WO1997003661A1 - Use of non-estrogen polycyclic phenol compounds for the manufacture of a medicament for conferring neuroprotection to cells - Google Patents

Use of non-estrogen polycyclic phenol compounds for the manufacture of a medicament for conferring neuroprotection to cells Download PDF

Info

Publication number
WO1997003661A1
WO1997003661A1 PCT/US1996/012146 US9612146W WO9703661A1 WO 1997003661 A1 WO1997003661 A1 WO 1997003661A1 US 9612146 W US9612146 W US 9612146W WO 9703661 A1 WO9703661 A1 WO 9703661A1
Authority
WO
WIPO (PCT)
Prior art keywords
compound
ring
group
estradiol
cells
Prior art date
Application number
PCT/US1996/012146
Other languages
French (fr)
Inventor
James W. Simpkins
Patti S. Green
Katherine Gordon
Original Assignee
University Of Florida Research Foundation, Incorporated
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University Of Florida Research Foundation, Incorporated filed Critical University Of Florida Research Foundation, Incorporated
Priority to EP96924692A priority Critical patent/EP0841906A1/en
Priority to JP9506961A priority patent/JPH11510144A/en
Priority to AU65079/96A priority patent/AU6507996A/en
Publication of WO1997003661A1 publication Critical patent/WO1997003661A1/en

Links

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.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Steroid Compounds (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

The invention relates to the use of non-estrogen compounds having a terminal phenol group in a structure containing at least a second ring and having a molecular weight of less than 1000 Daltons (e.g. naphthols, phenanthrenes or steroid) for the manufacture of a medicament for conferring neuroprotection to cells in a subject.

Description

USE OF NON-ESTROGEN POLYCYCLIC PHENOL COMPOUNDS FOR THE MANUFACTURE OF A MEDI¬ CAMENT FOR CONFERRING NEUROPROTECTION TO CELLS
TECHNICAL FIELD
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.
BACKGROUND
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. At present, 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, Behl et al. ( 1995) Biochem. Biophys. Res. Commun. 216: 473-482;: Bishop et al. (1994) Molecular and Cellular Neuroscience 5: 303-308; Simpkins et al. (1994) Neurobiology of Aging 15: sl95-sl97). Furthermore, 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.
The mechanisms by which 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). However, the potential effect of free radicals on neurons per se is unproven. Droeschler et al. describes a cell free 'in vitro' assay systems using lipid peroxidation as an endpoint in which several estrogens as well as vitamin E were shown to have activity. Estradiol has also been reported to reduce lipid peroxidation of membranes (Niki (1987) Chem. Phys. Lipids 44: 227; Nakano et al. Biochem. Biophys. Res. Comm. (1987) 142: 919; Hall et al. J. Cer. Blood Flow Metab. (1991)1 1: 292. Other compounds including certain 2 l-amino steroids and a glucocorticosteroid have been found to act as anti-oxidants and have been examined for their use in spinal cord injury as well as head trauma, ischemia, and stroke. (Wilson et al. ( 1995) J. Trauma 39: 473; Levitt et al. (1994) J. Cardiovasc. Pharmacol 23: 136; Akhter et al. (1994) Stroke 25; 418). As described above, a number of factors may be involved in neuroprotection. Therapeutic agents that are selected on the basis of a single biochemical mechanism may have limited generalized utility in treating disease or trauma in patients. For example, in order to achieve an anti-oxidant effect in vitro using estrogen, 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.
It would be beneficial to identify a class of compounds that are non-sex related and have demonstrated biological efficacy in protecting neurons from cell death, where such compounds could be used in the treatment of the chronic as well as the acute conditions caused by neurodegenerative diseases, trauma, and aging at non-toxic dosages. An understanding of the structural requirements for compositions capable of inducing neuroprotection would provide the basis for designing novel drugs that have enhanced neuroprotective properties while at the same time having reduced adverse side effects .
SUMMARY
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.
In a preferred embodiment of the invention, a method is provided 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.
In another embodiment of the invention, a method of treating a neurodegenerative condition in a subject is provided, which 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.
In another embodiment of the invention, 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.
In another embodiment of the invention, 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.
In another embodiment of the invention, 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.
In an embodiment of the invention, the terminal phenol ring includes non-steroidal compounds . Embodiments of the invention utilize a compound having a phenolic A ring. In a further embodiment of the invention, 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.
DRAWINGS
These and other features of the invention will be better understood with reference to the following description, appended claims, and accompanying drawings where
Figure 1 shows the effects of 3J7β -estradiol and 3J7β-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
(=100%). *=p<0.05 versus serum-free controls. Data are expressed as mean + SEM for 6 wells per group.
Figure 2 shows the effects of 3J7β-estradiol, estra-2,3, 17 β-triol (2-OH- estradiol), l,3,5(10)-estratriene-3-ol (estratriene-3-ol) and 2,3-O-methyl estradiol, all at a concentration of 2nM. on live SK-N-SH cell number at 48 hours of serum deprivation. *=p<0.05 versus serum free controls. **=p<0.05 versus serum free controls and the respective 2,3-O-methyl estradiol. Data are expressed as mean + SEM for 5 wells/group.
Figure 3 shows the effects of 3J7β-estradiol, estriol, and 17α ethynyl 3J7β- 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 serum free controls and the respective 3-O-methyl steroid. **=p<0.05 versus all other groups. Data are expressed as mean + SEM for 5 wells/group.
Figure 4 shows the effects of 3J7β-estradiol, diethylstilbestrol (DES), DES mono- O-methyl ether (DES mono-O-ME) and DES di-O-methyl ether (DES Di-O-ME) all at a concentration of 2nM, on live SK-N-SH cell number at 48 hours of serum deprivation. *=p<0.05 versus serum free controls and DES di-O-methyl ether groups. Data are expressed as mean + SEM for 6 wells/group.
Figure 5 shows the effects of 3J7β-estradiol, estrone, [2S-(2a,4aα, 10aβ]- 1,2,3,4 ,4a,9J0J0a-octahydro-7-hydroxy-2-methyl-2-phenanthrenemethanol (PAM), and [2S-(2a,4aα, 1 Oaβ]- 1 ,2,3,4,4a,9, 10, 10a-octahydro-7-hydroxy-2-methyl-2- phenanthrenecarboxyaldehyde (PACA) at a concentration of 2nM on SK-N-SH dead cell number at 48 hrs of serum deprivation. *=p,0.05 versus serum free controls (SF). Data are expressed as mean + SEM for 6 wells/group.
Figure 6 shows the effects of 3J7β-estradiol, estrone, [2S-(2a,4aα, 10aβ]- l,2,3,4,4a,9J0J0a-octahydro-7-hydroxy-2-methyl-2-phenanthrenemethanol (PAM) and [2S-(2a,4aα, 10aβ]-l,2,3,4,4a,9J0J0a-octahydro-7-hydroxy-2-methyl-2- phenanthrenecarboxyaldehyde (PACA) at 2nM on SK-N-SH live cell number at 48 hrs of serum deprivation. *=p<0.05 versus serum free controls (SF). Data are expressed as mean + SEM for 6 wells/group
Figure 6a shows the effects of [2S-(2a,4a . 10aβ]- 1.2.3,4,4a,9J0J0a-octahydro-
7-hydrosy-2-methyl-2-phenanthrenemethanol (PAM) at 2nM on the percent increase of live cell number over the serum free controls at 48 hrs of serum deprivation. *=p<0.05 versus serum free controls (SF). Statistical analysis was performed on raw data. Data are expressed as mean + SEM for 8 wells/group.
Figure 6b shows the effects of [2S-(2a,4a . 10aβ]-l ,2,3,4,4a,9J0J0a-octahydro- 7-hydroxy-2-methyl-2-phenanthrenecarboxyaldehyde (PACA) on the percent increase of live cell number over the serum free controls at 48 hrs of serum deprivation. *=p<0.05 versus serum free controls (SF). Statistical analysis was performed on raw data. Data are expressed as mean + SEM for 8 wells/group.
Figure 7 shows the effects of treatment of 3J7 β-estradiol (0.2 or 2nM) and 3J7α- estradiol (2nM) on the toxicity induced by the neurotoxic fragment of the β amyloid protein (Aβ 25-35) (20μm) on neuronal cells. SK-N-SH neurons were exposed to 3J7β estradiol, 3J7 α-estradiol and Aβ 25-35 alone or in combination. After a four-day exposure, live cell number was determined. *=p,0.05 versus serum free controls (SF). Data are expressed as mean + SEM for 6 wells/group. Separate groups were exposed to 3J7β-estradiol and 3J7α-estradiol without the addition of Aβ. The steroid addition had no effect on cell number in the absence of insult.
Figure 8 shows the effect of treatment of ovariectomized rats with sham injection (oil) or with 3J7β-estradiol at 100 μg/kg body weight by subcutaneous injection, at 2 hours before occlusion of the middle cerebral artery. The maximum lesion size in brain Cross sections of sacrificed animals 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,4aαJ0aβ)]- 1,2,3,4 ,4a,9J0J0a-octahydro-7-hydroxy-2-methyl-2-phenanthrenemethanol (PAM) and [2S-(2a,4aαJ0aβ)]-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). DETAILED DESCRIPTION
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. 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."
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. For Alzheimer's patients, 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.
According to the invention, 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. 3J7β-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 β-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
500nM and more particularly between OJnM and InM. The relatively low effective dose of neuroprotective compounds capable of causing a neuroprotective effect according to the invention, is in stark contrast with the findings of others who have tested estrogens in in vitro assays. For example, Droescher et al. describe an IC50 of 12.8 μM 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 lOOμg/kg body weight, (a dose of compound at lOOμ /kg body weight provides approximately 0.4nM-2nM plasma concentration of the compound. (Figure 8).
According to the neuronal cell assay described in Example 1, it has been here demonstrated that the hydroxyl group on the phenolic A ring is required for neuroprotection. Following replacement of the hydroxyl group on the terminal phenol group with, for example, a methyl group, a significant loss of neuroprotective properties of the compound was observed (Figure 1-6, Tables I and II). Furthermore, compounds that normally lack a hydroxyl group on the terminal carbon ring, such as progesterone and corticosterone, show little or no neuroprotection (Figures 9 and 10). Applicants have further determined that the hydroxyl group on the terminal phenolic group may be located on any available carbon in order that neuroprotection to be maintained.
It has been determined that 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 β 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 π, Figure 4). This compound has a terminal phenolic ring structure that is associated with a second phenolic ring via a linkage group.
Removal of the hydroxyl group on the terminal phenolic ring results in a loss of neuroprotective activity. Furthermore, 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,4aαJ0αβ)]- 1,2,3, 4,4a,9J0J Oa-octahydro- 7hydroxy-2-methyl-2-phenanthrenemethanol (PAM) and [2S-(2a, 4aα, 10 αβ)]- 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 3J7β-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. In addition to these required structures, 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. These 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.
(a) 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. For example, 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, sulfoethylamide, thionitrosyl, thiophosphoryl, p-toluenesulfonate, amino, imino, cyano, carbamoyl, acetamido, hydroxyamino, nitroso, nitro, cyanato, selecyanato, arccosine, pyridinium, hydrazide, semicarbazone, carboxymethylamide, oxime, hydrazone, sulfurtrimethylammonium, semicarbazone, o-carboxymethyloxime, aldehyde hemiacetate, methylether, ethylether, propylether, butylether, benzylether, methylcarbonate, carboxylate, acetate, chloroacetate, trimethylacetate, cyclopentylpropionate, propionate, phenylpropionate, carboxylic acid methylether, formate, benzoate, butyrate, caprylate, cinnamate, decylate, heptylate, enanthate, glucosiduronate, succinate, hemisuccinate, palmitate, nonanoate, stearate, tosylate, valerate, valproate, decanoate, hexahydrobenzoate, laurate, myristate, phthalate, hydroxyl, ethyleneketal, diethyleneketal, formate, chloroformate, formyl, dichloroacetate, keto, difluoroacetate, ethoxycarbonyl, trichloroformate, hydroxymethylene, epoxy, peroxy, dimethyl ketal, acetonide, cyclohexyl, benzyl, phenyl, diphenyl, benzylidene, and cyclopropyl groups. 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.
(b) 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-isoxazine, p-isoxazine, 1,2,5-oxathiazine, 1,2,6-oxathiazine, 1,4,2-oxadiazine, 1,3,5,2-oxadiazine, moφholine (tetrahydro-p-isoxazine), any of the six ringed structure listed above being a terminal group in the compound. Additionally, 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-dioxazole; 1,2,4-dioxazole; 1,3,2-dioxazole; 1,3,4-dioxazole; 1,2,5-oxathiazole; 1,3-oxathiole, cyclopentane. These compounds in turn may have associated R groups selected from section (a) or section (b) above that are substituted on the carbon ring at any of the available sites.
(c) Any compound including those listed above, that may form a cyclopentanophen(a)anthrene ring compound and which, for example, may be selected from the group consisting of 1,3,5 (10), 6,8-estrapentaene, 1,3,5 (10), 6,8, 11- estrapentaene, 1,3,5 (10) 6,8,15-estrapentaene, 1,3,5 (10), 6.-estratetraene, 1,3,5 (10), 7- estratetraene, 1,3,5 (10)8-estratetraene, 1,3,5 (lθ)lό-estratetraene, 1,3,5 (10)15- estratetraene, 1,3,5 (10)- estratriene, 1,3,5 (10) 15- estratriene. (d) Any compound including precursors or derivatives selected from raloxifen, tamoxifen, androgenic compounds, and their salts where an intact phenol ring is present with a hydroxyl group present on carbons 1,2,3 and 4 of the terminal phenol ring.
(e) Any compound in the form of a prodrug, that may be metabolized to form an active polycyclic phenolic compound having neuroprotective activity. Administration of any of the compounds of the invention 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
37°C and under 5% CO: and 95% air. Media was changed three times weekly; SK-N-SH cells were backcultured every five to seven days to maintain cell lines and cells used in the following experiments were in passages seven to twelve. The growth media were initially decanted and the cells were rinsed with 0.02% EDTA that was subsequently discarded. Another aliquot of 0.02% EDTA was then added and after a 30 min incubation of 37°C, the cells were counted on a Neubauer hemacytometer (Fisher Scientific Inc., Orlando, FL) and resuspended in appropriate media. Experiments were initiated by the backculturing of SK-N-SH at a concentration of 1.0' X IO6 cells/ml in the appropriate treatment media. Cell density did not influence the response to 3J7β-estradiol or 3J7α- estradiol.
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 3J7β- and 3J7 -estradiol . 3J7β-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 ED50 of 0J3 nM and significant neuroprotection at the 0.2nM concentration (Fig 1). This effect was robust with the 2 nM concentration of 3J7β- 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 β-estradiol group for each study using the following calculation:
% Neuroprotectivity = Test cell # - Serum-Free Cell #
17β E2 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,
0.02% EDTA at 37°C for 30 min. Cells were suspended by repeated pipetting of the EDTA over the cells. One hundred ul aliquots from each cell suspension was incubated with 100 ul of 0.4% Trypan Blue stain (Sigma Chemical Co.) for 5 minutes at room temperature. All suspensions were counted on a Neubauer hemacytometer within 15 minutes of addition of Trypan Blue. Two independent counts of live cells were made for each aliquot.
Example 2: Comparison of the neuroprotection afforded by different four-ring and two- ring compositions.
3,17 α-estradiol, l l,3,5(10)-estratrien-3-ol and l ,3,5(10)estra-2,3J7β-triol (2,3- catecholestradiol) were found to be equivalent to 3J7β-estradiol in their neuroprotectivity (Figure 2, Table 1). Estrone, estriol and 17 α-ethynyl-3J7β estradiol, while significantly neuroprotective, were less active than 3J7β-estradiol (Figures 3 and Table 13). The two- ring non-fused diethylstilbestrerol (DES), was active as a neuroprotectant and retained nearly full neuroprotectivity when one, both not both, of the phenolic hydroxyl functions were replaced with an O-methyl ether function (Figure 4 and Table 2). Similarly, all steroids were rendered inactive when the 3-hydroxyl group was replaced with an O- methyl ether group (Figures 1-4 and Tables I and II), a substitution that eliminates the acid, hydrophilic properties of the A ring. The 3-0 methyl ether of 3J7β-estradiol was inactive even at concentrations as high as 20nM (Fig 1 ). These data demonstrate that C-3 hydroxylated estratrienes are neuroprotective. A similarly positioned phenolic hydroxyl group in the diphenols may serve the same function.
Two 19-carbon steroids were evaluated at a 2nM concentration for neuroprotection in the assay. The following results were obtained for live cells/ml. (mean + SEM X lOVml) for 5 to 6 cultures/group; Study 1 ; serum free controls = 94 + 7, testosterone = 87+6, dihydrotestosterone = 90+7, cholesterol = 65+4; Study 2; serum free controls = 177+18, 3J7 β-estradiol = 329 +33 (p<0.05 vs serum free controls), prednisolone = 187+16, 6 α-methylprednisolone = 173+13, aldosterone = 132+18. There was no neuroprotective effects of any non-phenolic steroids.
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. Similarly, all five of the 21- carbon pregnane derivatives that were tested contained a C-3 ketone function, and three Δ4 -steroids, progesterone and aldosterone and two Δ1,4-steroids, prednisolone and 6- methylprednisolone were inactive . Finally, 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.
In all studies, cells were cultured in RPMI- 1640 media (Serum Free, SF group), RPMI- 1640 media supplemented with 10% FBS (FBS group), or RPMI- 1640 media supplemented one of the following steroids at a concentration of 2nM (unless otherwise noted): 3,17β-estradiol (l,3,5(10)-estratriene-3J7β-diol); 3,17 α-estradiol (1,3,5(10)- estratriene-3βJ7α-diol); 3J7β-estradiol 3-O-methyl ether (l,3,5(10)-estratriene-3J7β- diol 3-O-methyl ether); 3J7α-estradiol 3 acetate (l,3,5(10)-estratriene-3β,17α-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-one 3-O-methyl ether); estriol (l,3,5(10)estratriene-3β,16αJ7β- triol);estriol 3-O-methyl ether (l,3,5(10)estratriene-3βJ6α, 17β-triol 3-O-methyl ether); 17 -ethynyl estradiol (l,3,5(10)-estratriene-17α-ethynyl-3β, 17α-diol); ethynyl estradiol 3-O-methyl ether (l,3,5(10)-estratriene-17α-ethynyl-3β, 17α-diol 3-O-methyl ether); 2- hydroxyestradiol (l,3,5(10)estratriene-2,3J7β-triol); 2,3-methoxyestradiol (l,3,5(10)estratrien-2,3J7β-triol 2,3-O-methyl ether) or estratriene-3-ol (l,3,5(10)estratrien-3-ol); cortisone, progesterone, prednisolone ( 1 ,4-pregnadience-
I lβ,17,21-triol-3,20-dione); methylprednisolone (6α-methyl- 1 ,4-pregnadiene-
I I lβ,17,21-triol-3,20-dione); and aldosterone (4-pregnen-l lβ,21-diol-3J8,20-trione). All steroids were from Steraloids, Inc., Wilton, NH and were initially dissolved at 1 mg/ml in absolute ethanol and diluted in RPMI- 1640 media to a final concentration of 2 nM. To control for possible ethanol effects in the treated wells, both the serum-free media (SF group) and FBS media (FBS group) were supplemented with absolute ethanol at a concentration of 544 pl/ml. In all studies, at least 4 and usually 6 replicate well were treated with each media.
Example 3: Three ring structures with neuroprotective properties.
[2S-(2a, 4a , 10 αβ)]-l,2,3,4,4a,9J0, 10a-octaydro-7hydroxy-2-methyl-2- phenanthrenemethanol (PAM) and [2S-(2a, 4aα, 10 αβ)]-l,2,3,4,4a,9J0, lOa-octaydro- 7hydroxy-2-methyl-2-phenanthrenecarboxyaldehyde (PACA) were added to media containing SK-N-SH cells concurrent with serum deprivation. 24 or 48 hrs later, cell viability was determined by a dye production method that required the activity of mitochondria for reduction of the dye to a colored reagent. (Goodwin et al. (1995) J. Immunol. Methods, 179: 95). Both PACA and PAM showed neuroprotective activity with peak responses at the 2nM concentration. (Figures 5a, 6a) Neuroprotective activity was similar to the positive control, 3,17β estradiol. (Figures 5 and 6)
Example 4: "In vivo" dosage studies
A number of compounds were tested at doses of lOOug/kg body weight in rats injected at 2 hrs prior to occlusion of the middle cerebral artery. The injection produced plasma estradiol concentrations of 100 to 200 pg/ml (0.4 to 0.8nM) at the time of the occlusion. In ovariectomized rats, maximal lesions of 25% of the brain cross-sectional area was observed. When treated with 3J7β-estradiol, maximal lesion area was reduced by about 50%. These data demonstrate that only low nM concentrations of 3J7β estradiol are required to protect from ischemic lesions in vivo.
16/1
Table I. Phenolic A Ring Requirement for the Neuroprotectivity of Estratrienes
Namet % of l"β-Estradiol Neuroprotection
3,17β-Estra ol 100* 3,17β-Estra iol 3-O-ME -1.5
Estratriene-3-ol 103*t
3,17α-Estradiol 8P
3,17α-Estraiol 3-acetate -20
2-Hydroxy- 17 β-estradiol 70*
17β-Estra o! 2,3-O-ME 7
Estrone 58*
Estrone 3-O-ME -11
Estriol 46*
Estriol 3-O-ME 2
Ethynyl Estradiol 41' Mestranol -6
Figure imgf000019_0001
*p < 0.05 vs serum-free control groups as described in Figures 1 to 4 and note 8. t = trivial name is indicated here and structural name in note 10. φ=No 3-0-conjugates were available for evaluation. 16/2
Table II. Neuroprotectivity of Estradiol, Phenol, Napthol and Dipenols
Name % of 17β-Estradiol Neuroprotection
3J7β-Estradiol 100*
Phenol -27
Figure imgf000020_0001
Diethylstilbesterol 74*
Diethylstilbesterol-mono-O-ME 60*
Diethylstilbesterol-di-O-ME
Figure imgf000020_0002
''P'CO.OS vs serum-free control groups, as described in note 8. 16/3
TABLE m
THE EFFECTS OF BRANCHED CHAIN
SUBSTITUTED PHENOLS ON SK-N-SH LIVE
CELL NUMBERS AT 48 HOURS EXPOSURE
Treatment Live Cell Number ± SEM (IO3 cells/ml)
Serum free controls 203 ± 16
Butylated Hydroxyanisol 182 ± 16
Butylated hydroxytoluene 175 ± 11

Claims

- 17 -We claim :
1. A method for conferring neuroprotection on a population of cells in a subject, comprising: (i) providing a non-estrogen compound, having a terminal phenol group in a structure containing at least a second ring, having a molecular weight of less than 1000 Daltons; and
(ii) administering the compound in an effective dose to the population of cells so as to confer neuroprotection.
2. A method according to claim 1, wherein the compound comprises a four-ring structure.
3. A method according to claim 2, wherein the effective dose achieves a plasma concentration of less than 500nM.
4. A method according to claim 1, wherein the compound is comprises a three-ring structure.
5. A method according to claim 4, wherein the three-ring compound is non- steroidal.
6. A method according to claim 1, wherein the compound comprises a two-ring structure.
7. A method according to claim 6, wherein the two-ring compound is non-steroidal
8. A method according to claim 5, wherein the three compound is a phenanthrene compound.
9. A method according to claim 8, wherein the phenanthrene compound is selected from the group consisting of a tetrahydrophenanthrene and a octahydrophenanthrene. - 18 -
10. A method according to claim 8, wherein the phenanthrene compound is selected from the group consisting of phenanthrenemethanol and phenanthrenecarboxyaldehyde.
11. A method according to claim 6, wherein the two ring structure is selected from the group consisting of naphthol and naphthalene.
12. A method according to claim 1, wherein the terminal phenol group has a hydroxyl group on any of carbons 1-4.
13. A method according to claim 1, wherein the at least second ring structure is linked to the terminal phenol group via a linkage group.
14. A method according to claim 1, wherein the at least second ring structure is covalently linked to the terminal phenol group in the absence of a linkage group.
15. A method according to claim 1, wherein the effective dose achieves a plasma concentration of less than 500nM.
16. A method according to claim 15, wherein the effective dose achieves a plasma concentration in the range 0.02nM-500nM.
17. A method according to claim 16, wherein the effective dose achieves a plasma concentration in the range OJnM-lnM.
18. A method according to claim 1, wherein the compound has a molecular weight that is greater than 170 Daltons.
19. A method according to claim 1, wherein the terminal phenol group is a phenolic A ring. - 19 -
20. A method of treating a neurodegenerative disease in a subject, comprising:
(a) preparing an effective dose of a formulation containing a compound having a terminal phenol and at least a second ring structure, having a molecular weight less than 1000 Daltons; and (b) administering the dose to the subject.
21. A method according to claim 17, wherein the compound has a molecular weight that is greater than 170 Daltons
22. A method according to claim 20, wherein the terminal phenol is a phenolic A ring.
PCT/US1996/012146 1995-07-24 1996-07-24 Use of non-estrogen polycyclic phenol compounds for the manufacture of a medicament for conferring neuroprotection to cells WO1997003661A1 (en)

Priority Applications (3)

Application Number Priority Date Filing Date Title
EP96924692A EP0841906A1 (en) 1995-07-24 1996-07-24 Use of non-estrogen polycyclic phenol compounds for the manufacture of a medicament for conferring neuroprotection to cells
JP9506961A JPH11510144A (en) 1995-07-24 1996-07-24 Use of non-estrogenic polycyclic phenolic compounds for the manufacture of a medicament for imparting neuroprotection to cells
AU65079/96A AU6507996A (en) 1995-07-24 1996-07-24 Use of non-estrogen polycyclic phenol compounds for the manufacture of a medicament for conferring neuroprotection to cells

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US139495P 1995-07-24 1995-07-24
US60/001,394 1995-07-24

Publications (1)

Publication Number Publication Date
WO1997003661A1 true WO1997003661A1 (en) 1997-02-06

Family

ID=21695818

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1996/012146 WO1997003661A1 (en) 1995-07-24 1996-07-24 Use of non-estrogen polycyclic phenol compounds for the manufacture of a medicament for conferring neuroprotection to cells

Country Status (5)

Country Link
EP (1) EP0841906A1 (en)
JP (1) JPH11510144A (en)
AU (1) AU6507996A (en)
CA (1) CA2227634A1 (en)
WO (1) WO1997003661A1 (en)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999042108A1 (en) * 1998-02-20 1999-08-26 Jenapharm Gmbh & Co. Kg Pharmaceutical preparations for selectively supplementing oestrogen deficiency in the central nervous system
US6245756B1 (en) 1998-02-27 2001-06-12 Jenapharm Gmbh & Co. Kg Pharmaceutical preparations for treatment of estrogen deficiency in the central nervous system
WO2002005813A2 (en) * 2000-07-17 2002-01-24 The Salk Institute For Biological Studies Method for preventing and/or decreasing amyloid production with polycyclic compounds
WO2002013870A2 (en) * 2000-08-17 2002-02-21 Mitokor, Inc. Formulation for administering therapeutic lipophilic molecules
WO2002040032A2 (en) * 2000-11-17 2002-05-23 Washington University Cytoprotective estrogen derivatives
US6844456B2 (en) 2000-11-03 2005-01-18 Washington University Modified, hydroxy-substituted aromatic structures having cytoprotective activity
US11969351B2 (en) 2022-02-03 2024-04-30 James M. Jackson Systems and methods for magnetic joints

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE2803582A1 (en) * 1978-01-27 1979-08-02 Sandoz Ag 2-Amino-tetra:hydro-naphthol derivs. - used as adrenergic and dopamine receptor stimulants useful for treating heart failure, cardiac infarct, hypertension and Parkinsonism
WO1987002666A2 (en) * 1985-10-31 1987-05-07 Maggioni-Winthrop S.P.A. Bicyclic alkoxy- and alkylthio-substituted aminoalcohols
EP0339579A2 (en) * 1988-04-28 1989-11-02 Novo Nordisk A/S Piperidine compositions and their preparation and use
WO1992003049A1 (en) * 1990-08-23 1992-03-05 New York University Methods and compositions for treating t-cell mediated diseases
WO1992007855A1 (en) * 1990-10-30 1992-05-14 Warner-Lambert Company Tetracyclic amines having pharmaceutical activity
EP0606661A1 (en) * 1993-01-15 1994-07-20 F. Hoffmann-La Roche Ag Octahydrophenanthrene derivatives and their use as NMDA-Antagonists
EP0659418A1 (en) * 1993-12-21 1995-06-28 Eli Lilly And Company 2-Phenyl-3-azoylbenzothiophene for treating Alzheimer's disease

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE2803582A1 (en) * 1978-01-27 1979-08-02 Sandoz Ag 2-Amino-tetra:hydro-naphthol derivs. - used as adrenergic and dopamine receptor stimulants useful for treating heart failure, cardiac infarct, hypertension and Parkinsonism
WO1987002666A2 (en) * 1985-10-31 1987-05-07 Maggioni-Winthrop S.P.A. Bicyclic alkoxy- and alkylthio-substituted aminoalcohols
EP0339579A2 (en) * 1988-04-28 1989-11-02 Novo Nordisk A/S Piperidine compositions and their preparation and use
WO1992003049A1 (en) * 1990-08-23 1992-03-05 New York University Methods and compositions for treating t-cell mediated diseases
WO1992007855A1 (en) * 1990-10-30 1992-05-14 Warner-Lambert Company Tetracyclic amines having pharmaceutical activity
EP0606661A1 (en) * 1993-01-15 1994-07-20 F. Hoffmann-La Roche Ag Octahydrophenanthrene derivatives and their use as NMDA-Antagonists
EP0659418A1 (en) * 1993-12-21 1995-06-28 Eli Lilly And Company 2-Phenyl-3-azoylbenzothiophene for treating Alzheimer's disease

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CHEMICAL ABSTRACTS, vol. 96, no. 7, 15 February 1982, Columbus, Ohio, US; abstract no. 46388, XP002014896 *
WAEFELAER, ADOLPHE: "Structural analogs of apomorhine", ING.CHIM, vol. 63, no. 299, 1981, pages 12 - 24 *

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU739071C (en) * 1998-02-20 2002-04-11 Jenapharm Gmbh & Co. Kg Pharmaceutical preparations for the selectively supplementing estrogen deficiency in the central nervous system
AU739071B2 (en) * 1998-02-20 2001-10-04 Jenapharm Gmbh & Co. Kg Pharmaceutical preparations for the selectively supplementing estrogen deficiency in the central nervous system
WO1999042108A1 (en) * 1998-02-20 1999-08-26 Jenapharm Gmbh & Co. Kg Pharmaceutical preparations for selectively supplementing oestrogen deficiency in the central nervous system
US6245756B1 (en) 1998-02-27 2001-06-12 Jenapharm Gmbh & Co. Kg Pharmaceutical preparations for treatment of estrogen deficiency in the central nervous system
WO2002005813A3 (en) * 2000-07-17 2003-07-10 Salk Inst For Biological Studi Method for preventing and/or decreasing amyloid production with polycyclic compounds
US6472436B1 (en) * 2000-07-17 2002-10-29 The Salk Institute For Biological Studies Methods for protecting cells from amyloid toxicity and for inhibiting amyloid protein production
WO2002005813A2 (en) * 2000-07-17 2002-01-24 The Salk Institute For Biological Studies Method for preventing and/or decreasing amyloid production with polycyclic compounds
US7053116B2 (en) * 2000-07-17 2006-05-30 The Salk Institute For Biological Studies Methods for protecting cells from amyloid toxicity and for inhibiting amyloid protein production
WO2002013870A2 (en) * 2000-08-17 2002-02-21 Mitokor, Inc. Formulation for administering therapeutic lipophilic molecules
WO2002013870A3 (en) * 2000-08-17 2003-02-06 Apollo Biopharmaceutics Inc Formulation for administering therapeutic lipophilic molecules
US6844456B2 (en) 2000-11-03 2005-01-18 Washington University Modified, hydroxy-substituted aromatic structures having cytoprotective activity
US9156876B2 (en) 2000-11-03 2015-10-13 Washington University Modified, hydroxy-substituted aromatic structures having cytoprotective activity
WO2002040032A2 (en) * 2000-11-17 2002-05-23 Washington University Cytoprotective estrogen derivatives
WO2002040032A3 (en) * 2000-11-17 2003-08-28 Univ Washington Cytoprotective estrogen derivatives
US11969351B2 (en) 2022-02-03 2024-04-30 James M. Jackson Systems and methods for magnetic joints

Also Published As

Publication number Publication date
AU6507996A (en) 1997-02-18
EP0841906A1 (en) 1998-05-20
CA2227634A1 (en) 1997-02-06
JPH11510144A (en) 1999-09-07

Similar Documents

Publication Publication Date Title
US5859001A (en) Neuroprotective effects of polycyclic phenolic compounds
US6197833B1 (en) Neuroprotective effects of polycyclic phenolic compounds
US9156876B2 (en) Modified, hydroxy-substituted aromatic structures having cytoprotective activity
Green et al. 17α-Estradiol exerts neuroprotective effects on SK-N-SH cells
Green et al. Phenolic A ring requirement for the neuroprotective effects of steroids
Simpkins et al. Mitochondrial mechanisms of estrogen neuroprotection
Morissette et al. Estrogen and SERM neuroprotection in animal models of Parkinson's disease
US6319914B1 (en) Cytoprotective effect of polycyclic phenolic compounds
EP0977578B1 (en) Compositions to enhance the cytoprotective effects of polycyclic phenolic compounds through the synergistic interaction with anti-oxidants
EP1143947A2 (en) Use of estrogen compounds for prevention and treatment of ischemic damage
EP0841906A1 (en) Use of non-estrogen polycyclic phenol compounds for the manufacture of a medicament for conferring neuroprotection to cells
Perez et al. Neuroprotective effects of estratriene analogs: structure‐activity relationships and molecular optimization
DE69912247T2 (en) E-2- (4- (CHLORO-1,2-DIPHENYLBUT-1-ENYL) PHENOXY) ETHANOL AND PHARMACEUTICAL COMPOSITIONS THEREOF
EP1834959B1 (en) Tert-butyl-substituted aromatic steroids having cytoprotective activity
CZ200377A3 (en) 7-Hydroxyepiandrosterone exhibiting neuroprotective activity
EP1415653A2 (en) Use of progesterone or an agonist thereof for the inhibition of steroid synthesis
US20020132802A1 (en) Cytoprotective polycyclic compounds
Jameie et al. Neuroapoptosis Signaling Pathways in Hippocampus Following Ovariectomy and Its Inhibition by Systemic Estradiol Replacement Therapy
Ahmed et al. Impact of castration and sex hormones on some hormonal and biochemical parameters of male rabbits.
Simpkins et al. Structure–Nongenomic Neuroprotection Relationship of Estrogens and Estrogen‐Derived Compounds

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU CA JP KR

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
ENP Entry into the national phase

Ref document number: 2227634

Country of ref document: CA

Ref country code: CA

Ref document number: 2227634

Kind code of ref document: A

Format of ref document f/p: F

ENP Entry into the national phase

Ref country code: JP

Ref document number: 1997 506961

Kind code of ref document: A

Format of ref document f/p: F

WWE Wipo information: entry into national phase

Ref document number: 1996924692

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 1996924692

Country of ref document: EP

WWR Wipo information: refused in national office

Ref document number: 1996924692

Country of ref document: EP

WWW Wipo information: withdrawn in national office

Ref document number: 1996924692

Country of ref document: EP