U.S. patent application number 09/839905 was filed with the patent office on 2002-11-28 for method of treating, preventing or inhibiting central nervous system injuries and diseases.
Invention is credited to Koenig, Michael L., Meyerhoff, James L., Yourick, Debra L..
Application Number | 20020177558 09/839905 |
Document ID | / |
Family ID | 26894315 |
Filed Date | 2002-11-28 |
United States Patent
Application |
20020177558 |
Kind Code |
A1 |
Meyerhoff, James L. ; et
al. |
November 28, 2002 |
METHOD OF TREATING, PREVENTING OR INHIBITING CENTRAL NERVOUS SYSTEM
INJURIES AND DISEASES
Abstract
Methods of preventing, treating, or both preventing and treating
CNS injury, disease, neurotoxicity or memory deficit in a subject
by the administration of at least one lipoic acid compound to the
subject are disclosed. Examples of CNS injuries or disease include
traumatic brain injury (TBI), posttraumatic epilepsy (PTE), stroke,
cerebral ischemia, neurodegenerative diseases of the brain such as
Parkinson's disease, Dementia Pugilistica, Huntington's disease and
Alzheimer's disease, brain injuries secondary to seizures which are
induced by radiation, exposure to ionizing or iron plasma, nerve
agents, cyanide, toxic concentrations of oxygen, neurotoxicity due
to CNS malaria or treatment with anti-malaria agents, and other CNS
traumas. Examples of lipoic acid compounds include alpha-lipoic
acid (ALA), dihydrolipoic acid (DHLA), 2-(N,N-dimethylamine)
ethylamido lipoate-HCL (LA-plus), the oxidized or reduced R- or
S-isomers thereof, the metabolites of alpha-lipoic acid such as
6,8-bisnorlipoic acid and tetranorlipoic acid and analogs thereof.
Also disclosed are pharmaceutical compositions and kits comprising
at least one lipoic acid compound.
Inventors: |
Meyerhoff, James L.; (Silver
Spring, MD) ; Yourick, Debra L.; (Linthicum Heights,
MD) ; Koenig, Michael L.; (Silver Spring,
MD) |
Correspondence
Address: |
Office of the Staff Judge Advocate
U.S. Army Medical Research and Materiel Command
ATTN: MCMR-JA (Ms. Elizabeth Arwine)
504 Scott Street
Fort Detrick
MD
21702-5012
US
|
Family ID: |
26894315 |
Appl. No.: |
09/839905 |
Filed: |
April 20, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60198958 |
Apr 21, 2000 |
|
|
|
Current U.S.
Class: |
514/440 ;
424/94.1; 514/15.1; 514/18.2; 514/21.9; 514/356; 514/4.4;
514/8.4 |
Current CPC
Class: |
Y02A 50/411 20180101;
A61K 31/455 20130101; A61K 31/385 20130101; A61K 31/19
20130101 |
Class at
Publication: |
514/18 ;
424/94.1; 514/356; 514/440 |
International
Class: |
A61K 038/06; A61K
038/43; A61K 031/455; A61K 031/385 |
Goverment Interests
[0002] This invention was made by employees of the United States
Army. The government has rights in the invention.
Claims
1. A method of treating a subject suffering from a central nervous
system injury or disease comprising administering to the subject a
composition comprising a therapeutically effective amount of at
least one lipoic acid compound.
2. The method of claim 1, wherein the central nervous system injury
or disease is traumatic brain injury, posttraumatic epilepsy,
stroke, cerebral ischemia, or a neurodegenerative disease.
3. The method of claim 1, wherein the central nervous system injury
is induced by fluid percussion, a blunt object impacting the head
of the subject, an object which penetrates the head of the subject,
or exposure to radiation, ionizing or iron plasma,
electroconvulsive shock therapy, a nerve agent, cyanide, toxic
concentrations of oxygen, central nervous system malaria, or an
anti-malaria agent.
4. The method of claim 1, wherein the subject is a mammal.
5. The method of claim 4, wherein the mammal is human.
6. The method of claim 1, wherein the lipoic acid compound is
alpha-lipoic acid, dihydrolipoic acid, 2-(N,N-dimethylamine)
ethylamido lipoate-HCL, the oxidized or reduced R- or S-isomers
thereof, a metabolite of alpha-lipoic acid, or an analog
thereof.
7. The method of claim 1, wherein the composition is alpha-lipoic
acid, dihydrolipoic acid, or 2-(N,N-dimethylamine) ethylamido
lipoate-HCL.
8. The method of claim 1, wherein the composition further comprises
at least one reactive oxygen species scavenger or at least one
neurotrophic factor.
9. The method of claim 8, wherein the reactive oxygen species
scavenger is coenzyme Q, vitamin E, vitamin C, pyruvate, melatonin,
niacinamide, N-acetylcysteine, GSH, or a nitrone.
10. The method of claim 1, wherein the therapeutically effective
amount is about 0.001 mg to about 20 mg per kg of the subject.
11. The method of claim 10, wherein the therapeutically effective
amount is about 1 mg to about 10 mg per kg of the subject.
12. The method of claim 11, wherein the therapeutically effective
amount is about 3 mg to about 10 mg per kg of the subject.
13. The method of claim 1, further comprising a pharmaceutically
acceptable excipient.
14. The method of claim 1, wherein the composition is administered
intravenously, intradermally, subcutaneously, orally,
transdermally, transmucosally or rectally.
15. The method of claim 14, wherein the composition is administered
intravenously.
16. The method of claim 14, wherein the composition is administered
orally.
17. The method of claim 14, wherein the composition is administered
subcutaneously.
18. A method of preventing or inhibiting a central nervous system
injury or disease in a subject comprising administering to the
subject a composition comprising a therapeutically effective amount
of at least one lipoic acid compound.
19. The method of claim 18, wherein the central nervous system
injury or disease is traumatic brain injury, posttraumatic
epilepsy, stroke, cerebral ischemia, or a neurodegenerative
disease.
20. The method of claim 18, wherein the central nervous system
injury is induced by fluid percussion, a blunt object impacting the
head of the subject, an object which penetrates the head of the
subject, or exposure to radiation, ionizing or iron plasma,
electroconvulsive shock therapy, a nerve agent, cyanide, toxic
concentrations of oxygen. central nervous system malaria, or an
anti-malaria agent.
21. The method of claim 18, wherein the lipoic acid compound is
alpha-lipoic acid, dihydrolipoic acid, 2-(N,N-dimethylamine)
ethylamido lipoate-HCL, the oxidized or reduced R- or S-isomers
thereof, a metabolite of alpha-lipoic acid, or an analog
thereof.
22. The method of claim 19, wherein the lipoic acid compound is
alpha-lipoic acid, dihydrolipoic acid, or 2-(N,N-dimethylamine)
ethylamido lipoate-HCL.
23. The method of claim 18, wherein the composition further
comprises at least one reactive oxygen species scavenger or at
least one neurotrophic factor.
24. The method of claim 23, wherein the reactive oxygen species
scavenger is coenzyme Q, vitamin E, vitamin C, pyruvate, melatonin,
niacinamide, N-acetylcysteine, GSH, or a nitrone.
25. The method of claim 18, wherein the therapeutically effective
amount is about 0.001 mg to about 20 mg per kg of the subject.
26. The method of claim 25, wherein the therapeutically effective
amount is about 1 mg to about 10 mg per kg of the subject.
27. The method of claim 26, wherein the therapeutically effective
amount is about 3 mg to about 10 mg per kg of the subject.
28. The method of claim 18, further comprising a pharmaceutically
acceptable excipient.
29. The method of claim 18, wherein the composition is administered
intravenously, intradermally, subcutaneously, orally,
transdermally, transmucosally or rectally.
30. The method of claim 29, wherein the composition is administered
intravenously.
31. The method of claim 29, wherein the composition is administered
orally.
32. The method of claim 29, wherein the composition is administered
subcutaneously.
33. A method of preventing, inhibiting or treating a central
nervous system injury or disease, neurotoxicity or memory deficit
in a subject comprising administering to the subject a composition
comprising a therapeutically effective amount of at least one
lipoic acid compound.
34. The method of claim 33, wherein the lipoic acid compound is
alpha-lipoic acid, dihydrolipoic acid, 2-(N,N-dimethylamine)
ethylamido lipoate-HCL, the oxidized or reduced R- or S-isomers
thereof, a metabolite of alpha-lipoic acid, or an analog
thereof.
35. The method of claim 33, wherein the composition further
comprises at least one reactive oxygen species scavenger or at
least one neurotrophic factor.
36. The method of claim 35, wherein the reactive oxygen species
scavenger is coenzyme Q, vitamin E, vitamin C, pyruvate, melatonin,
niacinamide, N-acetylcysteine, GSH, or a nitrone.
37. A pharmaceutical composition for preventing, inhibiting, or
treating a central nervous system injury, disease or neurotoxicity
in a subject comprising a therapeutically effective amount of at
least one lipoic acid compound and a pharmaceutically acceptable
excipient.
38. The pharmaceutical composition of claim 37, wherein the lipoic
acid compound is alpha-lipoic acid, dihydrolipoic acid,
2-(N,N-dimethylamine) ethylamido lipoate-HCL, the oxidized or
reduced R- or S-isomers thereof, a metabolite of alpha-lipoic acid,
or an analog thereof.
39. The pharmaceutical composition of claim 37, wherein the
composition further comprises at least one reactive oxygen species
scavenger.
40. The pharmaceutical composition of claim 39, wherein the
reactive oxygen species scavenger is coenzyme Q, vitamin E, vitamin
C, pyruvate, melatonin, niacinamide, N-acetylcysteine, GSH, or a
nitrone.
41. A kit comprising a composition comprising a therapeutically
effective amount of at least one lipoic acid compound.
42. The kit of claim 41, wherein the lipoic acid compound is
alpha-lipoic acid, dihydrolipoic acid, 2-(N,N-dimethylamine)
ethylamido lipoate-HCL, the oxidized or reduced R- or S-isomers
thereof, a metabolite of alpha-lipoic acid, or an analog
thereof.
43. The kit of claim 41, wherein the kit or the composition further
comprises at least one reactive oxygen species scavenger or at
least one neurotrophic factor.
44. The kit of claim 42, wherein the reactive oxygen species
scavenger is coenzyme Q, vitamin E, vitamin C, pyruvate, melatonin,
niacinamide, N-acetylcysteine, GSH, or a nitrone.
45. The kit of claim 41, further comprising a device for
administrating the composition.
46. The kit of claim 41, further comprising instructions for
use.
47. The method of claim 33, wherein the lipoic acid compound is
administered before the central nervous system injury or disease,
neurotoxicity or memory deficit.
48. The method of claim 33, wherein the lipoic acid compound is
administered after the central nervous system injury or disease,
neurotoxicity or memory deficit.
Description
RELATED APPLICATION DATA
[0001] This application claims the benefit of U.S. Provisional
Patent Application No. 60/198,958, filed Apr. 21, 2000, naming
James L. Meyerhoff, Debra L. Yourick and Michael L. Koenig as
inventors, which is herein incorporated by reference.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention.
[0004] The invention relates to a method of treating, preventing or
inhibiting central nervous system (CNS) injuries and diseases. In
particular, the invention relates to a method of treating,
preventing or inhibiting a CNS injury or disease in a subject by
the administration of at least one lipoic acid compound to the
subject.
[0005] 2. Description of the Related Art.
[0006] Traumatic brain injury (TBI) can initiate a cascade of
events which may lead to dramatic elevation of intracranial
pressure (ICP), cerebral edema, ischemia, intracranial hemorrhage
and dysfunction of cerebrovascular regulatory mechanisms essential
for survival. Deficits in memory, attention, and perception,
emotional disorders, social behavioral problems, seizures
(including non-convulsive seizures), paralysis, aphasia,
post-traumatic epilepsy (PTE), and oxidative stress-induced
neurotoxicity may result from TBI.
[0007] In several studies of severely head-injured patients, over
80% had ischemic damage in the hippocampus. See McIntosh, T. K., et
al., (1996) Laboratory Investigation 74(2):315-342. The hippocampal
damage may explain the prevalence of memory defects in survivors of
TBI. Generally, the two main stages in the development of TBI are
(1) primary, including contusion, laceration, intracranial
hemorrhage and diffuse axonal injury; and (2) secondary, including
delayed effects such as seizures, ischemia, edema, and biochemical
reactions, which lead to necrosis and apoptosis.
[0008] Penetrating brain injuries, associated with retained
intracranial ferric metal fragments and inevitably associated with
hemorrhage, are highly likely to produce posttraumatic epilepsy
(PTE). See Salazar, A. M., et al., (1985) Neurology 35:1406-1414.
Development of seizures following penetrating craniocerebral trauma
has been associated with the presence of hematoma, total brain
volume loss, presence of retained metal fragments, hemiparesis,
aphasia, visual field loss, organic mental disorder, headache and a
history of seizures during the first year post injury.
[0009] Initial events in TBI such as hemorrhage and ischemia can
elicit activation of leukocytes and excessive release of the
excitatory neurotransmitter glutamic acid with resulting excess
influx of calcium. These events can trigger a number of interactive
intermediate reactions which can lead ultimately to neurotoxicity.
These include decompartmentalization of iron, and the activation of
several enzymes including phospholipases, xanthine oxidase,
intraneuronal nitric oxide synthase and poly[ADP-ribose]polymerase
(PARP). Formation of neurotoxic reactive oxygen species (ROS)
appears to be a result common to many of these "initiator" pathways
and is a major "perpetrator" in mediating necrotic neuronal death.
For example, it is well-known that glutamate, acting via both NMDA
and non-NMDA receptors, leads to increased intraneuronal calcium,
which in turn may activate (a) phospholipase A2, triggering
arachidonic acid production, or (b) xanthine oxidase. Both pathways
lead to the production of free radicals, such as superoxide.
Additional pathways leading to free radical formation include
liberation of "catalytic" iron from extravasated hemoglobin and
decompartmentalization of iron or copper from damaged mitochondria.
Thus, although the immediate mechanisms of pathologic responses to
nervous system may vary, many forms of neurotoxicity are believed
to share a common final pathway via formation of ROS, reactive
nitrogen species, or both. ROS are most aggressively damaging in
the central nervous system (CNS) as ROS attack double bonds in the
unsaturated fatty acids, which are abundant in CNS membranes, to
form carbon-centered radicals (R) or (R-HC-R) wherein "R" generally
refers to any carbon chain which length may vary. These radicals
initiate a chain reaction of lipid peroxidation, which continues
until arrested by the formation of a relatively non-reactive
species such as oxidized vitamin E or vitamin C. Scavengers, such
as vitamin E, also known as alpha-tocopherol, donate a hydrogen
atom to a radical, thereby becoming a secondary radical. Since the
tocopherol radical is rather stable, it breaks the chain reaction,
hence these scavengers are known as "chain-breaking
antioxidants".
[0010] The several neurotoxic pathways can produce a variety of
small, diffusible ROS, including superoxide, nitric oxide, peroxyl,
perhydroxyl, peroxynitrite, hypochlorous and singlet oxygen. The
antioxidant enzyme superoxide dismutase converts the superoxide
radical to hydrogen peroxide, a non-radical oxidizing agent that
can engage in a number of iron-catalyzed reactions producing the
very toxic hydroxyl ion. For example, the ferrous ion can trigger
the Fenton reaction with hydrogen peroxide to form hydroxyl ions
plus a ferric ion. Iron ions can also catalyze the Haber-Weiss
reaction, in which superoxide and hydrogen peroxide react to form
hydroxyl ions and molecular oxygen. Superoxide can also react with
nitric oxide to produce the intermediate peroxynitrite, which
subsequently yields the hydroxyl radical.
[0011] Although a particular neurotoxic reaction might predominate
initially, other pathways may rapidly be recruited, thereby
exacerbating damage. For example, hemoglobin, a potential source of
catalytic iron, potentiates excitatory amino acid-induced
neurotoxic injury in cortical cell culture. Ischemia is a secondary
effect of TBI and causes a metabolic imbalance wherein mitochondria
increase production of ROS while decreasing production of energy
required for neuronal homeostasis, engendering oxidative stress.
Injury-induced activation of PARP can deplete NAD+, and
consequently also deplete ATP. Depletion of energy sources such as
ATP transforms glutamic acid from neurotransmitter to neurotoxin.
Moreover, ROS exacerbate the excitotoxic pathways by increasing the
release of glutamate and inhibiting its reuptake inactivation.
[0012] There are important endogenous antioxidant defenses in the
central nervous system which are essential in providing cellular
resilience in response to injury. These protective mechanisms
include glutathione (GSH), alpha-lipoic acid (ALA), dihydrolipoic
acid (DHLA), coenzyme Q, vitamin E, vitamin C, pyruvate, melatonin,
and niacinamide. Some of these are synthesized endogenously and
some are dietary requirements. The sulfhydryl group of GSH is
particularly important in protecting cell membranes against
peroxidative stress. GSH peroxidase, using GSH as a co-substrate
and selenium as a metallic cofactor, reduces intracellular
formation of hydrogen peroxide and free radicals.
[0013] Unfortunately, these endogenous antioxidant defenses in the
central nervous system are not sufficient to prevent or inhibit
TBI, PTE and other related CNS traumas. Consequently, various
compounds and treatments have been developed.
[0014] Additionally, recent clinical trials of Tirilazad.TM.
(Upjohn) and Peg-SOD (superoxide dimutase linked to polyethylene
glycol) have been disappointing, their design has been
controversial, and leaves the question of the value of antioxidants
unresolved. See Marshall, S. B., et al., (1998) J. Neurosurg.
89(4):519-525. Tirilazad.TM. is an antioxidant aminosteroid which
proved to be clinically unsuccessful against stroke because it did
not readily cross the blood brain barrier and it failed to protect
the hippocampus. Furthermore, a number of large clinical trials
have failed to demonstrate a benefit of administering phenytoin,
phenobarbital, carbamazepine or valproate as a way to prevent the
onset of PTE.
[0015] Clearly, a need exists for effectively treating and
preventing TBI, PTE and other CNS traumas.
SUMMARY OF THE INVENTION
[0016] In some embodiments, the present invention relates to a
method of treating a subject suffering from a CNS injury or disease
comprising administering to the subject a composition comprising a
therapeutically effective amount of at least one lipoic acid
compound.
[0017] In some embodiments, the present invention relates to a
method of preventing or inhibiting a CNS injury or disease in a
subject comprising administering to the subject a composition
comprising a therapeutically effective amount of at least one
lipoic acid compound.
[0018] In some embodiments, the invention relates to a method of
preventing, inhibiting or treating neurotoxicity or memory deficit
in a subject comprising administering to the subject a composition
comprising a therapeutically effective amount of at least one
lipoic acid compound.
[0019] Where the memory deficit may be induced by electroconvulsive
shock therapy for treating diseases and disorders such as
depression and schizophrenia, the composition may be administered
before the electroconvulsive shock therapy to mitigate memory
loss.
[0020] In some embodiments, the CNS injury or disease may be
traumatic brain injury (TBI), posttraumatic epilepsy (PTE), stroke,
cerebral ischemia, or a neurodegenerative disease. In some
embodiments, CNS injury may be induced by fluid percussion, a blunt
object impacting the head of the subject, an object which
penetrates the head of the subject, or exposure to radiation,
ionizing or iron plasma, a nerve agent, cyanide, toxic
concentrations of oxygen, CNS malaria, or an anti-malaria
agent.
[0021] In the embodiments of the invention, the lipoic acid
compound may be alpha-lipoic acid (ALA), dihydrolipoic acid (DHLA
or DHL), 2-(N,N-dimethylamine) ethylamido lipoate-HCL (LA-plus),
the oxidized or reduced R- or S-isomers, a metabolite of
.alpha.-lipoic acid, or an analog thereof. In preferred
embodiments, the lipoic acid compound is ALA, DHLA, or LA-plus.
[0022] In some embodiments of the invention, the composition may
further comprise at least one ROS scavenger. Suitable ROS
scavengers include coenzyme Q, vitamin E, vitamin C, pyruvate,
melatonin, niacinamide, N-acetylcysteine, GSH, and nitrones.
[0023] In the embodiments of the present invention, the
therapeutically effective amount of the lipoic acid compound
administered to the subject is about 0.001 mg to about 20 mg per kg
of the subject, preferably about 1 mg to about 10 mg per kg of the
subject, more preferably about 3 mg to about 10 mg per kg of the
subject.
[0024] In some preferred embodiments, the total daily amount of the
lipoic acid compound administered to the subject is about 50 mg to
about 1200 mg, preferably about 100 mg to about 1000 mg, more
preferably about 200 mg to about 800 mg, even more preferably about
300 mg to about 600 mg.
[0025] In some embodiments, the invention relates to administering
the lipoic acid compound to a subject a period of time before the
subject is exposed or likely to be exposed to a risk of CNS injury
or damage or before the subject is exposed to conditions likely to
cause neurotoxicity or memory deficit or both. The conditions
likely to cause CNS injury or damage, neurotoxicity or memory
deficit include electroconvulsive shock therapy, traumatic brain
injury (TBI), posttraumatic epilepsy (PTE), stroke, cerebral
ischemia, neurodegenerative diseases, fluid percussion, a blunt
object impacting the head of the subject, an object penetrating the
head of the subject, radiation, ionizing or iron plasma, nerve
agents, cyanide, toxic concentrations of oxygen, CNS malaria, and
anti-malaria agents. Other conditions likely to cause CNS injury or
damage, neurotoxicity or memory deficit include certain medical
procedures or conditions associated with risk for CNS ischemia,
hypoxia or embolism such as brain tumor, brain surgery, open heart
surgery, carotid endarterectomy, repair of aortic aneurysm, atrial
fibrillation, cardiac arrest, cardiac or other catheterization,
phlebitis, thrombosis, prolonged bed rest, prolonged stasis (such
as during space travel or long trips via airplane, rail, car or
other transportation), CNS injury secondary to air/gas embolism or
decompression sickness.
[0026] The period of time may be about 72 hours to about the time
of expected exposure, preferably about 48 hours to about the time
of expected exposure, more preferably about 12 hours to about the
time of expected exposure, even more preferably about 4 hours to
about the time of expected exposure, and most preferably about 2
hours to about the time of expected exposure.
[0027] The administration of the lipoic acid compound may be
continuous from the initial time of treatment to the end of
treatment. For example, a transdermal patch or a slow-release
formulation may be used to continually administer the lipoic acid
compound to the subject for a given period of time. Alternatively,
the lipoic acid compound may be administered to the subject
periodically. For example, the lipoic acid compound may be first
administered at about 24 hours before the time of expected exposure
and then administered at about every 2 hours thereafter. In some
embodiments, at least one ROS scavenger such as coenzyme Q, vitamin
E, vitamin C, pyruvate, melatonin, niacinamide, N-acetylcysteine,
GSH, or a nitrone, is administered prophylactically in combination
with the prophylactic administration of the lipoic acid
compound.
[0028] In the embodiments of the invention, the composition may
further comprise a pharmaceutically acceptable excipient. The
composition may be administered intravenously, intradermally,
subcutaneously, orally, transdermally, transmucosally or rectally.
Preferably, the composition is orally administered.
[0029] In some embodiments, the invention relates to a
pharmaceutical composition for treating or preventing CNS injury,
disease or neurotoxicity in a subject comprising a therapeutically
effective amount of at least one lipoic acid compound and a
pharmaceutically acceptable excipient. The lipoic acid compound may
be ALA, DHLA, LA-plus, the oxidized or reduced R- or S-isomers, a
metabolite of ALA, or an analog thereof. The pharmaceutical
composition may further comprise at least one ROS scavenger.
Examples of suitable ROS scavengers include coenzyme Q, vitamin E,
vitamin C, pyruvate, melatonin, niacinamide, N-acetylcysteine, GSH,
and nitrones.
[0030] In some embodiments, the invention relates to a kit
comprising a composition comprising a therapeutically effective
amount of at least one lipoic acid compound. The lipoic acid
compound may be ALA, DHLA, LA-plus, the oxidized or reduced R- or
S-isomers, a metabolite of ALA, or an analog thereof. In a
preferred embodiment, the lipoic acid compound is ALA, DHLA, or
LA-plus. The kit or the composition may further comprise at least
one ROS scavenger. Suitable ROS scavengers include coenzyme Q,
vitamin E, vitamin C, pyruvate, melatonin, niacinamide,
N-acetylcysteine, GSH, and nitrones. The kit may further comprise a
device for administering the composition to a subject such as an
injection needle, an inhaler, a transdermal patch. The kit may also
comprise instructions for use.
DESCRIPTION OF THE DRAWINGS
[0031] This invention is further understood by reference to the
drawings wherein:
[0032] FIG. 1 illustrates that DHLA is protective in a
concentration-related manner against hemoglobin-induced toxicity in
cultured neurons in vitro.
[0033] FIG. 2 illustrates that DHLA is protective against
experimental spinal cord injury in rats in vivo.
[0034] FIG. 3a is an example of an iron-induced paroxysmal spiking
seizure on electroencephalogram (EEG) in rat.
[0035] FIG. 3b is an example of an iron-induced spike-and-wave
seizure on electroencephalogram (EEG) in rat.
[0036] FIG. 4 illustrates that pretreatment with ALA and
co-treatment with DHLA reduces iron chloride-induced seizure
activity by about 55%.
[0037] FIG. 5 illustrates the neuroprotective effects of DHL and
SPBN.
[0038] FIG. 6 illustrates the neuroprotective effects of DHL and
PBN.
DETAILED DESCRIPTION OF THE INVENTION
[0039] The present invention relates to a method of preventing,
treating, or both preventing and treating a CNS injury or disease
by the administration of at least one lipoic acid compound.
[0040] Examples of CNS injuries or disease include TBI, stroke,
concussion (including post-concussion syndrome), cerebral ischemia,
neurodegenerative diseases of the brain such as Parkinson's
disease, Dementia Pugilistica, Huntington's disease and Alzheimer's
disease, brain injuries secondary to seizures which are induced by
radiation, exposure to ionizing or iron plasma, nerve agents,
cyanide, toxic concentrations of oxygen, neurotoxicity due to CNS
malaria or treatment with anti-malaria agents, and other CNS
traumas. Examples of lipoic acid compounds include alpha-lipoic
acid (ALA), dihydrolipoic acid (DHLA or DHL), 2-(N,N-dimethylamine)
ethylamido lipoate-HCL (LA-plus), the oxidized or reduced R- or
S-isomers, the metabolites of ALA such as 6,8-bisnorlipoic acid and
tetranorlipoic acid and analogs thereof. Analogs of lipoic acid
compounds include lipoamides.
[0041] ALA is a physiological constituent of mitochondrial
membranes and is an essential cofactor in the oxidative
decarboxylation of alpha-keto acids such as pyruvate and
alpha-ketoglutarate. ALA is present in food and in mammalian
tissues. ALA is clinically safe, well-tolerated and is currently
used to treat peripheral neuropathy in patients with diabetes. ALA
is stable in tablet form, can be given orally, has good
bioavailability in humans, is well-tolerated and is endogenously
converted to DHLA.
[0042] Both ALA and DHLA are effective scavengers of numerous ROS
and have been shown to be neuroprotective in several rodent models
of cerebral ischemia. They are rapidly distributed to the central
nervous system, improve memory, are active in both lipid and
aqueous phases, and increase intracellular levels of GSH and
ATP.
[0043] ALA and DHLA may be used in methods for treating or
prevention TBI, PTE and other CNS traumas. In particular, they
protect neurons, among other things, by (1) acting as direct
chain-breaking antioxidants; (2) recycling the antioxidant vitamins
C and E; (3) scavenging at least 8 types of free radicals; (4)
chelating decompartmentalized iron ions; (5) chelating copper ions;
(6) increasing intracellular energy stores which include ATP; (7)
regenerating and increasing intracellular levels of the endogenous
antioxidant, glutathione; (8) increasing the ratio of reduced to
oxidized coenzyme Q; and (9) providing methionine sulfoxide
reductase with reducing equivalents. DHLA also enhances the repair
of oxidatively damaged proteins.
[0044] As illustrated in FIG. 1, DHLA was recently demonstrated to
be protective in a concentration-related manner (about 0.3 to about
10 micromolar) against hemoglobin-induced toxicity in cultured
neurons in vitro. See Koenig et al., (1999) Neurosci. Abs., 25,
which is herein incorporated by reference. Here, primary cultures
of neurons derived from the forebrains of E-15 rat fetuses were
pre-incubated with DHLA at several different concentrations for
about 4 hours. The neurons were then exposed to about 10 micromoles
of hemoglobin and resulted in about 50% viability after about a 24
hour exposure. Neuronal viability was assessed by using the
colorimetric MTT assay of succinate dehydrogenase activity. MTT is
a colorimetric dye, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-
zolium bromide, and is available from Sigma. Neuroprotection
increased with increasing concentrations of DHLA. There was no
evidence of neuroprotection where DHLA was coapplied with
hemoglobin.
[0045] As illustrated in FIG. 2, DHLA was also demonstrated to be
protective against experimental spinal cord injury in rats in vivo.
Here, about 5 micromoles of DHLA was administered intrathecally as
a 2 hour pretreatment. Flaccid paralysis was acutely evident about
10 minutes after injection with dynorphin A and where there was no
pretreatment with DHLA or co-treatment with DHLA. Pretreatment with
DHLA and treatment after injury with DHLA was observed to improve
motor scores. See Long et al., (1999) Neurosci. Abs. 25, which is
herein incorporated reference.
[0046] A modified Willmore model of ferric chloride-induced
epilepsy may be used to automatically record and quantify seizure
activity on a 24 hour basis as shown in FIGS. 3a and 3b. Here,
ferric chloride was injected into the cerebral cortex of a rat
which elicits intense seizures. FIG. 3a shows an example of an
electroencephalograph (EEG) paroxysmal spiking seizure pattern.
FIG. 3b shows an example of an EEG "spike and wave" seizure
pattern. By using this modified neurotoxic model, as illustrated in
FIG. 4, pretreatment with (.alpha.-LA and co-treatment with DHLA
reduced iron chloride-induced seizure activity by about 55%. In
this experiment, male Sprague Dawley rats were injected with 100
mg/kg of ALA i.p. once daily for 48 hours then anesthetized and 50
mg/kg of DHLA was injected i.p. after which 600 micromolar of
ferric chloride was injected unilaterally intracortically. EEG was
recorded for 24 hours and sampled for 10 seconds for each 60
seconds over a period of 24 hours. To achieve uniformly
quantitative information, the number of seconds of seizure activity
was measured in every sampled 10 seconds. As shown in FIG. 4, rats
pretreated with alpha-LA and DHLA had about 55% less seizure
activity than rats that were not pretreated.
[0047] LA-plus is a positively charged water soluble lipoic acid
amide analog which shows a higher rate of intracellular reduction
and retention than is seen with ALA, as well as better in vitro
protection against glutamate-induced loss of GSH, formation of
peroxide and neurotoxicity in cultured mouse hippocampal HT4 cells.
See Tirosh et al., (1999) Free Rad. Biol & Med.
26(11/12):1418-1426, which is herein incorporated by reference. In
addition to the administration of a lipoic acid compound, at least
one ROS scavenger may also be administered. Examples of ROS
scavengers include coenzyme Q, vitamin E, vitamin C, pyruvate,
melatonin, niacinamide, N-acetylcysteine, GSH and nitrones.
[0048] The neuroprotective efficacy of the lipoates may be enhanced
by supplementary compounds. For example, the neuroprotective
efficacy of the lipoates might be enhanced by (1) combination with
other free radical scavenger with slightly differing mechanisms of
action (e.g. coenzyme Q; or nitrones); or (2) with other classes of
neuroprotectants with fundamentally different mechanisms, such as
the neurotrophic factors brain-derived neurotrophic factor (BDNF),
nerve growth factor (NGF), neurotrophins (e.g. NT-3) and/or the
neuroprotective endogenous TRH analog, pyroGlu-Glu-Pro (known by
its abbreviation, EEP).
[0049] Coenzyme Q10 (CoQ10), also known as ubiquinone, is an
endogenous molecule that transports electrons in mitochondria, as
part of the process of ATP generation. CoQ10 levels in spinal cord
have been reported to be decreased following injury and the changes
were found to be reflective of the degree of trauma. Ubiquinone 10
has been found to be a lipid-soluble antioxidant which can prevent
lipid peroxidation in brain synaptosomes and mitochondria, recycle
vitamin E and scavenge ROS. Pretreatment with coenzyme Q reduced
33malonate-induced neurotoxicity in vivo. See Beal et al., (1994)
Eur. J. Pharmacology-Molec. Pharm. Section 266:291-300, which is
herein incorporated by reference. Although some studies reported
that it was therapeutically efficacious in certain clinical
situations, others have found it ineffective. See Nishikawa et al.,
(1989) Neurol. 39:399-403; Matthews, et al., (1993) Neurol.
43:884-890, which are herein incorporated by reference. However, it
is clearly an antioxidant neuroprotectant which may be synergistic
with alpha-LA much as it synergizes with niacinamide. See Gotz et
aL, (1994) Eur. J. Pharmacology - Molec. Pharm. Section
266:291-300; Beal, et al., (1994) Ann. Neurol. 36:882-888, which
are herein incorporated by reference. Accordingly, coenzyme Q may
be co-administered with the lipoic acids of the invention.
[0050] Nitrone-based free radical traps (nitrones) such as
alpha-Phenyl-N-tert-butylnitrone (PBN),
N-tert-Butyl-.alpha.-(2-sulfophen- yl)nitrone (SPBN), Azulenyl
nitrones, and NXY-059, offer a ROS scavenging mechanism which
differs from vitamin E, the lipoic acids and other endogenous
compounds. The nitrones react covalently with ROS to form stable
nitroxides. They have also been shown to be neuroprotective against
glutamate-induced toxicity in cultured neurons as well as in
several rodent models of cerebral ischemia, including transient
global ischemia, transient and permanent occlusion of the middle
cerebral artery. Therefore, nitrones may be co-administered with
the lipoic acids of the invention.
[0051] SPBN is an effective neuroprotectant without significant
toxicity. The free radical scavenger SPBN is synergistic with
brain-derived neurotrophic factor (BDNF) in survival of axotomized
retinal ganglion cells. See Klocker et al., (1998) J. Neurosci.
18(3):1038-1046, which are herein incorporated by reference. Thus,
BDNF may be co-administered with the lipoic acids of the invention.
PBN is a nitrone which has been shown to be neuroprotective in
animal models of ischemia, even when administered post injury. PBN
is also effective as a pre-treatment against DFP-induced
convulsions.
[0052] As described in Example 7 and shown in FIGS. 5 and 6, since
nitrones have more than one mode of action, as do the lipoic acids,
and since the scavenging mechanisms of nitrones and lipoic acids
differ, nitrones may be co-administered with lipoic acids to
provide a synergistic neuroprotective effect.
[0053] Since one group of compounds such as lipoic acids might be
more suitable for pretreatment, and another group such as nitrones
might be more suitable for post-injury treatment, the times and
amounts that the lipoic acid compounds and the supplementary
compounds are administered may be varied. For example, an
individual known to be at risk for certain injuries may be
administered a lipoate. After an injury occurs, the individual
would be administered a supplementary active compound such as a
nitrone, trophin, or other free radical scavenger. The lipoate
administered before the injury would confer a protection at the
time of injury and afterwards. The post-injury administration of
the supplementary active compound would provide a synergistic
neuroprotective effect with the lipoate.
[0054] A tripeptide comprising three linked amino acids,
pyroglutamate-glutamate-proline-amide, which is referred to as EEP,
is structurally similar to thyrotropin releasing hormone (TRH)
which comprises pyroglutamate-histidine-proline. Both EEP and TRH
are found in the brain. Like TRH, EEP is neuroprotective against
Glutamate-induced neurotroxicity. But unlike TRH, EEP is not
hydrolyzed in plasma by the TRH-hydrolyzing enzyme thyroliberinase.
Thus, EEP has better clinical neuroprotective potential than TRH.
However, either EEP or TRH or both may be administered in
combination with the lipoic acids of the invention.
[0055] Vitamin E, alpha-Tocopherol, is a normal dietary component
and prevents peroxidative injury of sulfhydryl groups of
glycolipids and glycoproteins by augmenting the antioxidant effects
of enzyme systems such as glutathione peroxidase. It stimulates the
synthesis of ATP, decreases lipid peroxidation, attenuates
neurotoxic effects of iron in vitro, and prevents iron-induced
seizures in vivo. Although vitamin E exhibits a slow rate of
absorption through the blood brain barrier and is not suitable in
methods to rapidly treat a brain injury, vitamin E may be suitable
for prophylactic treatments and treatments over long periods of
time.
[0056] As ischemia induces a significant decrease of the activity
of E1 component of the pyruvate dehydrogenase complex and DHLA
protects the E1 component from chemical inactivation, the lipoic
acid compounds of the present invention may be coupled via an amide
linkage to one of the three enzymes of the pyruvate dehydrogenase
complex.
[0057] In the method of the present invention, a therapeutically
effective amount of at least one lipoic acid compound or an analog
thereof may be given prophylactically to a subject having a high
risk for obtaining a CNS injury or disease. A subject pretreated
with a lipoic acid compound or analog thereof would have an
increased antioxidant reserve and would therefore have an increased
resistance to CNS injuries and diseases. Administration of the
lipoic acid compound may be continued after obtaining the CNS
injury or disease.
[0058] Lipoic acid compounds may be administered to a subject as a
method of preventing or treating CNS damage induced by exposure to
nerve agents such as Soman, sarin, VX and the like. Soman elicits a
sustained increase in extracellular glutamate levels in the
amygdala, induces the de-compartmentalization of iron in brain
tissue and catalyzes the formation of ROS.
[0059] Lipoic acid compounds may be administered to a subject as a
method of preventing or treating radiation-induced CNS damage as
inhibition of free radical formation provides neuroprotection to
cultured cortical neurons exposed to ionizing radiation and
injections of lipoic acid protects hematopoietic tissues in
gamma-irradiated mice. Lipoic acid should also protect against
injury to CNS, cardiac and hematopoeitic tissues secondary to
cancer chemotherapy.
[0060] Additionally, lipoic acid compounds may be administered to a
subject suffering from CNS malaria or a subject being treated with
Arteether and related antimalarial compounds as a method to prevent
or treat CNS damage.
[0061] Further, the lipoic acid compounds may be administered to a
subject exposed to high oxygen under pressure (OHP) as the
pathophysiological mechanism in toxicity induced by OHP may involve
free radical generation. OHP causes seizures and convulsions.
[0062] The lipoic acid compounds may be administered
prophylactically to a subject at risk for CNS injury or disease.
Subjects at risk for CNS injury or disease include participants in
contact sports such as football and boxing, military personnel,
astronauts and others at risk for exposure to blast overpressure,
blunt head trauma, and penetrating brain injury. The lipoic acid
compounds may be administered intravenously, intradermally,
subcutaneously, orally, transdermally, transmucosally or
rectally.
[0063] A therapeutically effective amount of the lipoic acid
compound of the invention may be administered to a subject a period
of time before the subject is exposed to a risk of CNS injury or
damage or before the subject is exposed or likely to be exposed to
conditions likely to cause neurotoxicity or memory deficit or both.
The conditions likely to cause CNS injury or damage, neurotoxicity
or memory deficit include electroconvulsive shock therapy,
traumatic brain injury (TBI), posttraumatic epilepsy (PTE), stroke,
cerebral ischemia, neurodegenerative diseases, fluid percussion, a
blunt object impacting the head of the subject, an object
penetrating the head of the subject, radiation, ionizing or iron
plasma, nerve agents, cyanide, toxic concentrations of oxygen, CNS
malaria, and anti-malaria agents. The period of time may be about
72 hours to about the time of expected exposure, preferably about
48 hours to about the time of expected exposure, more preferably
about 12 hours to about the time of expected exposure, even more
preferably about 4 hours to about the time of expected exposure,
and most preferably about 2 hours to about the time of expected
exposure. It is to be understood, however, that the lipoic acid
compound may be administered at a period of time which is more than
about 72 hours before the time of expected exposure. For example,
the period of time may be about 7 days before the expected
exposure. The administration of the lipoic acid compound may be
continuous from the initial time of treatment to the end of
treatment. For example, a transdermal patch or a slow-release
formulation may be used to continually administer the lipoic acid
compound to the subject for a given period of time. Alternatively,
the lipoic acid compound may be administered to the subject
periodically. For example, the lipoic acid compound may be first
administered at about 24 hours before the time of expected exposure
and then administered at about every 2 hours thereafter.
[0064] ROS scavengers such as coenzyme Q, vitamin E, vitamin C,
pyruvate, melatonin, niacinamide, N-acetylcysteine, GSH, and
nitrones may also be administered prophylactically along with the
lipoic acid compound. For example, as described in Beal, M. F., et
al., (1994) Ann. Neurol. 36:882-888, which is herein incorporated
by reference, therapeutically effective doses of coenzyme Q may be
administered in combination with therapeutically effective doses of
a lipoic acid compound. Additionally, neurotrophic factors such as
brain-derived neurotrophic factor (BDNF), nerve growth factor
(NGF), neurotrophins, and analogs thereof, may be administered
prophylactically along with the lipoic acid compound.
[0065] The therapeutically effective amount of the lipoic acid
compound administered prophylactically to the subject may be about
0.001 mg to about 20 mg per kg of the subject, preferably about 1
mg to about 10 mg per kg of the subject, more preferably about 3 mg
to about 10 mg per kg of the subject. The total daily amount of the
lipoic acid compound administered prophylactically to the subject
may be about 50 mg to about 1200 mg, preferably about 100 mg to
about 1000 mg, more preferably about 200 mg to about 800 mg, even
more preferably about 300 mg to about 600 mg.
[0066] The lipoic acid compounds of the invention may be
incorporated into pharmaceutical compositions suitable for
administration. Such compositions typically comprise at least one
lipoic acid or at least one analog thereof and a pharmaceutically
acceptable carrier. As used herein the language "pharmaceutically
acceptable carrier" is intended to include any and all solvents,
dispersion media, coatings, antibacterial and antifungal agents,
isotonic and absorption delaying agents, and the like, compatible
with pharmaceutical administration. The use of such media and
agents for pharmaceutically active substances is well known in the
art. Except insofar as any conventional media or agent is
incompatible with the active compound, use thereof in the
compositions is contemplated. Supplementary active compounds such
as ROS scavengers may also be incorporated into the compositions.
Other supplementary active compounds include neurotrophic factors
such as brain-derived neurotrophic factor (BDNF), nerve growth
factor (NGF), neurotrophins, and analogs thereof.
[0067] A pharmaceutical composition of the invention is formulated
to be compatible with its intended route of administration.
Examples of routes of administration include parenteral, e.g.,
intravenous, intradermal, subcutaneous, oral, transdermal,
transmucosal, and rectal administration. Solutions or suspensions
used for parenteral, intradermal, or subcutaneous application may
include a sterile diluent such as water for injection, a saline
solution, a fixed oil, a polyethylene glycol, glycerine, propylene
glycol or other synthetic solvent, an antibacterial agent such as
benzyl alcohol or methyl paraben, an antioxidant such as ascorbic
acid or sodium bisulfite, a chelating agent such as
ethylenediaminetetraacetic acid, a buffer such as an acetate,
citrate or phosphate and an agent for the adjustment of tonicity
such as sodium chloride or dextrose. pH may be adjusted with acids
or bases, such as hydrochloric acid or sodium hydroxide. Parenteral
preparations may be enclosed in ampoules, disposable syringes or
multiple dose vials made of glass or plastic.
[0068] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions or dispersions and sterile
powders for the extemporaneous preparation of sterile injectable
solutions or dispersion. For intravenous administration, suitable
carriers include physiological saline, bacteriostatic water,
Cremophor EL.TM. (BASF, Parsippany, N.J.) or phosphate buffered
saline (PBS). In all cases, the composition must be sterile and
should be fluid to the extent that easy syringability exists. It
must be stable under the conditions of manufacture and storage and
must be preserved against the contaminating action of
microorganisms such as bacteria and fungi. The carrier may be a
solvent or dispersion medium containing water, ethanol, a polyol
such as glycerol, propylene glycol, and liquid polyetheylene
glycol, and the like, and suitable mixtures thereof. The proper
fluidity may be maintained by the use of a coating such as
lecithin, by the maintenance of the required particle size in the
case of dispersion or by the use of surfactants. Prevention of the
action of microorganisms may be achieved by various antibacterial
and antifungal agents such as parabens, chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. In many cases, isotonic
agents such as sugars, polyalcohols such as manitol, sorbitol,
sodium chloride may be used in the composition. Prolonged
absorption of the injectable compositions may be brought about by
including an agent which delays absorption such as aluminum
monostearate and gelatin.
[0069] Sterile injectable solutions may be prepared by
incorporating the lipoic acid compound in the required amount in an
appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by filtered sterilization.
Generally, dispersions are prepared by incorporating the lipoic
acid compound into a sterile vehicle which contains a basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum drying and freeze-drying which yields a
powder of the active ingredient plus any additional desired
ingredient from a previously sterile-filtered solution thereof.
[0070] Oral compositions generally include an inert diluent or an
edible carrier. They can be enclosed in gelatin capsules or
compressed into tablets. For the purpose of oral therapeutic
administration, the lipoic acid compound may be incorporated with
excipients and used in the form of tablets, troches, or capsules.
Pharmaceutically compatible binding agents, adjuvants, or both may
be included as part of the composition. The tablets, pills,
capsules, troches and the like may contain a binder such as
microcrystalline cellulose, gum tragacanth or gelatin, an excipient
such as starch or lactose, a disintegrating agent such as alginic
acid, Primogel, or corn starch, a lubricant such as magnesium
stearate or Sterotes, a glidant such as colloidal silicon dioxide,
a sweetening agent such as sucrose or saccharin, or a flavoring
agent such as peppermint, methyl salicylate, or orange
flavoring.
[0071] For administration by inhalation, the compounds may be
delivered in the form of an aerosol spray from pressured container
or dispenser which contains a suitable propellant, e.g., a gas such
as carbon dioxide, or a nebulizer.
[0072] Systemic administration may also be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art,
and include, for example, for transmucosal administration,
detergents, bile salts, and fusidic acid derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays
or suppositories. For transdermal administration, the active
compounds are formulated into ointments, salves, gels, or creams as
generally known in the art.
[0073] The active compounds may be prepared with carriers which
protect the lipoic acid compounds against rapid elimination from
the body, such as a controlled release formulation, implants and
microencapsulated delivery systems. Biodegradable, biocompatible
polymers such as ethylene vinyl acetate, polyanhydrides,
polyglycolic acid, collagen, polyorthoesters, and polylactic acid
may be used. Methods of preparing these formulations will be
apparent to those skilled in the art. The materials may also be
obtained commercially from Alza Corporation and Nova
Pharmaceuticals, Inc. Liposomal suspensions can also be used as
pharmaceutically acceptable carriers. These can be prepared
according to methods known to those skilled in the art.
[0074] The pharmaceutical compositions may be formulated in dosage
unit forms for ease of administration and uniformity of dosage. A
dosage unit form, as used herein, refers to a physically discrete
unit suitable for use as a unitary dosage for the subject to be
treated. Each unit may contain a predetermined quantity of the
lipoic acid compound calculated to produce the desired therapeutic
effect in association with the required pharmaceutical carrier. The
specification for the dosage unit form of the invention is dictated
by and directly dependent on the unique characteristics of the
particular lipoic acid compound and the particular therapeutic
effect to be achieved.
[0075] Toxicity and therapeutic efficacy of the particular lipoic
acid compound may be determined by standard pharmaceutical
procedures in cell cultures or experimental animals, e.g., for
determining the LD50 (the dose lethal to 50% of the population) and
the ED50 (the dose therapeutically effective in 50% of the
population). The dose ratio between toxic and therapeutic effects
is the therapeutic index and it can be expressed as the ratio
LD50/ED50. Lipoic acid compound which exhibit large therapeutic
indices are preferred. While compounds that exhibit toxic side
effects may be used, care should be taken to design a delivery
system that targets such compounds to the site of affected tissue
in order to minimize potential damage to uninfected cells and,
thereby, reduce side effects.
[0076] The data obtained from the cell culture assays and animal
studies may be used in formulating a range of dosage for use in
humans. The dosage of such compounds lies preferably within a range
of circulating concentrations that include the ED50 with little or
no toxicity. The dosage may vary within this range depending upon
the dosage form employed and the route of administration utilized.
For any compound used in the method of the invention, the
therapeutically effective dose can be estimated initially from cell
culture assays. A dose may be formulated in animal models to
achieve a circulating plasma concentration range that includes the
IC50 (i.e., the concentration of the test compound which achieves a
half-maximal inhibition of symptoms) as determined in cell culture.
Such information can be used to more accurately determine useful
doses in humans. Levels in plasma may be measured, for example, by
high performance liquid chromatography.
[0077] As defined herein, a therapeutically effective amount of the
lipoic acid compound of the present invention (i.e., an effective
dosage) ranges from about 0.001 mg to about 200 mg per kg of the
subject, preferably about 25 mg to about 125 mg per kg of the
subject, more preferably about 50 mg to about 100 mg per kg of the
subject. Where the subject is human, the therapeutically effective
amount ranges from about 0.001 mg to about 20 mg per kg of the
human, preferably about 1 mg to about 10 mg per kg of the human,
more preferably about 3 mg to about 10 mg per kg of the human.
[0078] The skilled artisan will appreciate that certain factors may
influence the dosage required to effectively treat a subject,
including but not limited to the severity of the disease or
disorder, previous treatments, the general health and/or age of the
subject, and other diseases present. Moreover, treatment of a
subject with a therapeutically effective amount of a lipoic acid
compound can include a single treatment or a series of treatments.
Administration of the lipoic acid compound may be administered
prophylactically before CNS trauma, in individuals at risk. The
lipoic acid compound may be administered during and after CNS
trauma. It is appreciated that the effective dosage of the lipoic
acid compound may increase or decrease over the course of a
particular treatment.
[0079] The following examples are intended to illustrate but not to
limit the invention.
EXAMPLE 1
Cultured Cerebral Rat Neurons
[0080] Primary cultures may be established following the procedures
outlined by Koenig, M. L., et al., (1996) Brain Res. 730:143-149,
which is herein incorporated by reference. The forebrains of fetal
rat pups (embryonic day 15) are isolated and the cells are
dispersed by repeated trituration in neuronal culture medium (NCM).
NCM comprises a 1:1 ratio of Ham's F-12 (Biofluids, Rockville, Md.)
to Basal Medium Eagle (Sigma, St. Louis, Mo.), supplemented with
0.6 g/L of dextrose, 0.35% glutamine and 1% Pen-Strep (Biofluids,
Rockville, Md.). Following centrifugation at 900.times. g for 5
minutes, the cells are plated onto poly-L-lysine-coated 48 well
plates at a density of 10.sup.6 cells/ml. To suppress glial growth,
the cultures are treated with cytosine arabinoside (10.sup.-5 M)
for 4 days. All cultures are maintained in an incubator comprising
5% CO.sub.2 at 37.degree. C. for about 6 days to about 15 days
prior to use.
[0081] The cultured rat forebrain neurons are subjected to
oxidative stress using hydrogen peroxide following the
well-characterized paradigm by Desagher, S., et al., (1997) J.
Neuroscience 17(23):9060-9067, which is herein incorporated by
reference.
[0082] Oxidative injury is assessed by using an MTT assay (Sigma,
St. Louis, Mo.). About 24 h after exposure to either hemoglobin or
an oxidative insult, MTT is added to each test well such that the
final concentration of the dye is about 0.15 mg/ml. Plates are
returned to the incubator for about 1 hr at which time
unincorporated MTT is removed and the plates are allowed to air
dry. The purple formazan product indicative of viable cells is then
dissolved by adding about 250 .mu.l of acidified isopropanol
comprising about 95% isopropanol and about 5% 2N HCI. About 200
.mu.l aliquots are collected and the absorbance of these aliquots
are measured on an ELISA plate reader at about 540 nm. Control
(untreated) wells are included in each experiment, and viability is
calculated as the percentage of mean control values for each
plate.
[0083] The concentrations of ALA and LA-plus are about 25, about 50
and about 100 micromolar. The concentrations of selenium are about
10 and about 20 micromolar. The concentrations of pyruvate are
about 0.3, about 1.0, and about 3.0 mM. The concentrations of SPBN
ranges from about 1 to about 50 mM.
[0084] These studies are evaluated by multi-way analysis of
variance. When a significant F score is found, an a posteriori or a
priori test involving a multiple comparison of means, whichever is
appropriate, is used to compare individual treatment groups.
EXAMPLE 2
Fluid Percussion
[0085] Male Sprague-Dawley rats weighing about 250 g to about 350
g, pretrained in neurobehavioral tasks described below, are
surgically prepared for fluid percussion (FP) injuries using
standard surgical procedures known in the art. See Long, J. B., et
al., (1996) J. Neurotrauma 13:149-162, which is herein incorporated
by reference. Surgical instruments and materials are
steam-sterilized using an autoclave and are resterilized between
repetitive surgeries using heated glass beads such as Steri 350,
which are available from Inotech Biosystems Inc. (Lansing,
Mich.).
[0086] Briefly, about 24 hr prior to the experiment, rats are
anesthetized with about 70 mg/kg of ketamine and about 6 mg/kg
xylazine administered i.m. and the scalp is shaved. The
anesthetized rat is then placed in a stereotaxic apparatus and a
surgical scrub of the scalp is performed. Using a one cc syringe
and a 27 ga needle, the scalp is infiltrated with about 0.2 ml of a
1% lidocaine solution, a midline incision is made, and the scalp
and temporal muscles are reflected.
[0087] Using a 4.8 mm diameter trephine, a craniotomy centered over
the right parietal cortex is performed, leaving the dura intact. A
stainless steel screw is inserted into the skull about 1 mm rostral
to bregma. A plastic luer adapter (2.6 mm I.D., about 4.8 mm O.D.)
is snugly seated into the craniotomy over the exposed dura, secured
to the skull with cyanoacrylate adhesive, and embedded in dental
acrylic. The connector is packed with sterile cotton and the
surgical site is treated with bacitracin and lidocaine ointments.
Rats are observed post-operatively and kept warm with a Vetko
(Stoelting, Wood Dale, Ill.) thermal barrier until recovery from
anesthesia (i.e. ambulatory), at which time they are returned to
their home cages.
[0088] After being retested in the visual discrimination task to
ensure a return to presurgery levels of accuracy the rats are
anesthetized with halothane and prepared with femoral vein and tail
artery catheters in experiments employing secondary hypoxia. Under
aseptic conditions, the vessels are exposed by blunt dissection and
PE 50 catheters filled with a sterile heparinized saline, about 10
USP units/ml, are inserted and secured using 3-O nylon sutures.
Patency of the i.v. cannulae is maintained with about 0.2 ml
flushes with heparinized saline immediately following drug or
vehicle injections. The halothane anesthesia is maintained using
compressed air and a continuous flow anesthesia system available
from Stoelting (Wood Dale, Ill.) in combination with a Fluovac
(Stoelting, Wood Dale, Ill.) halothane scavenger, which recaptures
anesthetic gases by vacuum and thereby minimizes the escape of
halothane to the ambient air.
[0089] The spontaneously breathing, anesthetized rats are then
positioned in a stereotaxic apparatus, and the luer cranial
connector is exposed, filled with sterile saline, and attached to a
fluid percussion device, available from Dragonfly (Ridgeley, W.
Va.). Briefly, with this device a fluid pressure pulse is generated
in a 1 {fraction (1/16)} in. bore stainless steel cylinder with a 3
in. piston stroke that is filled with sterile water and connected
to the cranial luer adapters using flexible high pressure tubing
(0.089 in. i.d.). The injury is induced by striking the opposing
piston with a weighted metal pendulum released from a predetermined
height. The resultant rapid injection of a small volume of saline
into the closed cranial cavity causes a pulse of increased
intracranial pressure that is associated with a brief deformation
of brain tissue. The pressure pulse is measured extracranially by a
pressure transducer, Model 211B4, available from Kistler
Instruments Corp. (Amherst, N.Y.) and recorded on a digital storage
oscilloscope, Tecktronix 2212, (Tektronix Beaverton, Oreg.) and
based upon prior instrument calibration is expressed in
atmospheres. Core temperature are maintained using a homeothermic
blanket available from Harvard Apparatus (South Natick, Mass.).
Temperature of the temporalis muscle is continuously monitored
using a thermistor probe available from Physiotemp Systems
(Clifton, N.J.). Halothane administration is maintained at about
1.0%.
[0090] Arterial blood samples are removed immediately preceding and
at about 30 minutes following sham treatments or fluid percussion
injuries for analysis of blood gases, electrolytes, pH, lactic
acid, and hematocrit. Fluid percussion pressures are about 4.0 to
about 5.0 atm, and typically yield moderately severe brain
injuries. Cranial luer connectors are disconnected immediately
following delivery of the fluid percussion pulse. Sham control rats
are handled in an identical manner with the exception of the
delivery of the fluid pressure pulse.
[0091] Immediately following fluid percussion injury,
post-traumatic hypoxia is induced by substituting a 10% oxygen
source to deliver the halothane for continued anesthesia. At the
conclusion of about 30 minutes of posttraumatic hypoxia, an
arterial blood sample is removed for blood gas analysis. The
arterial catheters are then removed and the surgical wound sites
are closed with nylon sutures and treated with the topical
antibacterial furizolidine. The rats are kept warm with a Vetko
thermal barrier until recovered from anesthesia (i.e. ambulatory),
at which time they are returned to the operant conditioning
chambers or to their home cages. In all experimental groups, at the
conclusion of acute monitoring, arterial and venous catheters are
removed, the surgical wound sites are closed with nylon sutures and
treated with the topical antibacterial furizolidine.
[0092] The dosages of the lipoic acid compounds may be adjusted
based on evaluation of initial studies. ALA will be given s.c., as
per Wolz & Kriglstein (1996) Neuropharmacology 35:369-375,
which is herein incorporated by reference. For example, about 50
mg/kg of ALA and about 25 mg/kg of DHLA is administered s.c. about
30 minutes after FP (immediately after the post-FP hypoxia).
Follow-up doses of about 100 mg/kg of ALA and about 50 mg/kg of
DHLA may be given s.c. at about 3, about 24, about 48 and about 72
hours after FP.
[0093] About 300 mg/kg of the nitrone, SPBN, is administered s.c.
about 30 minutes after FP, with repeated injections s.c. about
every four hours for about 72 hours. In studies examining the
effects of vitamin E, about 25 mg/kg of alpha-tocopherol, are
injected i.p. about 0.5, about 3, about 24, about 48 and about 72
hours after FP. About 50 .mu.g selenium is administered i.p.
whenever alpha-tocopherol is administered.
[0094] For studies evaluating prophylactic treatments, about 100
mg/kg of ALA are administered s.c. daily for three days prior to
FP, followed by about 50 mg/kg s.c. one hour prior to FP. If ALA is
administered only, then about 100 mg/kg of ALA are administered
s.c. about 72 hours, about 48 hours, about 24 hours, about 2 hours
or a combination thereof prior to FP. If coenzyme Q 10 is
administered, then about 200 mg/kg of coenzyme Q 10 is administered
p.o..times.10 days. If SPBN is administered, about 300 mg/kg is
administered s.c. q.i.d. during about 72 hours of pretreatment. If
alpha-tocopherol is administered, about 25 mg/kg is injected i.p.
about two hours prior to FP. About 50 micrograms i.p. of selenium
may be administered when alpha-tocopherol is administered.
EXAMPLE 3
Intracerebral Injection of Ferric Chloride
[0095] Sprague-Dawley rats weighing about 200 g to about 280 g from
Charles River (Wilmington, Mass.) are maintained either in hanging
cages or housed separately, on 12 hour light-dark cycles with
unlimited access to food and water. All procedures are performed
with aseptic techniques and sterile instruments and
electrodes/cannulae which are autoclaved or glass bead sterilized.
Surgical procedures are performed with about 37.5 mg/kg
intraperitoneal injection i.p. pentobarbital sodium anesthesia. At
the time of complete insensitivity to painful stimuli, e.g.
nonresponsive to tail pinch at about 20 minutes post-injection, the
scalp is shaved, a surgical scrub is performed, and the rat placed
in a stereotaxic frame. For stereotaxic surgery, the incisor bar is
set about 3.3 mm below the intraaural line. The skull is exposed
and the periosteum removed and the surface of the skull is dried
and marked for electrode placement.
[0096] A. Surgical preparation for Intracortical injection of
ferric chloride for acute seizures.
[0097] A burr hole is made in a location over the sensorimotor
cortex, about 2 mm posterior and lateral to bregma and either 300
mM iron chloride or saline is unilaterally stereotaxically injected
intracortically, 2.5 mm ventral to bregma, using a 30 gauge
Hamilton syringe over a period of about 2 minutes. After the
Hamilton syringe is withdrawn, gelfoam (Gelfoam, Kalamazoo, Mich.)
is packed into the burr hole. Extradural cortical recording
electrodes are implanted to evaluate the presence or absence of
seizure events (rhythmic ictal EEG). Burr holes for the EEG leads,
from the transmitter, are drilled in the top of the skull, about 2
mm on either side of the midline and about 2 mm anterior to lambda.
The silastic insulation on the EEG leads is removed to reveal about
5 to about 8 coils of the wire. The exposed wire is then turned
about 180.degree. in line with the remaining lead and placed
sideways into the hole in the skull. The leads are then attached to
the implanted electrodes with a lead connector kit or leads are
continuous to the transmitter. All electrodes and leads are coated
with Teflon.TM. except for contact points requiring conductance. A
cannula may also then be placed in the right lateral ventricle if
i.c.v. drug administration is planned. If an i.c.v. cannula is
included, an acrylic mount is constructed to hold the cannula in
place while dental acrylic is secured over set screws and the
electrodes (opening around the electrodes and cannula is closed
with gel foam). Otherwise, if no i.c.v. cannula is implanted, the
electrode assembly is completely subcutaneous.
[0098] B. Surgical implantation of miniature EEG radio transmitter
and connection to electrodes.
[0099] About a 2 cm incision is made in the midscapular area and a
pocket under the skin is created with a hemostat so that the
subcutaneous opening is large enough to accommodate the transmitter
and coiled electrodes. A transmitter is inserted in the opening. A
single channel transmitter, such as CTAF40 (Data Sciences
International, St. Paul, Minn.), is used for the acute seizure
study; a two-channel transmitter, such as TL10M3-F50-EET (Data
Sciences International, St. Paul, Minn.), is used for bilateral
hippocampal recording electrodes in the amygdalar delayed seizure
study. A sterile trocar with sleeve will be used to create a
subcutaneous channel opening up to the scalp incision, and, after
the trocar is removed, the transmitters are passed through the
sleeve to the appropriate burrholes in the skull surface making
contact with the dura and then covered with gelfoam. A small
acrylic mount is constructed to cover the electrodes and a set
screw in the skull for stability of the recording electrodes.
[0100] C. Surgical placement of amygdalar cannula for injection of
ferric chloride for induction of delayed, spontaneous clonic
seizures.
[0101] Burr holes are made in locations appropriate for stereotaxic
coordinates per atlas of Paxinos and Watson, THE RAT BRAIN IN
STEREOTAXIC COORDINATES. Academic Press, New York (1986), which is
herein incorporated by reference. Coordinates for the amygdaloid
body is about 3.3 mm posterior and about 5.0 mm lateral to the
bregma and about 8.5 mm below the surface of the skull. A 22 gauge
guide cannula, lumen occluded with stylet, is stereotaxically
placed about 0.5 mm above the left amygdala. Coordinates for the
dorsal hippocampus is about 3.3 mm posterior and about 1.5 mm
bilateral to the bregma and about 3.4 mm below the surface of the
skull. Teflon-coated stainless steel recording electrodes with less
than about 0.25-mm bare tips are positioned into dorsal hippocampi
bilaterally in the dorsal hippocampi to evaluate the presence or
absence of seizure events (rhythmic ictal EEG). Stainless steel
screws are positioned in the occipital bone to provide reference
and ground contacts. All electrodes are attached to wires with pin
adapters and then affixed to the skull with dental acrylic.
Implantation of miniature EEG radiotransmitter, connection to
electrodes and post-surgical analgesia are accomplished as
described in section B.
[0102] D. Intra-surgical and post-surgical care.
[0103] During surgery and anesthesia recovery, rats are monitored
by trained personnel and maintained on heating pads having a
temperature of about 37 .degree. C. to aid in the regulation of
body temperature. About 0.25 to about 0.5 mg/100 g of Butorphanol
tartrate (Torbugesic.TM., A. J. Buck. Owings Mills, Md.) is
administered s.c. at the onset of surgery and thereafter for the
treatment of pain as evidenced by the presence of signs of
discomfort such as piloerection, hunched posture, inactivity and/or
anorexia. Rats are observed until they are eating, drinking and
resting in a natural position after anesthesia. Rats are
individually housed in cages over receivers (RPC 1) and allowed
free access to food and water and are maintained on a 12 hour/12
hour light/dark cycle.
[0104] E. Intra-amygdalar ferric chloride injections for delayed
seizures.
[0105] After about 5 days of recovery the stylet is removed from
the guide cannula and replaced with an injection cannula comprising
a 24-gauge guide wire, the tip of which is about 0.5-mm length of
fused silica (0.075 mm i.d., 0.15 mm o.d.). Freely moving animals
are injected with an aqueous solution of about 1.5 .mu.l of about
100 mM ferric chloride (FeCI.sub.3) having a pH of about 2.2 at a
rate of about 1 .mu.l per minute using a microinfusion pump.
Control animals are injected with about 1.5 .mu.l of about 0.9%
NaCl, with pH adjusted to about 2.2.
[0106] F. 24 hour EEG and videotape recording.
[0107] EEG determinations are initiated by placing the entire
microisolator on top of the Data Sciences International (DSI)
Physiotel Receiver Model RPC-1 (St. Paul, Minn.) connected to a
Data Exchange Matrix (DSI, St. Paul, Minn.) and a Pentium III 733
Mhz computer using a DSI A.R.T. II System (DSI, St. Paul, Minn.)
for Windows NT. EEG is acquired and stored for analysis. The
transmitter is activated by swiping a magnet over the subcutaneous
implantation site, and EEG recording is initiated by placing the
rat over the top of an RPC-1 receiver (DSI, St. Paul, Minn.).
[0108] G. EEG for acute intracortical ferric chloride.
[0109] EEG is recorded only for 24 hours. Examples of epileptiform
spike and spike/wave patterns are illustrated in FIGS. 3a and 3b.
Specific parameters for data/statistical analysis include the
number of seizure events per unit time, seizure duration or
duration of rhythmic paroxysmal activity and overall slowing of
electrical activity in and power spectral analysis of EEG. In the
power spectral analysis, identification of a decrease in mean
frequency values of the amplitude spectrum of equal to or greater
than about 0.5 Hz in two or more of the following frequency bands
is indicative of meaningful lesions in tested regions:
.delta.(0.5-5 Hz), .theta.(5-10 Hz), .alpha.(10-16 Hz),
.beta.(16-48) and total (0-48). Twenty-four hour videotaping of
animals will be used to verify behaviors associated with seizures
identified by EEG. After about 24 hour EEG, rats are euthanized,
perfused and brains removed. The area of cortical lesion is
quantified by digital photographic imaging. Brains are then
prepared for histological analysis as described above.
[0110] ii. EEG recording in delayed seizure model.
[0111] EEG data is recorded as above on a round-the-clock basis for
about 30 days after intra-amygdalar ferric chloride injection.
Twenty-four hour videotaping of animals is used to verify behaviors
associated with seizures identified by EEG. Behaviors are scored
from review of the video images by using the criteria of set forth
by Racine, R. (1972) Electroencephalogr. Clin. Neurophysiol.
32:281-294, which is herein incorporated by reference, by an
observer blind to the nature of the injectate received by the
animals. After about 30 days, rats are tested for memory function
in the Morris water maze as described below. After behavioral
testing, rats are euthanized, perfused and brains removed,
sectioned and stained to ascertain amygdalar placement and identify
siderosis.
[0112] H. Assessment of pharmaceutical interventions.
[0113] For selected protective agents or combinations of agents,
treatment with ALA is given s.c., as per Wolz & Kriglstein
(1996) Neuropharmacology 35:369-375, which is herein incorporated
by reference. In the ferric chloride-induced seizure experiments,
about 100 mg/kg of ALA and about 50 mg/kg DHLA is administered s.c.
about 30 minutes after ferric injection. Follow-up doses of about
100 mg/kg of ALA and about 50 mg/kg of DHLA are given s.c. at about
3, about 24, about 48 and about 72 hours after ferric chloride.
About 300 mg/kg of SPBN or 150 mg/kg of PBN is administered s.c. 30
minutes after FP with repeated injections every four hours lasting
for about 72 hours. In studies examining the effects of vitamin E,
about 25 mg/kg of alpha-tocopherol is injected i.p. about 0.5,
about 3, about 24, about 48 and about 72 hours after FP. About 50
.mu.g of selenium is administered i.p. whenever alpha-tocopherol is
administered. In all experiments vehicle-treated groups and
sham-operated groups are studied which identifies any effects of
the compounds tested on EEG or memory. The dosages may be adjusted
as necessary.
[0114] For studies evaluating prophylactic treatments, about 100
mg/kg of ALA are administered s.c. daily for three days prior to
FP, followed by about 50 mg/kg s.c. one hour prior to FP. If ALA is
administered only, then about 100 mg/kg of ALA are administered
s.c. about 72 hours, about 48 hours, about 24 hours, about 2 hours
or a combination thereof prior to FP. If coenzyme Q 10 is
administered, then about 200 mg/kg of coenzyme Q 10 is administered
p.o. .times. 10 days. About 300 mg/kg of SPBN or about 100 mg/kg of
PBN is administered s.c. q.i.d. during about 72 hours of
pretreatment. If alpha-tocopherol is administered, about 25 mg/kg
is injected i.p. about two hours prior to FP. About 50 micrograms
i.p. of selenium may be administered when alpha-tocopherol is
administered.
[0115] These studies are evaluated by multi-way analysis of
variance. When a significant F score is found, an a posteriori or a
priori test involving a multiple comparison of means, whichever is
appropriate, will be used to compare individual treatment
groups.
EXAMPLE 4
Spatial Memory Assessment
[0116] The Morris water maze is used to evaluate the extent to
which fluid percussion injury or amygdalar ferric chloride
injection impairs a rat's ability to learn the location of a
submerged platform in a pool of water. During the two days that
precede sham treatment or fluid percussion injury, the training on
the visual recognition memory task is suspended and each rat is
trained to swim to the safety of a non-visible submerged platform
in a circular pool of water (26 .degree. C.). On each of these
days, a rat will be placed randomly at one of four locations
(north, south, east, or west) in a circular pool of water and
allowed to swim for about 60 seconds. If during that time the rat
locates the submerged platform, it is allowed to remain on the
platform for about 10 seconds. But, if the rat fails to locate the
platform, at the end of about 60 seconds, it is removed from the
pool. 12 learning trials are scheduled/day with about four minute
breaks between each learning trial. Similarly, water maze training
is accomplished two days prior to surgery for placement of
amygdalar ferric chloride cannulae.
[0117] Since injury induced motor impairments might interfere with
any retention tests of maze performance, the first water-maze
retention test does not occur until the 8.sup.th post-injury day.
However, daily tests of visual recognition memory are scheduled for
the first seven days after fluid percussion injury. On the 8.sup.th
post-injury day, visual discrimination assessments are discontinued
and five retention trials are scheduled. Retention trials are
identical in every respect to pre-injury learning trials, including
the location of the platform. On the 9.sup.th day, the platform is
removed and a probe trial is used to determine whether a rat's
preference was controlled by location of the platform or by
visibility. On days 11-14, the location of the platform is changed
on successive test days.
[0118] The rat's performance during all learning and retention
tests is tracked and quantified by the TSE VideoMot2
(www.TSE-Systems.de) video tracking system. For each rat, the
number of entries into the four quadrants (zones) of the pool, time
spent in each zone, distance traveled, swim speed, as well as
latency to find the platform from the initiation of a trial is
collected for each rat.
EXAMPLE 5
Visual Recognition Memory Test
[0119] In the visual non-matching-to-sample experiments, rats are
housed in individual test cages that contain three levers, a
dot-pattern module above each lever, and a food trough below the
center lever. Initially, a dot pattern is illuminated over one of
the side levers and rats are trained to press the lever beneath the
illuminated dot pattern. After rats learn to track the location of
the dot pattern above the side levers, one of several dot-patterns
is illuminated above the center lever. A press on the center lever
will turn the sample pattern off and simultaneously illuminate
comparison dot patterns above each of the side levers. The dot
pattern above one side lever matches the (sample) pattern and the
dot pattern above the other lever is different. A press on the
lever beneath the comparison dot pattern that is different from the
sample pattern results in the delivery of food and immediately
initiates a 10 second inter-trial interval. At the end of the
inter-trial interval, one of the seven dot-patterns is randomly
selected for presentation as a new sample.
[0120] However, a press on the lever beneath the comparison dot
pattern that matches the sample dot pattern ends the trial and
after a 10 second inter-trial interval, a correction trial is
initiated. The previous sample is represented as a flashing dot
pattern and a press on the center lever results in the
representation of the comparison stimuli that appeared on the
previous trial. On a correction trial, the comparison pattern that
matches the flashing sample does not flash and the probability is
0.50 that the previous sample will reappear above the same lever.
After a rat's non-matching performance stabilizes, a delay of
variable duration will be imposed between presentation of the
sample pattern and the presentation of the comparison patterns.
Prior to FP, different delays between offset of the sample and
presentation of the comparison patterns will be used to determine
the extent to which the passage of time or activity within the
delay interfere with recall of the sample.
[0121] In all experiments, accuracy and latency data from each
day's experimental session will be maintained in computer files.
Standard graphic display programs are used to prepare graphs and
the data from experimental and control groups are analyzed with a
computer program, such as SASr, using repeated measures analysis of
variance and appropriate contrast tests to identify significant
group differences on recovery days. See Bauman et al., (2000)
Journal of Neurotrauma 17(8):679-693, which is herein incorporated
by reference.
EXAMPLE 6
Neuroanatomical Evaluations
[0122] After the completion of the functional assessments described
above, the rats are anesthetized with about 70 mg/kg of ketamine
and about 6 mg/kg of xylazine i.m. After thoracotomy and laceration
of the right atrium of the heart, rats are sequentially perfused
transcardially with physiological saline which results in
euthanasia by exsanguination and about a 10% formalin solution for
fixation. Brains are removed and, after additional immersion
fixation, paraffin-embedded Nissl-stained tissue sections are
prepared.
[0123] In rats subjected to intracortical injection of ferric
chloride, the area of cortical lesion are quantified by digital
photographic imaging.
[0124] For rats subjected to amygdalar ferric chloride injection,
alternate sections are stained with Prussian Blue, to reveal the
extent and location of siderosis and cresyl violet stained sections
will be examined for cavitation and gliosis in the basolateral
amygdala. Placement of dorsal hippocampal electrodes are evaluated
in cresyl violet stained sections through that region.
[0125] Sections from FP-injured and sham rats are examined by
specifically focusing on anticipated injury-induced changes in
neuronal cell numbers within the hilar region of the dentate gyrus
of the hippocampus. Normal neurons are identified by he presence of
nuclei with clear nucleoplasm, surrounded by cytoplasm containing
Nissl substance. Hilar neuronal cell numbers are counted in six 30
micron non-adjacent sections per Lowenstein et al., (1992) J.
Neuroscience 12(12):4846-4853, which is herein incorporated by
reference, and compared among treatment groups in subsequent
statistical analysis. In addition, other neuroanatomical hallmarks
of fluid percussion injury, e.g. cortical contusion, hippocampal
CA1 and CA3 neurons, may be examined, and if suitably consistent
within experimental groups, are analyzed by nonparametric, e.g.
blinded rater score or parametric, e.g. cell number analysis. In
brains from rats subjected to FP injury, degenerative changes in
the lateral geniculate nucleus are determined as reported by Bauman
et al. (2000) Journal of Neurotrauma accepted pending revision,
which is herein incorporated by reference.
[0126] When the probability of an F score for an overall comparison
among groups is p<0.05, Dunnett's test is used for post-hoc
comparisons among group means. Cell counts in the hilar region of
the dentate gyrus are compared using analysis of variance.
Differences among nonparametric histopathological scores are
determined by means of the Kruskal-Wallis test.
EXAMPLE 7
Synergistic Neuroprotective Effect
[0127] The neuroprotective effect of the lipoic acids, such as DHL,
may be significantly enhanced by the co-administration of SPBN and
PBN.
[0128] A. DHL and SPBN.
[0129] Cultured neurons were incubated with four DHL
concentrations, 3.0, 10.0, 30.0, and 100.0 micromolar of DHL, with
or without 10 mM SPBN for four hours. After incubation, an
oxidative insult was applied by adding 50 micromolar of
H.sub.2O.sub.2 for 30 minutes after which the medium was removed
and replaced with Minimum Essential Medium (MEM) (Sigma, St. Louis,
Mo.). After 24 hours, the colormetric MTT assay as described above
was conduced to determine amount of neuroprotection.
[0130] As shown in Table 1 below and FIG. 5, SPBN significantly
increased the neuroprotective efficacy of DHL. The combination of
100 micromolar DHL and 10 mM SPBN provided almost complete
protection against H.sub.2O.sub.2-induced oxidative insult.
1 TABLE 1 Treatment % DHL uM SPBN mM Neuroprotection n 3 0 1.31
.+-. 5.23 8 3 10 10.48 .+-. 3.04 8 10 0 -3.68 .+-. 5.14 8 10 10
44.51 .+-. 11.98*** 8 30 0 7.04 .+-. 7.38 8 30 10 48.64 .+-. 6.09**
7 100 0 28.48 .+-. 5.83 8 100 10 86.44 .+-. 8.44*** 8
[0131] Data are depicted as means .+-.SEM (n=7 or 8). Significance
was determined by one way ANOVA and the differences between the
means was assessed by the Tukey-Kramer test. ** p<0.005; ***
p<0.001.
[0132] B. DHL and PBN.
[0133] Cultured neurons were incubated with four DHL
concentrations, 3.0, 10.0, 30.0, and 100.0 micromolar of DHL, with
or without 10 mM PBN for four hours. After incubation, an oxidative
insult was applied by adding 50 micromolar of H.sub.2O.sub.2 for 30
minutes after which the medium was removed and replaced with
Minimum Essential Medium (MEM) (Sigma, St. Louis, Mo.). After 24
hours, the colormetric MTT assay as described above was conduced to
determine amount of neuroprotection.
[0134] As shown in Table 2 below and FIG. 6, PBN significantly
increased the neuroprotective efficacy of DHL.
2 TABLE 2 Treatment % DHL uM SPBN mM Neuroprotection n 3 0 -1.3
.+-. 7.2 7 3 10 20.7 .+-. 10.5 6 10 0 22.2 .+-. 13.6 8 10 10 56.9
.+-. 11.6 8 30 0 22.1 .+-. 9.7 8 30 10 87.4 .+-. 12.0 8 100 0 36.9
.+-. 9.6 8 100 10 81.6 .+-. 11.2 6
[0135] Data are depicted as means .+-.SEM (n=6, 7, 8). Significance
was determined by one way ANOVA and the differences between the
means was assessed by the Tukey-Kramer test.** p<0.005; ***
p<0.001.
[0136] Incorporation by Reference
[0137] To the extent necessary to understand or complete the
disclosure of the present invention, all publications, patents, and
patent applications mentioned herein are expressly incorporated by
reference to the same extent as though each were individually so
incorporated,
* * * * *