Note: Descriptions are shown in the official language in which they were submitted.
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1
PROTECTINV NEURONS FROM ISCHEMIA
Background of the Invention
yl
The present invention is directed to the use of EGb 761 ~, more particularly
bilobalide, in protecting neurons from ischemic insults associated with
stimulation of
mitochondria) gene expression. l;schemia is caused by reduced or severely
blocked
blood flow, resulting in inadequate supply and loss of function of the
affected tissues.
1U The brain is very sensitive to ischemia because it has a high metabolic
rate and low
oxygen stores and small reservea of high-energy phosphates or carbohydrates. A
brief period of global brain ischerr~ia causes cell death in hippocampal CA1
pyramidal
neurons days after perfusion (Pulsinelli, W.A., et al., 1982, Ann. Neurol.,
11, 491-4.98
and Schmidt-Kastner, R., et al., 1991, Neuroscience 40, 599-636), whereas,
other
1 ~> neurons within the hippocampus, such as the CA3 sector and the dentate
gyrus are
largely resistant. Recently, factors of neuronal disorganization have been
suggested
to play an important role in causing selective neuronal death (Rothman, S.M.
and
Olney, J.W., 1986, Ann. Neurol., 19, 105-111; Widmann, R., et al., 1992, J.
Cereb.
Blood Flow Metab., 12, 425-433; Robin, O., et al., 1996, in Advances in
Ginkcro
20 biloba Extract Research, Vol. 5, Effect of Ginkgo biloba Extract (EGb 761~)
on
Neuronal Plasticity. Eds. Y. Christen, M.T. Droy-Lefaix and J. F. Macias-
Nunez,
Elsevier, Paris, pp. 7-19). These factors include massive accumulation of
calcium,
release of excitatory amino acids, increased release and incorporation of
arachidonic
acid and persistent disturbances ~of protein synthesis.
2:5 In a recent study, K. Abe et al., showed that the level of mRNA for
cytochrome c oxidase subunit 1 (COX 1 ), which is encoded by mitochrondrial
DNA
(mtDNA), progressively decreased in CA1 neurons of gerbils from 1 to 3 h of
the
reperfusion after 3.5 min transient forebrain ischemia and complete
disappeared at 7
days (Abe, K., et al., 1993, Mol. Brain Res., 19, pp. 69-75). The early
decrease in
30 COX I mRNA occurs in absence: of a decrease in mtDNA. Similar
disproportionate
decrease in levels in mtDNA-encoded COX subunit mRNA to the levels of mtDNA
are also observed in vulnerable brain regions in Alzheimer disease and due to
decreased neuronal activity in animal experiments (Chandrasekaran, K., et al.,
1994,
Mol. Brain Res., 24, 336-340; Hatanpaa, K., et al., 1996, Ann. Neurol., 40,
411-420,
CONFIRMATION COPY
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2
1996; Wong-Riley, M.T.T., 1989, Trends Neurosci., 12, pp. 94-101 ). These
results
suggest that disturbances in mtDNA gene expression occurs early in response to
acute changes in neuronal activity and energy demand. Pharmacological agents
which stimulate mitochondria) transcription may then help to protect or render
neurons less vulnerable to sudden metabolic insult such as ischemia and
excitotoxicity.
Recent studies of a leaf extract of Ginkgo biloba, termed EGb 761 ~, have
reported medicinal value of the product in the treatment of a variety of
clinical
disorders including cerebrovascular and peripheral vascular insufficiencies
associated with aging and senility. See e.g., Ginkgo biioba Extract (EGb 761~)
Pharmacological Activities and Clinical Applications, DeFeudis, F.V., Eds,
Elsevier,
1991; and Ullstein Medical 1998, Gingko biloba extract (EGb 761 ~), Eds.
Wiesbaden, DeFeudis, F.V. The' extract contains 24% ginkgo-flavone glycosides,
6%
terpene lactones (ginkgolides and bilobalide), about 7% proanthocyanidins and
~i 5 several other constituents. See Boralle, N., et al., In: Ginkgolides,
Chemistry,
Biology, Pharmacology and Clinical perspectives, Ed: Braquet, P., J.R. Prous
Science Publishers, 1988. Bilobalide accounts for about 3% of the total
extract. See
Drieu, K., Presse Med, 1986, 15, 1455-1457. Previously, a protective role by
bilobalide on mitochondria) respiration has been shown (Spinnewyn, B., Blavet,
N.
0 and Drieu, K. Effects of Ginkgo biloba extract (EGb 761 ~) on oxygen
consumption
by isolated cerebral mitochondria, Advances in Ginkgo biloba extract research;
Vol.
4, Eds. Y. Christen, Y. Courtoi:;, M-T Droy-Lefaix, pp. 17-22, Elsevier,
Paris, 1995).
In vivo administration of EGb 7(51~ was shown to increase mitochondria)
respiration
after cerebral ischemia in gerbils. The principal component in Ginkgo bifoba
extract
:25 that increased 'state 3' respiration (maximal rate of respiration achieved
in presence
of substrate, ADP and Pi) was identified to be bilobalide.
Summary of the lnventlon
30 In one aspect, this invention is directed to a method of protecting neurons
from ischemic insult in a patient in need thereof, which comprises
administering to
said patient an effective amount of bilobalide.
In a second aspect, this invention is directed to a pharmaceutical composition
useful for protecting neurons from ischemic insult in a patient in need
thereof, which
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3
comprises a pharmaceutically acceptable carrier and an effective amount of
bilobalide.
In another aspect, this invention is directed to a pharmaceutical composition
useful for protecting neurons from ischemic insult in a patient in need
thereof, which
:5 comprises a gingko biloba extract comprising an effective amount of
bilobalide.
In still another aspect, this invention is directed to a method of stimulating
mitochondria) gene expression in a patient in need thereof, which comprises
administering to said patient an effective amount of bilobalide.
In yet another aspect, this invention is directed to a pharmaceutical
composition useful for stimulating mitochondriai gene expression in a patient
in need
thereof, which comprises a pharmaceutically acceptable carrier and an
effective
amount of bilobalide.
In yet still another aspect, this invention is directed to a pharmaceutical
composition useful for stimulating mitochondria) gene expression in a patient
in need
thereof, which comprises a gingko biloba extract comprising an effective
amount of
bilobalide.
In a further aspect, this invention is directed to a method of protecting
neurons from ischemic insult in a patient in need thereof, which comprises
administering to said patient a gingko biloba extract comprising an effective
amount
a!0 of bilobalide.
In a further still another aspect, this invention is directed to a
pharmaceutical
composition useful for protecting neurons from ischemic insult in a patient in
need
thereof, which comprises a pharmaceutically acceptable carrier and an
effective
amount of bilobalide.
In an even further aspect, this invention is directed to a pharmaceutical
composition useful for protecting neurons from ischemic insult in a patient in
need
thereof, which comprises a gingko biloba extract comprising an effective
amount of
bilobalide.
In another further aspect, this invention is directed to a method of
stimulating
mitochondria) gene expression in a patient in need thereof, which comprises
administering to said patient a gingko biloba extract comprising an effective
amount
of bilobalide.
In a furthermore aspect, this invention is directed to a pharmaceutical
composition useful for stimulating mitochondria) gene expression in a patient
in need
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thereof, which comprises a pharmaceutically acceptable carrier and an
effective
amount of bilobalide.
In another furthermore aspect, this invention is directed to a pharmaceutical
composition useful for stimulating mitochondrial gene expression in a patient
in need
:5 thereof, which comprises a gingko biloba extract comprising an effective
amount of
bilobalide.
This invention is also directed to a method of treating stroke, head trauma,
spinal cord trauma, traumatic brain injury, multiinfarct dementia, Alzheimer's
disease,
senile dementia of the Alzheimer's type, Huntington's disease, Parkinson's
disease,
epilepsy, amyotrophic lateral sclerosis, pain, AIDS dementia, drug addictions,
or an
ischemic event arising from CPJS surgery, open heart surgery or any procedure
during which the function of the cardiovascular system is compromised, which
comprises administering to a patient in need thereof an effective amount of
biiobalide.
This invention is also furi:her directed to a pharmaceutical composition
useful
for treating stroke, head trauma, spinal cord trauma, traumatic brain injury,
multiinfarct dementia, AlzheimerAs disease, senile dementia of the Alzheimer's
type,
Huntington's disease, Parkinson's disease, epilepsy, amyotrophic lateral
sclerosis,
pain, AIDS dementia, drug addictions, or an ischemic event arising from CNS
2:0 surgery, open heart surgery or any procedure during which the function of
the
cardiovascular system is compromised, which comprises a pharmaceutically
acceptable carrier and an effective amount of bilobalide.
This invention is furthermore directed to, a pharmaceutical composition useful
for treating stroke, head trauma, spinal cord trauma, traumatic brain injury,
:!5 multiinfarct dementia, Alzheimer's disease, senile dementia of the
Alzheimer's type,
Huntington's disease, Parkinsoin's disease, epilepsy, amyotrophic lateral
sclerosis,
pain, AIDS dementia, drug addictions, or an ischemic event arising from CNS
surgery, open heart surgery or any procedure during which the function of the
cardiovascular system is compromised, which comprises a gingko biloba extract
:30 comprising an effective amount of biiobalide.
In the foregoing aspects of this invention, it is preferred that the gingko
biloba
extract comprising bilobalide is PGb 761 ~.
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Detailed Descriation
The term "ginkgo terpenoid" as used herein includes all of the naturally
5 occurring terpenes which are derived from the gymnosperms tree Ginkgo biioba
as
well as synthetically produced ginkgo terpenoids and pharmaceutically active
derivatives and salts thereof and mixtures thereof. Examples of ginkgo
terpenaids
include ginkgolides and bilobalide. Examples of ginkgo terpenoids are
disclosed in
Ginkgolides, Chemistry, Biology, Pharmacology, and Clinical Perspectives, J.R.
Prove. Science Publishers, Edited by P. Braguet (1988); F.V. DeFeudis, Ginkgo
Biloba Extract (EGb 761 ~); Pharmacological Activities and Clinical
Applications,
Elsevier, Chapter II (1991 ). Bilobalide has the following structure:
C 7H
O
t 5 The term "ginkgolide" and "bilobalide" herein include the various
ginkgolides
and bilobalide disclosed in the books cited above as well as non-toxic
pharmaceutically active derivatives thereof. Examples of ginkgolide and
bilobalide
derivatives include tetrahydro derivatives, acetyl derivatives, and alkyl
esters such as
the monoacetate derivatives and triacetate derivatives disclosed in Okabe, et
al., J.
Chem. Soc. (c), pp. 2201-2206 (1967).
The term "ginkgo bilob~a extract" as used herein includes a collection of
natural molecules, including terpenoids, derived from the ginkgo biloba tree.
Preferably, the extract is the ginkgo biloba extract EGb 761 ~.
Bilobalide can be administered by oral, parenteral (e.g., intramuscular,
intraperitoneal, intravenous or subcutaneous injection, or implant), nasal,
vaginal,
rectal, sublingual or topical routes of administration and can be formulated
with
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6
pharmaceutically acceptable carriers to provide dosage forms appropriate for
each
route of administration.
Solid dosage forms for oral administration include capsules, tablets, pills,
powders and granules. In such solid dosage forms, bilobalide is admixed with
at
least one inert pharmaceutically acceptable carrier such as sucrose, lactose,
or
starch. Such dosage forms can also comprise, as is normal practice, additional
substances other than such inert diluents, e.g., lubricating agents such as
magnesium stearate. In the case of capsules, tablets and pills, the dosage
forms
may also comprise buffering agents. Tablets and pills can additionally be
prepared
1 o with enteric coatings.
Liquid dosage forms for oral administration include pharmaceutically
acceptable emulsions, solutions, suspensions, syrups, the elixirs containing
inert
difuents commonly used in the art, such as water. Besides such inert diluents,
compositions can also include adjuvants, such as wetting agents, emulsifying
and
suspending agents, and sweetening, flavoring and perfuming agents.
Preparations according to this invention for parenteral administration include
sterile aqueous or non-aqueous solutions, suspensions, or emulsions. Examples
of
non-aqueous solvents or vehicles are propylene glycol, polyethylene glycol,
vegetable oils, such as olive oil and corn oil, gelatin, and injectable
organic esters
such as ethyl oleate. Such dosage forms may also contain adjuvants such as
preserving, wetting, emulsifying, and dispersing agents. They may be
sterilized by,
for example, filtration through ~~ bacteria-retaining fitter, by incorporating
sterilizing
agents into the compositions, by irradiating the compositions, or by heating
the
compositions. They can also be manufactured in the form of sterile solid
~~5 compositions which can be dissolved in sterile water, or some other
sterile injectable
medium immediately before use.
Compositions for rectal or vaginal administration are preferably suppositories
which may contain, in addition to the active substance, excipients such as
coca
butter or a suppository wax.
;30 Compositions for nasal or sublingual administration are also prepared with
standard excipients well known in the art.
The dosage of bilobalide in the compositions of this invention may be varied;
however, it is necessary that the amount of the active ingredient be such that
a
suitable dosage form is obtained. The selected dosage depends upon the desired
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therapeutic effect, on the route of administration, and on the duration of the
treatment. The dose can be administered as a single dose or divided into
multiple
doses.
An effective amount of bilobalide depends upon the condition being treated,
the route of administration chosen and ultimately will be decided by the
attending
physician or veterinarian. Bilobalide may be administered in an amount of 0.05
to 2
mg/kg body weight of the patient or, preferably, administered in an amount of
0.1 to 1
mg/kg body weight of the patient.
A pharmaceutical composition of bilobalide includes within its meaning an
extract of ginkgo biloba which contains bilobalide, such as EGb 761 ~. It
should be
noted that not all extracts of ginkgo biloba contains bilobafide, however, EGb
761~
(which is also known as TANA,KAN~, R_KAN~, TEBON1N~, TANAKENE~, and
TEBOFORTAN~) does.
The use of bilobalide in protecting neurons from ischemic insults associated
with stimulation of mitochondriall gene expression is evaluated using a cell
culture
model system. A morphological variant of rat pheochromocytoma PC12 cells,
called
PC12S (obtained from Dr. Sayeeda Zain, Dept. of Biochemistry, University of
Rochester, School of Medicine and Dentistry, 603 Elm St., Rochester, NY), that
retains the ability to grow attached to plastic tissue culture dish, is used.
PC12S cells
undergo differentiation in presence of nerve growth factor (NGF) and their
morphology resemble that of sympathetic neurons. In this cell culture system,
the
expression of mtDNA-encoded genes can be regulated by changes in intracellular
Na'. Thus, addition of ouabain, an inhibitor of the NaIK-ATPase or sodium
pump, or
monensin, a sodium ionophore, causes a significant decrease in levels of mtDNA-
encoded cytochrome c oxidase subunit ill (COX III) gene.
Cell Culture: a morphological variant of rat pheochromocytoma PC12 cells,
called
PC12S, that retains~the ability to grow attached to plastic tissue culture
dish, is used.
PC12S cells are grown in DMEM medium (Bio Fluids, Rockviile, Maryland)
containing
2 mM glutamine, 7.5% heat inactivated fetal calf serum, and 7.5% heat
inactivated
horse serum, with penicillin-streptomycin (Bio Fluids, Rockville, Maryland).
Nerve
growth factor (NGF); (Life Technologies, MD, USA) was added at a concentration
of
50 ng/ml. Two days after addition of NGF, PC12S cells demonstrate prominent
neurite outgrowth, and after 5 days, their morphology resembles that of
sympathetic
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8
neurons. All experiments are done on cells that are maintained in the presence
of
NGF for 10 days.
Chemicals: Ouabain and monensin were purchased from Research Biochemicais
(Natick, MA, USA). Bilobalide was obtained from Dr. Katy Drieu, {IPSEN,
France), it
;5 is also available from Sigma (S~t. Louis, Missouri). Ouabain is dissolved
in water,
whereas, bilobalide is dissolved in 95% ethanol. When ethanol is used as a
solvent,
appropriate control experiments are conducted using vehicle alone. Ethanol
concentrations are always <0.1 °/~. In studies related to the effect of
pretreatment of
bilobalide, cells are treated with 10 ~cg/ml for about 24 hours and then
ouabain is
added and total RNA is isolated at various time periods.
Experimental Procedure: PC12S cells grown in presence of NGF (on 10-cm
dishes) are used. Cells are treal:ed with drugs for various periods of time,
0, 1, 3, 6,
12, 24 and 48 hours after addition of drug. Cells are then washed once with
DPBS
(Dulbecco's Phosphate Buffered Saline; Bio-Fluids, Rockville, Maryland)
without
1'i calcium and magnesium and total RNA is isolated using the TRlzol reagent
(Life
Technologies, MD, USA). One nnl of TRlzol reagent is added directly to the
dish and
placed in a rocker for about 5 ruin. The suspension is then transferred to a 2
ml
eppendorf centrifuge tube containing 0.2 ml of chloroform. The tube is
vortexed for
about 15 seconds and is centrifuged for about 15 min at about 4 °C. The
aqueous
2~) phase is removed and total RNA is precipitated with half-volume of
isopropanol. The
total RNA is pelleted by centrifugation of about 13,000 g for about 15 min at
about 4
C, the pellet is washed once with 70% ethanol, dried and suspended in 100 ~c'I
of
DEPC (diethyl pyrocarbonate; Sic~ma, St. Louis, Missouri) treated water.
Northern blot analysis: Ten ~cg of total RNA is run on a 1.2% formaldehyde
2:i agarose gel and transferred onto a GeneScreen membrane as described by the
manufacturer (Dupont, New England Nuclear, Massachusetts, USA).
Prehybridization is done by about 76 hours at about 42 °C using the
hybridizoi
reagent (Oncor, MD, USA; Hybridizol I and Hybridizol II mixed in the ratio of
4:1 ).
Hybridization is done by about 48 hours at about 42 °C in the same
solution with the
3~) addition of [32P] labeled cytochn~me oxidase subunit Ill (COX III) probe.
Blots are
washed in 2 X SSC (1 X SSC == 150 mM sodium chloride, 15 mM sodium citrate),
0.1 % sodium dodecyl sulfate (SDS) at room temperature for about 30 min, 2 X
SSC,
0.1 % SDS at about 42 °C for about 1 hour with one change, and finally
at about 65 °
C with 0.2 X SSC, 0.1 % SDS for about 30 min. Blots are exposed to X-ray film
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9
(Biomax MS, Kodak, Rochester, NY, USA) with an intensifying screen for about
45
min to about 2 days at about -70 °C. The probe is removed form the
blots by placing
the blots in boiling DEPC-treated water for about 10 min. The blots are then
rehybridized with a ['2P] labeled control ø actin probe as described above.
The level
of RNA hybridized is quantified using an image analysis program (NIH image
1.54)
from autoradiograms of lower exposure than is used for photography. Ratios of
COX
III mRNA to ø actin mRNA are calculated.
Probe preparation and labeling: Cytochrome oxidase subunit III probe is
prepared
by polymerise chain reaction (PCR) using rat genomic DNA and primers
corresponding to rat mtDNA sequence. The PCR conditions are as follows: 30
cycles for about 1 min at about 94 °C, about 30 sec at about 55
°C, and about 1 min
at about 72 °C. The PCR product (565 bp) is purified by agarose gel
electrophoresis
separation, elution and alcohol precipitation (Quiagen, CA, USA). ø actin
probe is
prepared by isolating the cDNA insert from the plasmid clone (ATCC-# 78554,
American Type Culture Collection, MD, USA). The probes are labeled using the
random primer method and purified by gel-filtration (Pharmacia, NJ, USA).
Results: Expression of Mitochondria! DNA encoded Cytochrome oxidise subunit
III
mRNA in differentiated PC12S cells. To examine the effect of chemicals on
mtDNA-
encoded COX 111 gene expression, the basal level of COX III mRNA in PC12S
cells
:!0 treated with the vehicle, PC12S cells were differentiated with NGF for 10
days. The
cells were treated with the vehicle for various periods of time and total RNA
was
isolated. The total RNA samples were subjected to northern blot analysis with
a
probe for the mtDNA-encoded COX III gene and with a probe for p actin gene.
Northern blots showed discrete bands for COX III mRNA at 0.8 kb, and far the ø
actin
;z5 at 1.9 kb. Expression of nuclear DNA (nDNA)-encoded ø actin (1.9 kb} was
determined to ensure that equivalent amounts of RNA were loaded and
transferred to
each lane in Northern Blot analyses. In vehicle treated PC12S cells, the
amounts of
COX 111 mRNA and that of the ø actin mRNA were found to be roughly equivalent
among samples, see Table 1. No significant differences were observed in the
ratio of
30 COX III mRNA to ø actin mRNA among samples. Thus, in vehicle treated PC12S
cells, there was no significant change in the amount of COX III mRNA.
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Table 1
Ratio of COX III mRNA to Q actin mRNA in vehicle treated cells
5
Time in hours Ratio of COX III mRNA to ~i
actin mRNA
0 0.82
1 0.82
3 0.82
1.2
12 1.0
24 1.0
48 1.0
Expression of Mitochondrial DNA encoded Cytochrome oxidase subunit Ill
mRNA in differentiated PC12S cells in presence of bilobalide (Lot # BN52023;
CP
160.002, IPSEN). In Table 2, the results on COX III mRNA level obtained with
the
addition of increasing concentration of bilobalide (from 0.2~glml to 10uglml)
are
given. PC12S cells were treated for 6 hours with increasing concentrations of
bilobalide. Total RNA was then isolated and Northern blot analysis was done
for COX
III mRNA. Addition of 5 ~glml and 10 uglml bilobalide to differentiated PC12S
cells
caused a significant increase in the ratio of COX III mRNA to (3 actin mRNA.
The
r 5 stimulator effect of bilobalide reached two fold increase with these
concentrations.
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11
Table 2
Effect of addition of bilobalide on COX III mRNA in PC12S cells.
:i
Concentration of Bilobalide Ratio of COX III mRNA to (3
(uglml) actin mRNA
0 1.2
0.2 1.3
0.5 1.3
1 1.35
2 1.3
2.1
2.25
A time course on the expression of Mitochondrial DNA encoded Cytochrome
oxidase subunit III mRNA in differentiated PC12S cells in presence of
bilobalide (Lot
# BN52023; CP 160.002). The time course of the induction of mitochondrial gene
1i) expression by bilobalide was examined . Bilobalide was added at a
concentration of
10 ~cg/ml to differentiated PC12S cells and after various periods of
treatment, total
RNA was isolated. The total RNA samples were subjected to Northern Blot
analysis
with COX III probe and then wit6~ p actin probe. The results are shown in
Table 3.
Addition of 10 ~.g/ml bilobalide caused a significant increase in the amount
of COX ill
1.5 mRNA during the first 6 hours after addition and remained at a higher
level up to 48
hours.
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12
Table 3
Effect of Bilobalide on COX III mRNA levels - time course
Time in hours Ratio of COX Ili mRNA to p actin
mRNA
p 1.2
3 2.0
g 2.0
12 2.2
24 2.3
48 2.5
