Classifications

• • 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
  • A61K31/567—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 substituted in position 17 alpha, e.g. mestranol, norethandrolone
• • 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
  • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
  • A61K31/05—Phenols
• • 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
  • A61K31/075—Ethers or acetals
  • A61K31/085—Ethers or acetals having an ether linkage to aromatic ring nuclear carbon
• • 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
  • A61K31/11—Aldehydes
• • 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
• • 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
  • A61K31/566—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 having an oxo group in position 17, e.g. estrone

Definitions

• the present invention relates to compositions and methods for protecting cells in the central nervous system of subjects from cell death and for stimulating neuronal survival in subjects with neurodegenerative conditions.
• Pathological conditions resulting from the accelerated or ongoing death of neurons are prevalent in today's society and include chronic diseases such as Alzheimer's disease and Parkinson's disease, acute diseases such as stroke, brain cell loss that follows myocardial infarction, and acute neuronal injury associated with spinal cord trauma and head trauma.
• Chronic and acute neurodegenerative diseases and acute neuronal injury as well as associated mortality and morbidity are largely untreatable.
• the consequences of patient disability resulting from these conditions is a high cost to society of patient care as well as a significant reduction in quality of life. Effective therapeutic approaches directed to the prevention or reduction of neuron death or damage associated with the above conditions are needed.
• the greatest challenge in the development of therapeutic agents for treating conditions in the brain resulting from neuron loss include obtaining an efficacious drug that is relatively non-toxic, suitable for use in both females and males, and which can readily access the brain across the blood-brain barrier.
• Estrogen compounds have been found to protect neurons from cell death and have utility in retarding the progression of neurodegenerative diseases such as Alzheimer's disease.
• Simpkins et al. WO 95/12402 has shown that alpha isomers of estrogen compounds, previously thought to be biologically inert, are effective in retarding neurodegeneration. This demonstration provided for the first time an opportunity to administer estrogen therapeutically to men without associated sex-related side effects.
• estrogen compounds bring about a neuroprotective effect are unknown although these compounds have been shown to have a number of different physiological and biochemical effects on neurons. For example, estrogen has been shown to stimulate the production of neurotrophic agents that in turn stimulate neuronal growth. (REF) Estrogen compounds have also been found to inhibit NMDA- induced cell death in primary neuronal cultures (Bahl et al. Biochem. Biophys Res. Commun. (1995) 216: 973; Goodman et al. J. Neurochem (1996) 66: 1836), and further to be capable of removing oxygen free radicals and inhibiting lipid peroxidation. (Droescher et al. WO 95/13076).
• Droescher et al. used very high doses of estrogens. Such doses, even if effective on neurons in vivo, would have limited utility in treating chronic neurological conditions because of associated problems of toxicity that result from prolonged use of high dosages.
• the present invention satisfies the above stated need for a class of compounds that is effective in protecting neurons from deterioration and cell death arising from disease, trauma or aging and may be used to achieve a similar effect in male and female subjects with minimal adverse side effects.
• a method for conferring neuroprotection on a population of cells in a subject having the steps of providing a non ⁇ estrogen compound, having a terminal phenol group in a structure containing at least a second ring having a molecular weight that is less than 1000 Daltons; and administering the compound in an effective dose to the population of cells to confer neuroprotection.
• a method of treating a neurodegenerative condition in a subject includes the steps of selecting an effective dose of a compound having a terminal phenol ring having a molecular weight less than 1000D and at least one additional ring structure covalently linked to the phenol ring; and administering the compound to the subject.
• the compound used in the method may have a four-ring structure, a three-ring structure or a two-ring structure where the four- ring structure may be administered at an effective dose that achieves a plasma concentration of less than 500nM.
• the molecular weight of the compound may be greater than 170 D.
• the three ring structure is a phenanthrene compound which may further be selected from the group consisting of tetrahydro- phenanthrene and a octahydrophenanthrene more particularly a phenanthrenemethanol or a phenanthrencarboxyaldehyde.
• the two- ring structure may be fused and include a naphthol and naphthalene or may be a non-fused two ring structure having a linkage group.
• the terminal phenol ring includes non-steroidal compounds .
• Embodiments of the invention utilize a compound having a phenolic A ring.
• the dosage of the neuroprotective compound results in a plasma concentration of less than 500nM, more particularly in the range of .02nM-500nM and more particularly in the range of OJnM-lnM.
• Figure 1 shows the effects of 3J7 ⁇ -estradiol and 3J7 ⁇ -estradiol 3-O-methyl ether on the % change in live SK-N-SH cell number at 48 hours of serum deprivation.
• Raw data were compared to the respective serum-free control group by analysis of variance and Scheffe's F test and data were then normalized to the serum free group
• Figure 3 shows the effects of 3J7 ⁇ -estradiol, estriol, and 17 ⁇ ethynyl 3J7 ⁇ - estradiol (ethynyl estradiol) and their 3-O-methyl ethers; estriol 3-O-methyl ether (estriol 3-O-ME) and ethynyl estradiol 3-O-methyl ether (ethynyl estradiol 3-O-ME), all at a concentration of 2nM, on live SK-N-SH cell number at 48 hrs of serum deprivation.
• ** p ⁇ 0.05 versus all other groups. Data are expressed as mean + SEM for 5 wells/group.
• DES diethylstilbestrol
• DES mono-O-ME DES mono- O-methyl ether
• DES Di-O-ME DES di-O-methyl ether
• Figure 6a shows the effects of [2S-(2a,4a . 10a ⁇ ]- 1.2.3,4,4a,9J0J0a-octahydro-
• Figure 7 shows the effects of treatment of 3J7 ⁇ -estradiol (0.2 or 2nM) and 3J7 ⁇ - estradiol (2nM) on the toxicity induced by the neurotoxic fragment of the ⁇ amyloid protein (A ⁇ 25-35) (20 ⁇ m) on neuronal cells.
• Figure 8 shows the effect of treatment of ovariectomized rats with sham injection (oil) or with 3J7 ⁇ -estradiol at 100 ⁇ g/kg body weight by subcutaneous injection, at 2 hours before occlusion of the middle cerebral artery.
• the maximum lesion size in brain was recorded and is shown as % of the cross sectional area of the brain.
• A,B,C,D and E correspond to brain sections at 7,9,11,13, and 15 mm respectively posterior to the olfactory bulb.
• Figure 9 shows the effects of progesterone at 2nM on SK-N-SH live cell number at 48 hours of serum deprivation. Data are expressed as mean + SEM for 6 wells/group.
• Figure 10 shows the effects of corticosterone at 2nM on SK-N-SH live cell number at 48 hours of serum deprivation. Data are expressed as mean + SEM for 6 wells/group.
• Figure 11 shows the structures of 3-ring compounds: [2S-(2a,4a ⁇ J0a ⁇ )]- 1,2,3,4 ,4a,9J0J0a-octahydro-7-hydroxy-2-methyl-2-phenanthrenemethanol (PAM) and [2S-(2a,4a ⁇ J0a ⁇ )]-l,2,3,4,4a,9J0J0a-octahydro-7-hydroxy-2-methyl-2- phenanthrenecarboxaldehyde (PACA).
• Figure 12 shows the generalized core ring structures with numbered carbons (a) 4-ring structure, (b) 3-ring structure, (c) 2-ring structure (fused) (d) 2 ring structure (non- fused).
• Neuroprotection is defined here and in the claims as the inhibition of progressive deterioration of neurons that leads to cell death.
• a non-estrogen compound is defined here and in the claims as a compound, other than an estrogen compound, described in the 11 th Edition of "Steroids" from Steraloids Inc., Wilton, N.H.
• a phenol ring is referred to as "terminal" here and in the claims, when it is the ultimate carbon ring on a molecule consisting of more than 1 carbon ring.
• a steroid is defined here and in the claims as a compound having numbered carbon atoms arranged in a 4-ring structure (J. American Chemical Society 82: 5525-5581 (1960) and Pure and Applied Chemistry 31: 285-322 (1972)).
• 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.
• 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.”
• the present invention identifies a method of neuroprotection that utilizes a novel class of neuroprotective compounds, where the class of compounds is identified according to a set of features that has here been recognized for the first time as determinative of neuroprotection. This method is further suited for the treatment of neurodegenerative diseases, trauma and aging in human subjects.
• the protection of 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.
• the method of the invention may be of significant therapeutic use.
• Other diseases for which such a method 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 the observed cytoprotective properties, it is suggested that one pathway of action for the polycyclic phenolic compounds is the inhibition of apoptosis.
• the characteristic set of features that define the class of neuroprotective compounds include (a) the presence of two or more ring structures in the compound where the compound has a size range less than 1000 Daltons; (b) a terminal phenol ring and (c) an effective dose in vivo for causing a neuroprotective effect.
• the class of neuroprotective compounds described here includes (a) compounds that are characterized by two-carbon rings (Table II); (b) compounds that are characterized by three-carbon rings ( Figure 11, 12); and steroids that are characterized by 4-carbon rings, (Figure 12). Neuroprotective compounds may further comprise 5-carbon rings or more providing that the overall molecular weight of 1000 Daltons is not exceeded.
• an assay has been used as disclosed in Example 1, that utilizes SK-N-SH neuronal cells under stress to determine neuroprotection by test compounds.
• 3J7 ⁇ -estradiol has been selected as the control, because of its previously demonstrated neuroprotective effects (Simpkins WO 95/12402).
• the neuroprotective activity of the control compound is shown in Figure 1. It can be seen from Figure 1 , that at 48 hrs of serum deprivation, the percentage of live SK-N-SH cells increases by 100% in the presence of 2nM 3J7 ⁇ -estradiol.
• This invention teaches that neuroprotection can be achieved at an effective dose providing low plasma concentrations of polycyclic phenolic compounds. More specifically, a neuroprotective effect can be achieved at plasma concentrations of less than
• Droescher et al. describe an IC 50 of 12.8 ⁇ M estrogen to inhibit free radical oxidation in a lipid peroxidation assay.
• the indirect lipid peroxidation assay for cell protection used by Droescher et al. is a biochemical assay and is not comparable to the neuron cell assay used in the invention to directly measure a cellular response. Consequently, the two assays cannot be directly compared and the results of one assay cannot be extrapolated to the other.
• the advantage of the neuroprotection assay used here is that live and dying cells are utilized to determine neuroprotection directly. It is desirable that the amount of a neuroprotective agent to be used to treat a subject, should be within the range that is relatively non-toxic to the patient especially when the compound is used long-term. According to the invention, significant neuroprotection has been obtained in neuronal cell cultures at concentrations of 2nM ( Figures 1-6) and furthermore, significant neuroprotection has been achieved in rats at lOO ⁇ g/kg body weight, (a dose of compound at lOO ⁇ /kg body weight provides approximately 0.4nM-2nM plasma concentration of the compound. ( Figure 8).
• neuroprotective compounds for use in the method of the invention require a terminal phenolic ring. These compounds may have an R group substitution on any suitable carbon on the terminal phenolic ring other then the carbon bearing the hydroxyl group and these R groups may be present in or ⁇ isomeric configurations. Furthermore, phenol on its own is not neuroprotective nor are straight chain substitution of phenolic compounds (Table III). However, a compound having a terminal phenolic ring and at least one other carbon ring is neuroprotective. For example, the non-fused two- ring structure, diethyl stilbestrol, has demonstrated neuroprotective properties according to the invention (Table ⁇ , Figure 4). This compound has a terminal phenolic ring structure that is associated with a second phenolic ring via a linkage group.
• compounds that are non-steroidal and have a terminal phenolic ring and at least two additional carbon ring structures include three-ring compounds ( Figures 11, 12) such as exemplified by [2S-(2a,4a ⁇ J0 ⁇ )]- 1,2,3, 4,4a,9J0J Oa-octahydro- 7hydroxy-2-methyl-2-phenanthrenemethanol (PAM) and [2S-(2a, 4a ⁇ , 10 ⁇ )]- l,2,3,4,4a,9J0J0a-octahydro-7hydroxy-2-methyl-2-phenanthrenecarboxyaldehyde (PACA) have been demonstrated to have a neuroprotective effect (see Figure 5 and 6, 6a and 6b).
• the structure of these compounds are shown in Figure 12. Two namomolar concentrations of either PAM or PACA were found to permit an increase in neuron cell survival of about 15%. Compounds having a terminal phenolic ring and at least three additional carbon rings have been shown to have a neuroprotective effect as exemplified by 3J7 ⁇ -estradiol. ( Figure 1).
• the upper size limit of a compound of the invention that is neuroprotective and has a terminal phenolic ring depends not so much on the number of carbon rings in the structure but rather whether the compound is of a sufficiently small size to permit crossing of the blood brain barrier.
• the recommended route of administration of the neuroprotective compound includes oral, intramuscular, transdermal, buccal, intravenous and subcutaneous. Methods of administering the compound of the invention may be by dose or by controlled release vehicles.
• the preferred embodiment of the invention includes a compound having a terminal phenolic ring and at least a second carbon ring.
• the compound may have a number of R groups attached to any available site on the phenolic ring or elsewhere providing that the phenolic structure of the terminal ring is maintained.
• R-groups may be selected from inorganic or organic atoms or molecules. Below, examples of a number of different types of R groups have been provided although the invention is not limited by these examples.
• the R group may include any inorganic R group including any of a halogen, an amide, a sulfate, a nitrate, fluoro, chloro, or bromo groups . Additionally, R groups selected from sodium, potassium and /or ammonium salts may be attached to the alpha or beta positions to replace hydrogen on any available carbon in the structure.
• the R-group may be organic or may include a mixture of organic molecules and ions.
• Organic R groups may include alkanes, alkenes or alkynes containing up to six carbons in a linear or branched array.
• additional R group substituents may include methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, dimethyl, isobutyl, isopentyl, tert-butyl, sec-butyl, isobutyl, methylpentyl, neopentyl, isohexyl, hexenyl, hexadiene, 1 ,3-hexadiene-5-yne, vinyl, allyl, isopropenyl, ethynyl, ethylidine, vinylidine, isopropylidene; methylene, sulfate, mercapto, methylthio, ethylthio, propylthio, methylsulfinyl, methylsulfonyl, thiohexanyl, thiobenyl, thiopenol, thiocyanato, sulf
• R groups may be attached to any of the constituent rings to form a pyridine, pyriazine, pyrimidine, or v-triazine. Additional R group substituents may include any of the six member or five member rings itemized in section b below.
• any compound having in addition to the phenol A ring, a heterocyclic carbon ring which may be an aromatic or non-aromatic phenolic ring with any of the substitutions described in (a) above and further may be selected from for example, one or more of the following structures- phenanthrene, naphthalene, napthols, diphenyl, benzene, cyclohexane, 1,2-pyran, 1,4-Pyran, 1,2-pyrone, 1,4-pyrone, 1,2-dioxin, 1,3-dioxin (dihydro form), pyridine, pyridazine, pyrimidine, pyrazine, piperazine, s-triazine, as- triazine, v-triazine, 1,2,4-oxazine, 1,3,2-oxazine, 1,3,6-oxazine (pentoxazole), 1,2,6 oxazine, 1,4-oxazine, o
• any of the above carbon ring structure may be linked directly or via a linkage group to any further heterocyclic aromatic or non aromatic carbon ring including: furan; thiophene (thiofuran); pyrrole (azole); isopyrrole (isoazole); 3-isopyrrole (isoazole); pyrazole (1,2- daizole); 2-isoimidazole (1,3-isodiazole); 1,2,3-triazle; 1,2,4 triazole; 1,2-diothiole; 1,2,3- oxathiole, isoxazole (furo(a) monozole); oxazole (furo(b) monazole); thiazole; isothiazole; 1,2,3-oxadiazole; 1,2,4-oxadiazole, 1,2,5-oxadiazole, 1,3,5 oxadiazole, 1,2,3,4-oxatiazole; 1,2,3,5-oxatriazole; 1,2,3-d
• Any compound in the form of a prodrug, that may be metabolized to form an active polycyclic phenolic compound having neuroprotective activity may include the use of a single compound or a mixture of neuroprotective compounds.
• Example 1 Assay to identify neuroprotective compounds
• the cell line SK-N-SH, a well characterized human neuroblastoma cell line, was used to test potential neuroprotective drugs. This cell line is widely considered representative of neuronal cells and an appropriate assay system for evaluation of pharmaceutical neuroprotective drugs for human diseases and injury.
• SK-N-SH cells were obtained from American Type Tissue Collection (Rockville, MD). Cell cultures were grown to confluency in RPMI- 1640 media supplemented with 10% Fetal Bovine Serum (FBS), lOOU/ml penicillin G, and 100 mg/ml streptomycin at
• the neuroprotection assay involved continuous exposure of SK-N-SH cells for 48h to conditions of serum deprivation and to 2nM concentrations of each test compound. This concentration is at least 10-fold higher than that required for the neuroprotective effects of 3J7 ⁇ - and 3J7 -estradiol . 3J7 ⁇ -estradiol was used as a control. This compound caused a dose-dependent protection of SK-N-SH cells under conditions of serum deprivation with an ED 50 of 0J3 nM and significant neuroprotection at the 0.2nM concentration (Fig 1). This effect was robust with the 2 nM concentration of 3J7 ⁇ - estradiol showing neuroprotection in 8 separate trials ( Figures 1 to 4).
• Neuroprotection was determined by the magnitude of the difference in live cell number in the treated wells versus the cell number in the serum free wells. The statistical analysis was performed on the raw data in each experiment. The significance of differences among groups was determined by one way analysis of variance. Planned comparisons between groups were done using Scheffe's F-test. For all tests, p ⁇ 0.05 was considered significant. Following the analysis, data were normalized to the % response of the 3.17 ⁇ -estradiol group for each study using the following calculation:
• % Neuroprotectivity Test cell # - Serum-Free Cell #
• Cell viability was assessed at 48 h of treatment using the Trypan Blue dye- exclusion method. At the appropriate time, cell suspensions were made by decanting media, rinsing each well with 0.2 ml, 0.02% EDTA. and incubating cells with 0.2% ml,
• Example 2 Comparison of the neuroprotection afforded by different four-ring and two- ring compositions.
• the two androgens containing a C-3 ketone namely the partially unsaturated testosterone that lacks a phenolic A ring, and the saturated compound that lacks a phenolic A ring, dihydrotestosterone, were both inactive.
• all five of the 21- carbon pregnane derivatives that were tested contained a C-3 ketone function, and three ⁇ 4 -steroids, progesterone and aldosterone and two ⁇ 1,4 -steroids, prednisolone and 6- methylprednisolone were inactive .
• cholesterol was tested because it has a 3- hydroxyl function on a completely saturated A ring and was inactive.
• the conformational shape of the flat, phenolic ring and/or the enhanced acidity of phenols relative to cyclohexanols may be important in conferring the observed neuroprotective activity.
• RPMI- 1640 media serum Free, SF group
• RPMI- 1640 media supplemented with 10% FBS FBS group
• RPMI- 1640 media supplemented one of the following steroids at a concentration of 2nM (unless otherwise noted): 3,17 ⁇ -estradiol (l,3,5(10)-estratriene-3J7 ⁇ -diol); 3,17 ⁇ -estradiol (1,3,5(10)- estratriene-3 ⁇ J7 ⁇ -diol); 3J7 ⁇ -estradiol 3-O-methyl ether (l,3,5(10)-estratriene-3J7 ⁇ - diol 3-O-methyl ether); 3J7 ⁇ -estradiol 3 acetate (l,3,5(10)-estratriene-3 ⁇ ,17 ⁇ -diol 3- acetate); estrone (l,3,5(10)-estratriene-3-ol-17-one); estrone 3-O-methyl ether (1,3,5(10)-estratriene-3-ol-17
• Example 3 Three ring structures with neuroprotective properties.

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