U.S. patent application number 12/639708 was filed with the patent office on 2010-07-08 for 7-hydroxyepiandrosterone having neuroprotective activity.
This patent application is currently assigned to Hunter-Fleming Limited. Invention is credited to Ashley Ker Pringle, Lars Eric Sundstrom, Ernst Wulfert.
Application Number | 20100173883 12/639708 |
Document ID | / |
Family ID | 9894707 |
Filed Date | 2010-07-08 |
United States Patent
Application |
20100173883 |
Kind Code |
A1 |
Wulfert; Ernst ; et
al. |
July 8, 2010 |
7-HYDROXYEPIANDROSTERONE HAVING NEUROPROTECTIVE ACTIVITY
Abstract
3-Hydroxy-7.beta.-hydroxy steroids and 3-oxo-7.beta.-hydroxy
steroids and pharmaceutically acceptable esters thereof are useful
for protection against neuronal damage.
Inventors: |
Wulfert; Ernst; (Brussels,
BE) ; Pringle; Ashley Ker; (Southampton, GB) ;
Sundstrom; Lars Eric; (Old Alresford, GB) |
Correspondence
Address: |
DECHERT LLP
P.O. BOX 390460
MOUNTAIN VIEW
CA
94039-0460
US
|
Assignee: |
Hunter-Fleming Limited
Temple Quay
GB
|
Family ID: |
9894707 |
Appl. No.: |
12/639708 |
Filed: |
December 16, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10312533 |
Mar 26, 2003 |
7718639 |
|
|
PCT/GB01/02937 |
Jun 29, 2001 |
|
|
|
12639708 |
|
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|
Current U.S.
Class: |
514/178 |
Current CPC
Class: |
A61P 9/00 20180101; A61P
25/16 20180101; A61P 25/28 20180101; A61P 25/00 20180101; A61K
31/5685 20130101; A61P 25/02 20180101; A61P 9/10 20180101 |
Class at
Publication: |
514/178 |
International
Class: |
A61K 31/5685 20060101
A61K031/5685; A61P 9/10 20060101 A61P009/10; A61P 25/28 20060101
A61P025/28; A61P 25/02 20060101 A61P025/02; A61P 25/16 20060101
A61P025/16 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 29, 2000 |
GB |
GB0016022.6 |
Claims
1.-6. (canceled)
7. A method of treating neuronal damage in a mammal comprising
administering thereto an effective amount of
7.alpha.-hydroxyepiandrosterone.
8. The method according to claim 7, wherein the neuronal damage is
caused by stroke or brain trauma.
9. The method according to claim 7, wherein the neuronal damage is
caused by Alzheimer's Disease, Parkinson's Disease, Cognitive
Impairment No Dementia, spinal cord injury, or peripheral nerve
injury.
10. A method for reducing neuronal cell death following acute
neuronal damage in a mammal comprising administering thereto an
effective amount of 7.alpha.-hydroxyepiandrosterone.
11. The method according to claim 10, wherein the acute neuronal
damage is caused by stroke.
12. The method according to claim 10, wherein the acute neuronal
damage is caused by spinal cord injury.
13. A method for reducing neuronal cell death following chronic
neuronal damage in a mammal comprising administering thereto an
effective amount of 7.alpha.-hydroxyepiandrosterone.
14. The method according to claim 13, wherein the chronic neuronal
damage is caused by Alzheimer's Disease.
15. The method according to claim 13, wherein the chronic neuronal
damage is caused by Parkinson's Disease.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation of U.S. patent
application Ser. No. 10/312,523 filed May 29, 2003, which is the
371 national phase of Int'l App. No. PCT/GB01/02929, filed Jun. 29,
2001, which claims priority to GB0016027.5, filed Jun. 29, 2000,
each of which is incorporated by reference in its entirety.
[0002] The present invention relates to the use of certain
7-hydroxy-steroid compounds for protection against neuronal cell
death, and which are thus useful in the treatment and prevention of
such conditions or the sequelae of such conditions as Alzheimer's
Disease, Parkinson's Disease, Cognitive Impairment No Dementia
(CIND), stroke, brain trauma, spinal cord injury and peripheral
nerve injury; it is also useful for enhancing cognitive
function.
[0003] The production of 7.alpha.-hydroxylated metabolites of
dehydroepiandrosterone (DHEA) in vivo has been known since 1959
with the identification DHEA in urine [J J Schneider, M L Lewbart,
Recent Progr. Horm. Res. 15 (1959) 201-230; L Starka et al, Clin.
Chim. Acta. 7 (1961) 309-316)]. Since then, extensive
7.alpha.-hydroxylation of 3.beta.-hydroxysteroid substrates
(including DHEA and epiandrosterone EPIA) has been reported in
tissue preparations from many human organs, including adult and
foetal liver, testes, epididymus, skin, mammary tissue, prostate,
adipose stromal cells and tonsils. Hydroxylation of DHEA at the
7-position has also been demonstrated in rat liver and in numerous
mouse tissues and organs. In all these studies,
7.alpha.-hydroxy-DHEA was by far the major metabolite produced.
Indeed, Doostzadeh et al [Steroids 63 (1998) 608-614] reported that
the production rate of 7.alpha.-hydroxy-DHEA by mouse liver
microsomes was more than fifteen times the production rate of
7.beta.-hydroxy-DHEA.
[0004] EPIA, DHEA and pregnenolone have also been shown to be
rapidly and extensively transformed to their corresponding
7.alpha.-hydroxy metabolites in the rat brain [J M Guiraud et al,
Steroids 34 (1979) 241-248; M Warner et al, Endocrinology 124
(1989) 2699-2706; Y Akwa et al, Biochem. J. 288 (1992)
959-964)].
[0005] WO97/37664 discloses the use of certain specific compounds,
including hydroxy-substituted steroids, to treat neuropsychiatric,
immune or endocrine disorders. Among the disorders suggested in
WO97/37664 that these compounds may be used to treat is included
Alzheimer's Disease. However, the mechanism suggested for this
action is that the disorder is hypothesised to result from a
deficit of the 7.alpha.-hydroxy-substituted steroid in the brain,
and the treatment proposed in WO97/37664 thus rectifies this
deficit by the administration of a 7.alpha.-hydroxy-substituted
steroid to replace the missing compound. The procedure described in
WO97/37664 thus treats an existing condition, rather than
preventing the condition or preventing a worsening of the condition
by preventing further neuronal damage. WO97/37664 does not,
therefore, describe a neuroprotective effect. It also does not
suggest that the compounds may be used to prevent the damage caused
by sudden and traumatic events such as stroke.
BRIEF DESCRIPTION OF THE DRAWINGS
[0006] FIGS. 1A, 1B and 1C show graphs representing the number of
morphological intact hippocampal CA1 pyramidal cells 7 days after
global cerebral ischemia in rats and under the influence of
different compounds.
[0007] FIG. 1A, the data are presented as mean number.+-.SEM of
intact neurons per 400 .mu.m length of CA1 region.
[0008] FIG. 1B, the data are expressed as percentage of intact
neurons per 400 .mu.m length of CA1 region compared to sham
operated animals set as 100%.
[0009] FIG. 1C, the data are presented as absolute percentage of
neuroprotection when the number of surviving neurons in the
ischemia group was set to zero and those of the sham operated group
was set to 100%.
[0010] We have now surprisingly discovered that
7-hydroxy-epiandrosterone, whether .alpha., .beta. or a mixture,
can be used to protect against acute and chronic neuronal damage
caused by such events as stroke, brain trauma and cerebral
ischaemia such as may be induced by sub-arachnoid haemhorrage or
which occurs during heart bypass surgery etc.
[0011] In events such as prolonged hypoxia and ischaemia, which may
or may not be associated with hypoglycaemia, neuronal damage, to
varying degrees, is encountered.
[0012] Ischaemia typically occurs during heart attacks, but the
damage incurred at these times is substantially limited to the
heart tissues, and certain treatments have been developed. With
regard to the present invention, we are concerned with the effects
of more long term ischaemia on the brain, such as occurs with
stroke patients or as a result of head injury. The severity of the
ischaemia depends on the nature of the stroke or injury, but,
invariably, there is brain damage, and it is this which the present
invention addresses.
[0013] Various neuroprotective agents are known in the art which
attempt to alleviate the problem of brain damage, but all of those
currently known tend to be associated with adverse side effects.
For example, MK801 (dizocilpine maleate) is a fairly simple
molecule and is known to provide a level of neuroprotection to
ischaemic patients. However, MK801 is also associated with
"alarming psychotropic effects" (Martindale), as well as adverse
motor effects. The neuroprotective effects are detailed in Brain
Research 755 (1997) 36-46 (Pringle, A. K., et al), incorporated
herein by reference. These same authors also described the
neuroprotective effects of conotoxin in an earlier paper but,
despite the neuroprotective effects of this compound, adverse side
effects, in vivo, are observed. Thus, the present invention
consists in the use for the manufacture of a medicament for
protection against acute or chronic neuronal damage of
7-hydroxyepiandrosterone (7-hydroxy-EPIA). This compound may be
represented by the formula (I):
##STR00001##
[0014] and the 7.alpha. and 7.beta. isomers are, respectively:
##STR00002##
[0015] The invention also embraces the use of precursors of these
compounds and compounds which, in vivo, are metabolised to give
these compounds.
[0016] The .alpha. and .beta. isomers may be used alone or in
admixture, and, if in admixture, may be present in any proportions.
However, the 7.beta.-isomer appears to show greater activity and is
therefore presently preferred.
[0017] The compounds of the present invention may be prepared by a
variety of processes, well known in themselves, starting from the
parent steroids. For example, they may be prepared by the methods
described in the literature referred to above, which would give a
mixture of the 7.beta. and corresponding 7.alpha. compounds, which
may then be separated by well known techniques.
[0018] As an example, 7.alpha. and/or 7.beta.-hydroxy EPIA may be
obtained from DHEA by allylic oxidation after protection of the
3.beta.-hydroxy group and the 17-ketone group using conventional
methods. The product is then reduced with a soluble metal compound
catalyst (such as sodium hydride) and the 3.beta.-hydroxy and
17-ketone groups are deprotected. The 7.alpha.-hydroxy and
7.beta.-hydroxy epimers may then, if desired, be separated by
conventional means, for example column chromatography, and the
7.alpha.- and 7.beta.-hydroxy EPIA may be crystallised to
purity.
[0019] Alternatively, 7.alpha.- and 7.beta.-hydroxy-EPIA may be
prepared as illustrated by the following reaction scheme:
##STR00003##
[0020] In this reaction scheme, DHEA (II) is acetylated to give the
corresponding acetate of formula (III), which is then reacted with
ethylene glycol, to give the ketal of formula (IV). The ketal (IV)
is then oxidised as described in Example 3, to give the
corresponding 7-keto compound (V), which is then deacetylated, to
give the compound of formula (VI). This is reduced, to give
7-hydroxy-17-ketal-EPIA of formula (VII), which is then treated
with an acid to remove the ketal group and give 7-hydroxy-EPIA,
which is finally separated into the 7.beta.- and 7.alpha.-isomers
by chromatography, to give 7.alpha.-hydroxy-EPIA (IX) and
7.beta.-hydroxy-EPIA (X).
[0021] The compounds of the present invention may be applied to the
patient if it is suspected that they are in danger of an ischaemic
event, especially a stroke or head injury, or if they are suspected
of developing a chronic neurodegenerative disease, such as
Alzheimer's disease or CIND, which may be facilitated by chronic
sub-threshold brain ischaemia or by reduced neuronal energy
production, such as is frequently observed in the ageing brain.
Such prophylactic application may be exceedingly useful. However,
it has also been demonstrated that the compounds of the present
invention have useful activity, even if applied after an ischaemic
event, but it will be appreciated that it is preferred to
administer the compounds as soon as possible, in order to avoid as
much neuronal degeneration as possible. In some circumstances it
may be desirable to administer repeated doses, especially where the
patient remains in danger of an ischaemic event.
[0022] Suitable methods of administration are generally by
injection, in order to achieve the desired result as soon as
possible. Thus, intravenous injection is particularly preferred
but, in some circumstances it may be preferable to administer the
compound directly into the cerebrospinal fluid.
[0023] The dose of the compound of the present invention will vary
depending upon many factors, including the age, body weight and
general condition of the patient, as well as the mode, frequency
and route of administration. However, a dose of from 0.01 to 50
mg/kg body weight is generally recommended, a dose of from 0.05 to
20 mg/kg body weight being more preferred. This may be administered
in a single dose or in divided doses.
[0024] The invention is further illustrated by the following
non-limiting Examples, of which Examples 1 to 7 illustrate the
preparation of compounds of the present invention and Examples 8
and 9 illustrate their activity. In Examples 1 to 7, the Roman
numerals refer to the formulae in the reaction schemes shown
above.
EXAMPLE 1
DHEA-3-Acetate (III)
[0025] A solution of 50 ml of pyridine and 50 ml of acetic
anhydride containing 10 g of DHEA (II) (34.72 mmol) was heated to
reflux for 4 hours. The reaction medium was cooled, poured into
water and extracted with ethyl acetate. The organic phase was dried
over anhydrous sodium sulphate and evaporated to dryness. 11.0 g of
DHEA-3-acetate (III) (33.33 mmol, 96%), which was recrystallised
from ethanol, were obtained.
EXAMPLE 2
17-Ketal-DHEA-3-Acetate (IV)
[0026] A solution of 100 ml of toluene containing 5 g of
DHEA-3-acetate (15.15 mmol), 5 ml of ethylene glycol and a
catalytic amount of p-toluenesulphonic acid was heated to reflux
with steam distillation using a Dean-Stark apparatus for 24 hours.
The reaction medium was poured into 100 ml of a 10% w/v aqueous
potassium carbonate solution. The organic phase was decanted. The
aqueous phase was extracted with ethyl acetate. The organic phases
were combined and evaporated to dryness. 5.10 g of
17-ketal-3-DHEA-acetate (IV) (13.64 mmol, 90%), which was
recrystallised from ethanol, were obtained.
EXAMPLE 3
7-Keto-17-Ketal-DHEA-3-Acetate (V)
[0027] A solution of 70 ml of pyridine containing 5 g of
17-ketal-DHEA-3-acetate (IV) (13.37 mmol) and a catalytic amount of
Bengal Rose was irradiated using a medium-pressure mercury vapour
lamp with oxygen sparging. A catalytic amount of copper acetate was
added to the reaction medium after 24 hours. The reaction medium,
after 24 hours, was evaporated to dryness. The residue was purified
by flash chromatography (SiO.sub.2/ethyl acetate:cyclohexane 3/7).
3.11 g of 7-keto-17-ketal-DHEA-3-acetate (V) (8.02 mmol, 60%) were
obtained.
EXAMPLE 4
7-Keto-17-Ketal-DHEA (VI)
[0028] A solution of 50 ml of methanol containing 1% of potassium
hydroxide and 1 g of 7-keto-17-ketal-DHEA-3-acetate (V) (2.58 mmol)
was heated to reflux for 2 hours. The reaction medium was then
cooled, neutralised and then extracted with ethyl acetate. The
organic phase was dried over anhydrous sodium sulphate and then
evaporated to dryness. 802 mg of 7-keto-17-ketal-DHEA 5 (2.32 mmol,
90%), which was recrystallised from methanol, were obtained.
EXAMPLE 5
7-Hydroxy-17-Ketal-EPIA (VII)
[0029] 10 g of 7-keto-17-ketal-DHEA (VI) (28.90 mmol) were added to
a liquid ammonia solution at -33.degree. C. containing 2.65 g of
sodium. After 4 hours, ammonium chloride was added until the blue
colour disappeared. 2.65 g of sodium were then added. After 4
hours, ammonium chloride was again added until the blue colour
disappeared. Water was added and the ammonia was allowed to
evaporate. The reaction medium was extracted with ethyl acetate.
The organic phase was dried over anhydrous sodium sulphate and then
evaporated to dryness. 6.07 g of 7-hydroxy-17-ketal-EPIA (VII)
(17.34 mmol, 60%) were obtained.
EXAMPLE 6
7-Hydroxy-EPIA (VIII)
[0030] A solution of 100 ml of acetone containing 5 ml of water, 10
g of 7-hydroxy-17-ketal-EPIA (VIII) (28.57 mmol, 50%) and a
catalytic amount of p-toluenesulphonic acid was heated to reflux
for 4 hours. The reaction medium was cooled, poured into 100 ml of
a 10% w/v aqueous sodium carbonate solution and then extracted with
ethyl acetate. The organic phase was dried over anhydrous sodium
sulphate and then evaporated to dryness. The residue was purified
by flash chromatography (SiO.sub.2/ethyl acetate). 5.24 g of
7-hydroxy-EPIA (VIII) (17.14 mmol, 60%) were obtained.
EXAMPLE 7
7.alpha.-Hydroxy-EPIA (IX) & 7.beta.-Hydroxy-EPIA (X)
[0031] 7-Hydroxy-EPIA (VIII) (5 g) containing 7.alpha. and 7.beta.
epimers in a ratio 65/35 was purified by flash chromatography
(Al.sub.2O.sub.3/CHCl.sub.3). 7.beta.-Hydroxy-EPIA (X) (2.5 g) was
obtained first, before 7.alpha.-hydroxy-EPIA (IX) (1.34 g).
7.beta.-Hydroxy-EPIA (X) and 7.alpha.-hydroxy-EPIA (IX) were
recrystallised from ethyl acetate.
EXAMPLE 8
Protocol For Studying Hypoxic Neuronal Damage
[0032] Organotypic hippocampal slice cultures were prepared using
the basic method of Pringle et al (1996, 1997) modified as
follows:
[0033] Wistar rat pups (8-11 days old) were decapitated and the
hippocampus rapidly dissected into ice-cold Gey's balanced salt
solution supplemented with 4.5 mg/ml glucose. Slices were separated
and plated onto Millicell CM culture inserts (4 per well) and
maintained at 37.degree. C./5% CO.sub.2 for 14 days. Maintenance
medium consisted of 25% heat-inactivated horse serum, 25% Hank's
balanced salt solution (HBSS) and 50% minimum essential medium with
added Earle's salts (MEM) supplemented with 1 mM glutamine and 4.5
mg/ml glucose. Medium was changed every 3-4 days.
[0034] Experimental hypoxia was performed as described previously
(Pringle et al., 1996; 1997). Briefly, cultures were transferred to
serum free medium (SFM--75% MEM, 25% HBSS supplemented with 1 mM
glutamine and 4.5 mg/ml glucose) containing 5 .mu.g/ml of the
fluorescent exclusion dye propidium iodide (PI). Cultures were
allowed to equilibrate in SFM for 60 minutes prior to imaging. PI
fluorescence was detected using a Leica inverted microscope fitted
with a rhodamine filter set. Any cultures in which PI fluorescence
was detected at this stage were excluded from further study.
Hypoxia was induced by transferring cultures to SFM (+PI) which had
been saturated with 95% N.sub.2/5% CO.sub.2. Culture plates
(without lids) were then sealed into an airtight chamber in which
the atmosphere was saturated with 95% N.sub.2/5% CO.sub.2 by
continuously blowing through gas at 10 L/min for ten minutes before
being sealed and placed in the incubator for 170 mins (total time
of hypoxia was therefore 180 mins). At the end of the hypoxic
period cultures were returned to normoxic SFM containing PI and
placed back in the incubator for 24 hours.
[0035] Neuronal damage was assessed as described previously
(Pringle et al., 1996; 1997) using either NIH Image 1.60 running on
an Apple IIsi computer or OpenLab 2.1 (Improvision) running on a
Macintosh G4/400. Images were captured using a monochrome camera
and saved onto optical disk for offline analysis. Light
transmission images were captured prior to the addition of drugs,
and PI fluorescence images recorded at the end of the 24-hour
recovery period. The area of the CA1 cell layer was determined from
the transmission image. The area of PI fluorescence in CA1 was
measured using the density slice function within NIH Image or
Openlab, and neuronal damage expressed as the percentage of the CA1
in which PI fluorescence was detected above background.
[0036] Steroid compounds were prepared by making an initial 1 mg/ml
solution in ethanol and further diluting down in SFM. Compounds
were added to the cultures for 45 minutes prior to hypoxia, during
the hypoxic episode and during the post-hypoxic recovery period.
Control experiments consisted of cultures treated with vehicle
alone.
Results
Experiment 1:
[0037] An initial experiment was performed to determine whether
7.alpha.OH-EPIA and 7.beta.OH-EPIA were neuroprotective at a high
concentration of 100 nM. Hypoxia produced a lesion in 25.5.+-.6.4%
of CA1. This damage was significantly reduced by both
7.alpha.OH-EPIA and 7.beta.OH-EPIA when present pre-, during and
post-hypoxia as shown in Table I, below.
TABLE-US-00001 TABLE 1 Compound N % Damage in CA1 Control Hypoxia
17 25.5 .+-. 6.4 Hypoxia + 100 nM 7.alpha.OH-EPIA 16 4.0 .+-. 2.9**
Hypoxia + 100 nM 7.beta.OH-EPIA 16 9.0 .+-. 4.7**
Experiment 2:
[0038] Having determined that both the .alpha.- and .beta.-isomers
of 7OH-EPIA were neuroprotective, we assessed the
concentration-dependency of this effect. Control hypoxia resulted
in neuronal damage to 31.9.+-.4.7% of the CA1. 7.alpha.OH-EPIA was
significantly protective at 100 nM. A small, but not-statistically
significant reduction, in neuronal damage was observed at 10 nM,
and there was no effect at 1 nM. In contrast, 7.beta.OH-EPIA was
significantly neuroprotective at 10 nM and 100 nM, but activity was
lost if the concentration was reduced to 1 nM. (See Table 2).
TABLE-US-00002 TABLE 2 Compound N % Damage in CA1 Control Hypoxia
29 31.9 .+-. 4.7 Hypoxia + 1 nM 7.alpha.OH-EPIA 14 28.8 .+-. 5.8
Hypoxia + 10 nM 7.alpha.OH-EPIA 15 21.9 .+-. 8.1 Hypoxia + 100 nM
7.alpha.OH-EPIA 16 11.8 .+-. 2.8** Hypoxia + 1 nM 7.beta.OH-EPIA 15
20.6 .+-. 7.2 Hypoxia + 10 nM 7.beta.OH-EPIA 12 11.9 .+-. 4.7*
Hypoxia + 100 nM 7.beta.OH-EPIA 13 14.3 .+-. 5.0
EXAMPLE 9
Global Cerebral Ischemia in Rats (4 Vessel Occlusion)
[0039] Cerebral ischemia was induced by four-vessel-occlusion (4VO)
in male Wistar rats (250-280 g). Both vertebral arteries were
occluded by electrocauterization in pentobarbital anesthesia (60
mg/kg i.p.). The animals were allowed to recover for 24 hours with
free access to water but not food. The next day the carotid
arteries were exposed under 2% halothane in 30% oxygen/70% nitrous
oxide anesthesia and were occluded for 10 minutes using
microvascular claps. Subsequently, both clamps were removed and
both arteries were inspected for immediate reperfusion. During the
operation and the following 3 hours normothermia of the animals
(37.5.GAMMA.0.5.degree. C.) was maintained by using a
thermostatically controlled heating blanket connected to a rectal
thermometer. For control, in sham-operated animals both vertebral
arteries were cauterized in pentobarbital anesthesia and both
common carotid arteries were exposed but not clamped under 2%
halothane in 30% oxygen/70% nitrous oxide anesthesia the following
day. The wound was treated with lidocaine gel and then sutured. The
animals were kept under a heating tamp at 30.degree. C.
environmental temperature until they regained consciousness.
Seven groups of animals were investigated: [0040] 1. (n=8) steroid
compound, 7.beta.-OH EPIA (0.1 mg/kg, i.v. via tail vein, three
injections: 15 minutes prior to the induction of ischemia, during
ischemia and 5 minutes after reperfusion); [0041] 2. (n=8) steroid
compound, 7.beta.-OH EPIA (0.3 mg/kg, i.v. three injections as
described in 1.); [0042] 3. (n=8) steroid compound, 7.beta.-OH EPIA
(1 mg/kg, i.v., three injections as described in 1.); [0043] 4.
(n=8) NBQX (disodium salt, because more water soluble) as reference
substance and positive control (TOCRIS, Germany, 30 mg/kg, i.p.,
three injections as described in 1.); [0044] 5. (n=8) received
vehicle (0.9% NaCl, containing 100 .mu.l Ethanol) three injections
as described in 1.); [0045] 6. (n=8) ischemia alone; [0046] 7.
(n=8) sham operated controls.
[0047] NBQX is
2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline and is known
to have neuroprotective activity [Gill, R., Nordholm, L., Lodge D.:
The neuroprotective action of
2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline (NBQX) in a
rat focal ischaemia model. Brain Res. 580, 35-43, 1992].
[0048] 7.beta.-OH EPIA is a 7.beta.-hydroxyepiandrosterone, a
compound of the present invention.
[0049] The substances were dissolved in 100 .mu.l Ethanol and
finally diluted with 0.9% NaCl.
[0050] After a survival time of 7 days after ischemia, all animals
were perfusion fixed transcardially with 4% paraformaldehyde. The
brains were then removed carefully and postfixed in the same
fixative for 2 hours. After cryoprotection in 30% sucrose, the
brains were rapidly frozen in isopentane and stored at -80.degree.
C. Twenty-Micrometer cryostat sections comprising the hippocampal
formation were Nissl stained with toluidine blue or NeuroTrace
fluorescence.
Data Analysis:
[0051] The severity of neuronal damage in the hippocampal CA1
region after ischemia was evaluated by the number of surviving
neurons using Nissl staining. The mean number of morphologically
intact neurons per 400 .mu.m length was calculated in CA1 region
for each group. Cell counting was performed in 3-5 serial sections
per animal and 6 times 400 .mu.m CA1 area per section using a light
microscope equipped with a 20.times. objective. The data were
statistically analyzed by paired Student's t-test. Data were
presented as mean.+-.SEM.
Results and Discussion
[0052] The results are shown in FIGS. 1A, 1B and 1C of the
accompanying drawings.
[0053] Morphological intact hippocampal CA1 neurons were
characterized by Nissl staining (toluidine blue and NeuroTrace)
with the following criteria: clear shape of a neuronal perikarya,
large nucleus with a positive labeled nucleolus, a small cytoplasm
zone around the nucleus with positive Nissl staining, indicating
the intact rough endoplasmic reticulum with ribosomes and therefore
the intact protein synthesis machinery.
[0054] 10 minutes of global ischemia (mild ischemia) and a survival
time of 7 days leads to a neurodegeneration of pyramidal cells
selectively in the hippocampal CA1 region (FIG. 1A-1C). The mean
number of pyramidal cells in Ca1 of sham operated animals was
121.5.GAMMA.4.3 (set as 100%). Therefore, 60% of CA1 neurons died
after 10 minutes of global ischemia (FIG. 1B). The number of
neurons in the animal group of ischemia and i.v. injection of
vehicle (NaCl plus 100 .mu.l Ethanol) applied as described in the
experiment was comparable to that of the ischemia group alone
(FIGS. 1A, 1B). NBQX (30 mg/kg, i.v., three injections as described
in the experiment) showed a significant (p=0.03) neuroprotection in
CA1 pyramidal cells compared to the ischemia group. Compared to the
ischemia alone NBQX leads to a 47.5% neuroprotection while compared
to the sham operated animals the protective effect was 68.5%. The
neuroprotection caused by NBQX was in agreement with Gill et al.,
1992 and Gill 1994 demonstrating the validity of the global
ischemia model we used in our experiments. 7.beta.-OH EPIA leads to
a concentration dependent neuroprotection of hippocampal CA1
pyramidal cells after 10 minutes of global ischemia and a survival
time of 7 days (FIG. 1A). T-test analysis revealed a highly
significant neuroprotective effect 7.beta.-OH EPIA in
concentrations of 0.1 mg/kg (p=0.01) and 0.3 mg/kg (p=0.0008).
Compared to the sham operated group EPIA showed a 74.8% (0.1 mg/kg)
and a 83.9% (0.3 mg/kg) neuroprotective effect on CA1 pyramidal
cells, respectively (FIG. 1C). 7.beta.-OH EPIA in a concentration
of 1.0 mg/kg showed only a tendency to neuroprotection, but the
effect was not significant.
[0055] In all experiments with 7.beta.-OH EPIA injected i.v. prior,
during and after ischemia we never observed any behavioral
abnormalities of the animals.
* * * * *