specific embodiments presented herein and should
 not be construed so narrowly as to exclude embodiments that fall within
 the scope and spirit of the invention, which invention is limited solely
 by the following claims.