Note: Descriptions are shown in the official language in which they were submitted.
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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.
1 5 BACKGROUND
Pathological conditions resulting from the accelerated or ongoing death of neurons
are prevalent in today's society and include chronic dise;l.ses such as Alzheimer's disease
and Parkinson's disease, acute diseases such as strol;e. br;lin cell loss that follows
myocardial infarction, and acute neuronal injury associate(l with spinal cord trauma and
2 0 head trauma. Chronic and acute neurodegenerative disea.~es 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
25 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
3 0 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:
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303-308; Simpkins et al. (1994) Neurobiology of Aging 15: s195-s197). 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 ~lmini~ter estrogen
5 therapeutically to men without associated sex-related side effects.
The mech~ni.cm.c 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 stim~ te the production of neurotrophic agents that in turn stim~ te
1 0 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
1 5 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)11: 292. Other
2 0 compounds including certain 21-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. tl994) J. Cardiovasc. Pharmacol 23: 136; Akhter et al. (1994) Stroke 25; 418).
As described above, a number of factors may be involved in neuroprotection.
2 5 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
3 0 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
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compounds could be used in the treatment of the cnronic as well as the acute conditions
caused by neurodegenerative diseases, trauma, and aging at non-toxic dosages. Anunderstanding of the structural requirements for compositions capable of inducing
neuroprotection would provide the basis for designing novel drugs that have enhanced
5 neuroprotective pLo~ ies while at the same time having reduced adverse side effects
SUMl\~ARY
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,
10 trauma or aging and may be used to achieve a similar effect in male and female subjects
with minim~l 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 cont~ining at least a
15 second ring having a molecular weight that is less than lO00 Daltons; and ~lministering
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 neurodegenerativecondition 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 lOOOD
2 0 and at least one additional ring structure covalently linked to the phenol ring; and
~lministering 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 ~-lmini.stered at an effective dose that achieves a plasma2 5 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
3 0 a phenanthrencarboxyaldehyde.
In another embodiment of the invention, the two- ring structure may be fused andinclude a naphthol and naphthalene or may be a non-fused two ring structure having a
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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
5 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 0.1 nM- 1 nM.
DRAWINGS
These and other features of the invention will be better understood with reference
1 0 to the following description, appended claims, and accompanying drawings where
Figure 1 shows the effects of 3,17~ -estradiol and 3,17~-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 of15 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 3,17,(3-estradiol, estra-2,3,17 ,~-triol (2-OH-
20 estradiol), 1,3,5(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 3,17,1~-estradiol, estriol, and 17cc ethynyl 3,17~-
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
3 0 versus all other groups. Data are expressed as mean + SEM for 5 wells/group.
Figure 4 shows the effects of 3,17,13-estradiol, diethylstilbestrol (DES), DES mono-
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O-methyl ether (DES mono-O-ME) and D~S 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<O.OS versus sel~m 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 3,17~-estradior, estrone, [2S-(2a,4aa, lOa~]-
1,2,3,4,4a,9,10,10a-octahydro-7-hydroxy-2-methyl-2-phenanthrenemethanol (PAM), and
[2S-(2a,4aa, 1 Oa~] -1,2,3 ,4,4a,9, 10,1 Oa-octahydro-7-hydroxy-2-methyl-2-
phenanthrenecarboxyaldehyde (PACA) at a concentration of 2nM on SK-N-SH dead cell
10 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 3,17,B-estradiol, estrone, [2S-(2a,4aa, lOa,(3]-
1,2,3,4,4a,9,10,10a-octahydro-7-hydroxy-2-methyl-2-phenanthrenemethanol (PAM) and
15 [2S-(2a,4a~, lOa,B]-1,2,3,4,4a,9,10,10a-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 control. (SF). Data are expressed as mean
+ SEM for 6 wells/group
2 0 Figure 6a shows the effects of [2S-(2a,4ac~. 1 Oa~3]- 1 .'.3,4,4a,9, 10, lOa-octahydro-
7-hydrosy-2-methyl-2-ph~n~n~hrenemethanol (PAM) at 'nM on the percent increase of
live cell number over the serum free controls at 48 hns of serum deprivation. *=pcO.05
versus serum free controls ~SF). Statistical analysis w~s pcrformed on raw data. Data are
expressed as mean + SEM for 8 wells/group.
Figure 6b shows the effects of [2S-(2a,4aa~ 1Oa~]- 1 ,''.3,4,4a,9, 10,10a-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 perforrned on raw data. Data are
3 0 expressed as mean + SEM for 8 wells/group.
Figure 7 shows the effects of treatment of 3.17 ,(~-estradiol (0.2 or 2nM) and 3,170~-
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estradiol (2nM) on the toxicity in~ cec~ by the neurotoxic fragment of the ,(~ amyloid
protein (A~ 25-35) (20ym) on neuronal cells. SK-N-SH neurons were exposed to 3,17
estradiol, 3,17 a-estradiol and A,3 25-35 alone or in combination. After a four-day
exposure, live cell number was determined. *=p,0.05 versus serum free controls (SF).
5 Data are expressed as mean + SEM for 6 wells/group. Separate groups were exposed to
3,17,(~-estradiol and 3,170~-estradiol without the addition of A,~. 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
10 (oil) or with 3,17,1~-estradiol at 100 llg/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 ~nim"l.c 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 at48 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
2 0 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,4aa,10a,B)]-
1,2,3,4,4a,9,10,10a-octahydro-7-hydroxy-2-methyl-2-phenanthrenemethanol (PAM) and
2 5 [2S-(2a,4aa,1 Oa,(3)] - 1,2,3,4,4a,9,10,1 Oa-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-
3 0 fused).
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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
5 than an estrogen compound, described in the l 1 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 l carbon ring.
A steroid is defined here and in the claims as a compound having numbered
10 carbon atoms arranged in a 4-ring structure (J. American Chemical Society 82: 5525-558 l
(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: chronic15 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.
2 0 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
2 5 neuroprotection. This method is further suited for the treatment of neurodegenerative
~lice~ces, trauma and aging in human subjects.
The protection of neurons from severe degeneration is an important aspect of
tre~tmPnt 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
3 0 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,
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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 t
brain cell loss; and peripheral neuropathy. Because of the observed cytoprotective
5 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
10 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
15 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. 3,17~-estradiol has been selected as the controk because of its previously
demonstrated neuroprotective effects (Simpkins WO 95/12402). The neuroprotective2 0 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 3~17 ~-estradiol.
This invention teaches that neuroprotection can be achieved at an effective doseproviding low plasma concentrations of polycyclic phenolic compounds. More
2 5 specifically, a neuroprotective effect can be achieved at plasma concentrations of less than
SOOnM and more particularly between O.lnM and lnM. 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 lC50 of 12.8 ,uM estrogen to
3 0 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
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_ g _
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 asubject, should be within the range that is relatively non-toxic to the patient especially
when the compound is used long-terrn. According to the invention, significant
neuroprotection has been obtained in neuronal cell cultures at concentrations of 2nM
(Figures 1-6) and furtherrnore, significant neuroprotection has been achieved in rats at
lOO,ug/kg body weight. (a dose of compound at lOO~lg/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 terrninal phenol
15 group with, for example, a methyl group, a signi~lcant loss of neuroprotective properties
of the compound was observed (Figure 1-6, Tables I and ll). Furthermore, compounds
that normally lack a hydroxyl group on the terminal carhon ring, such as progesterone
and corticosterone, show little or no neuroprotection (Fi~ure~ 9 and 10). Applicants have
further determined that the hydroxyl group on the terminal phenolic group may be located
2 0 on any available carbon in order that neuroprotection to he maintained.
It has been determined that neuroprotective compounds for use in the method of
the invention require a terrninal 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 c~ or ~3 isomeric
2 5 configurations. Furthermore, phenol on its own is not neuroprotective nor are straight
chain substitution of phenolic compounds (Table III). However, a compound having a
terrninal phenolic ring and at least one other carbon ring is neuroprotective. For example~
the non-fused two- ring structure, diethylstilbestrol. has demonstrated neuroprotective
properties according to the invention (Table II, Figure 4). This compound has a terminal
3 0 phenolic ring structure that is associated with a second phenolic ring via a linkage group.
Removal of the hydroxyl group on the terrninal phenolic ring results in a loss of
neuroprotective activity.
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Furthermore, compounds that are non-steroidal and have a termirral phenolic ringand at least two additional carbon ring structures include three-ring compounds (Figures
11, 12) such as exemplified by [2S-(2a,4ao~,100~)]-1,2,3,4,4a,9,10,10a-octahydro-
7hydroxy-2-methyl-2-phenanthrenemethanol (PAM) and [2S-(2a, 4ao~, 10 o~l3)]-
5 1 ,2,3,4,4a,9, 10,1 Oa-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
10 additional carbon rings have been shown to have a neuroprotective effect as exemplif1ed
by 3,17,B-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 ~f1mini.stration of the neuroprotective compound
includes oral, intramuscular, transdermal, buccal, intravenous and subcutaneous.Methods of atlmini.ctering 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
2 0 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
m~int~ine~l 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
2 5 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
3 0 may be organic or may include a mixture of organic molecules and ions. Organic R
groups may include alkanes, alkenes or alkynes cont~ining up to six carbons in a linear or
branched array. For example, additional R group substituents may include methyl, ethyl,
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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,
5 thiohexanyl, thiobenyl, thiopenol, thiocyanato, sulfoethylamide, thionitrosyl,thiophosphoryl, p-toluenesulfonate, amino, imino, cyano, carbamoyl, ~et~mido,
hydroxyamino, nitroso, nitro, cyanato, selecyanato, arccosine, pyridinium, hydrazide,
semicarbazone, carboxymethylamide, oxime, hydrazone, sulfurtrimethylammonium,
semicarb~one, o-carboxymethyloxime, aldehyde hemiacetate, methylether, ethylether,
10 propylether, butylether, benzylether, methylcarbonate, carboxylate, acetate, chloroacetate,
trimethylacetate, cyclopentylpropionate, propionate, phenylpropionate, carboxylic acid
rhethylether, formate, benzoate, butyrate, caprylate, cinn~m~te, decylate, heptylate,
en:~nth~te, glucosiduronate, succinate, hemisuccinate, palmitate, nonanoate, stearate,
tosylate, valerate, valproate, decanoate, hexahydrobenzoate, laurate, myristate, phth~l~t~,
15 hydroxyl, ethyleneketal, diethyleneketal, forrnate, 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
2 0 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,
25 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-tri~7ine, 1,2,4-oxazine, 1,3,2-oxazine, 1,3,6-oxazine (pentoxazole), 1,2,6
oxazine, 1,4-ox~ine, o-isoxazine, p-isoxazine, 1,2,5-o~c~thi~7ine, 1,2,6-oxathiazine,
1,4,2-ox~ 7ine, 1,3,5,2-o~ 7ine, morpholine (tetrahydro-p-isoxazine), any of the six
3 0 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
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- 12 -
(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,
5 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
10 cyclopentanophen(a)anthrene ring compound and which, for example. may be selected
from the group consisting of 1,3,5 (lO), 6,8-estrapentaene, 1,3,5 (10), 6,8, l l-
estrapentaene, 1,3,5 (10) 6,8,15-estrapentaene, 1,3,5 (10), 6,-estratetraene, 1,3,5 (lO), 7-
estratetraene, 1,3,5 (10)8-estratetraene, 1,3,5 (10)16-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 folm of a prodrug, that may be metabolized to form an
active polycyclic phenolic compound having neuroprotective activity.
2 0 Administration of any of the compounds of the invention may include the use of a
single compo~lnd 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
2 5 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
3 0 10% Fetal Bovine Serum (FBS), lOOU/ml penicillin G, and lO0 mg/ml streptomycin at
37~C and under 5% CO~ and 95% air. Media was changed three times weekly; SK-N-SHcells were backcultured every five to seven days to maintain cell lines and cells used in
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the following experiments were in passages seven to twelve. The growth media were
initially de~-~nteri 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.,
5 Orlando, FL) and resuspended in a~ iate media. Experiments were initiated by the
backculturing of SK-N-SH at a concentration of 1.(~ X 106 cells/ml in the ~l~lo~liate
treatment media. Cell density did not influence the response to 3,1713-estradiol or 3,17c~-
estradiol.
The neuroprotection assay involved continuou.~ exposure of SK-N-SH cells for
10 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 3,1713- and 3,170c-estradiol . 3,17~3-estradiol was used as a control. This
compound caused a dose-dependent protection of SK-N-SH cells under conditions ofserum deprivation with an ED50 of 0.13 nM and significant neuroprotection at the 0.2nM
15 concentration (Fig 1). This effect was robust with the ~ nM concentration of 3,17~3-
estradiol showing neuroprotection in 8 separate trials (Fi~ure.s I to 4). Neuroprotection
was deterrnined by the magnitude of the difference in li~e eell number in the treated wells
versus the cell number in the serum free wells. The .statistic;ll analysis was performed on
the raw data in each experiment. The significance of dift'erences among groups was
2 0 determined by one way analysis of variance. Planned compari!ion.s between group.s were
done using Scheffe's F-test. For all tests, p < 0.05 ~as considered significant. Following
the analysis, data were normalized to the % response of the :~.17 ~-estradiol group for
each study using the following calculation:
2 5 % Neuroprotectivity = Test cell # - Serum-Free Cell #
17,(~ 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 ~e.c~nting
3 0 media, rinsing each ~vell 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 theEDTA over the cells. One hundred ul aliquots from each cell suspension was incubated
with 100 ul of 0.4~ Trypan
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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 TrypanBlue. Two independent counts of live cells were made for each aliquot.
5 Example 2: Comparison of the neuroprotection afforded by different
four-ring and two- ring compositions.
3 ,17 a-estradiol, 1 1,3,5(1 0)-estratrien-3-ol and 1,3 ,5(1 O)estra-2,3 ,1 7~-triol (2,3-
catecholestradiol) were found to be equivalent to 3,1 7~-estradiol in their neuroprotectivity
10 (Figure 2, Table 1). Estrone, estriol and 17 a-ethynyl-3,17~ estradiol, while significantly
neuroprotective, were less active than 3,1713-estradiol (Figures 3 and Table II). The two-
ring non-fused diethylstilbestrerol (DES), was active ax 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
15 steroids were rendered inactive when the 3-hydroxyl group was replaced with an O-
methyl ether group (Figures 1-4 andl Tables I and II), a substitution that elimin~tes the
acid, hydrophilic properties of the A ring. The 3-0 methyl ether of 3,1713-estradiol was
inactive even at concentrations as high as 20nM (Fig I ). These data demonstrate that C-3
hydroxylated estratrienes are neuroprotective. A similarly positioned phenolic hydroxyl
2 0 group in the diphenols may serve the same function.
Two l9-carbon steroids were evaluated at a 2nM concentration for neuroprotectionin the assay. The following results were obtained for live cells/ml. (mean _ SEM X
103/ml) for 5 to 6 cultures/group; Study 1; serum free controh; - 94 _ 7, testosterone =
87_6, dihydrotestosterone = 90+7, cholesterol = 65+4; Study 2; serum free controls =
25 177_18, 3,17 ~3-estradiol = 329 _33 (p<0.05 vs serum free controls), prednisolone =
187_16, 6 a-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
3 0 phenolic A ring, dihydrotestosterone, were both inactive. Similarly, all five of the 2 l -
carbon pregnane derivatives that were tested contained a C-3 ketone function, and three
-steroids, progesterone and aldosterone and two ~ ~-steroids, prednisolone and 6-
methylprednisolone were inactive . Finally, cholesterol was tested because it has a 3-
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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),
5 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): 3,17~3-estradiol (1,3,5(10)-estratriene-3,17~(~-diol); 3,17 o~-estradiol (1,3,5(10)-
estratriene-3~,170~-diol); 3,1713-estradiol 3-0-methyl ether (1,3,5(10)-estratriene-3,17~-
diol 3-0-methyl ether); 3,17c~-estradiol 3 acetate (1,3,5(10)-estratriene-3,1~,17~-diol 3-
10 acetate); estrone (1,3,5(10)-estratriene-3-ol-17-one); estrone 3-0-methyl ether (1,3,5(10)-
estratriene-3-ol- 17-one 3-0-methyl ether); estriol (1,3,5(10)estratriene-313,16c~,17~-
triol);estriol 3-0-methyl ether (1,3,5(10)estratriene-3,13,160~, 17,(3-triol 3-0-methyl ether);
17c~-ethynyl estradiol (1,3,5(10)-estratriene-170~-ethynyl-3,(~, 17a-diol); ethynyl estradiol
3-0-methyl ether (1,3,5(10)-estratriene-170~-ethynyl-3,(~, 17a-diol 3-0-methyl ether); 2-
1 5 hydroxyestradiol (1,3,5(10)estratriene-2,3,17~-triol); 2,3-methoxyestradiol
(1,3,5(10)estratrien-2,3,17~-triol 2,3-0-methyl ether) or estratriene-3-ol
(1,3,5(10)estratrien-3-ol); cortisone, progesterone, prednisolone (1,4-pregnadience-
11~,17,21-triol-3,20-dione); methylprednisolone (60~-methyl-1,4-pregnadiene-
111~,17,21-triol-3,20-dione); and aldosterone (4-pregnen-1113,21-diol-3,18,20-trione).
2 0 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
2 5 treated with each media.
Example 3: Three ring structures with neuroprotective properties.
[2S-(2a, 4acc, 10 o~ )] - 1,2,3,4,4a,9,10, 1 Oa-octaydro-7hydroxy-2-methyl-2-
phenanthrenemethanol (PAM) and [2S-(2a, 4ao~, 10 o~ )]-1,2,3,4,4a,9,10, lOa-octaydro-
3 0 7hydroxy-2-methyl-2-phenanthrenecarboxyaldehyde (PACA) were added to media
cont~inin~ 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
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mitochondria for reduction of the dye to a colored reagent. (Goodwin et al. ( l 99S) J.
Immunol. Methods, 179: 95). Both PACA and PAM showed neuroprotective activity
with peak responses at the 2nM concentration. (Figures Sa, 6a) Neuroprotective activity
was similar to the positive control, 3,17,~ 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
10 occlusion. In ovariectomized rats, maximal lesions of 25% of the brain cross-sectional
area was observed. When treated with 3~17~1~-estradiol, maximal lesion area was reduced
by about 50%. These data demonstrate that only low nM concentrations of 3,17
estradiol are required to protect from ischemic lesions in vivo.
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TableI. Phenolic A Ring Re(lui~ t for the r~L~ Jlutectivity of Estrcttrienes.
Namet % of l~Estra~fiol
. . N~Llu~J~ut~Lion
~ R--H 3.17~-Estladiol 100*
RO~-- R=CH3 3,17~Est~3iol 3-O-ME -1.5
~ R=H F.~t~ nP 3 ol lû3*~
RO CX~
~ ~ R=H 3,170~Est~hol
R~ H R=CH3CO 3,17c~Est~ol 3-acetale -20
~ R---H 2-Hyd~xy-17~st~a~fiol 70*
RO~/ R=CH3 17~Esuadiol 2,3-O-ME 7
~) R=H Estrone 58*
R~bJ~~ R2CH3 Estrone 3-O-ME -11
QH
~ ~ R=H Estnol 46*
R~ ~ dOH R=CH Estriol 3-O-ME 2
~ c9cH R=H Ethynyl Esuadiol 41*
RO/~-- R=CH3 Mestranol -6
*p < 0.05 VS serum-free control groups as described in Figures 1 to 4 and note 8.t = trivial name iS
inr1ic~t~A here and ~illuClulcll name in note 10. ::=No 3-0-conjugates were available for evaluation.
SUBSTITUTE SHEET (RULE 26)
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Table II. r~.~ vtec~ ity of Estradiol, Phenol, Napthol and Dipenols
Name% of 17,B-Estradiol
Neuroprotection
,OH
~ 3,17~-Estradiol 100*
HOJ~--
HO~ Phenol -27
HO~ ~OH Diethylsdlbesrerol 74
HO~ / OCH3 Died ylsdlbo3rerol-n~ono-O ME 60
CH30J~J~ ~OCH3 Dicdlylsdlbe3terol-di-OMEi
*p<0.05 vs serum-free control groups, as described in note 8.
SUBSTITUTE SHEET (RULE 26)
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TABLE m
THE EFFECTS OF BRANCHED CHAIN
SUBSTITUTED PHENOLS ON SK-N-SH LIVE
CELL NUMBERS AT 48 HOURS EXPOSURE
Treatrnent Live Cell Number ~ SEM (103 cells/ml)
Serum free controls 203 + 16
Butylated Hydroxyanisol 182 + 16
Butylated hydroxytoluene 175 + 11
SUBSTITUTE SHEEt (RULE 26)
