Fred Lemonjello
07-05-2008, 07:35 AM
the liberty papers dot org
July 3, 2008
The left hand says <**link removed, see text below
Existing Legal Drugs Provide Superior Treatment for Serious Medical Conditions
The FDA has approved safe and effective medication for the treatment of glaucoma, nausea, wasting syndrome, cancer, and multiple sclerosis.
Marinol, the synthetic form of THC (the psychoactive ingredient contained in marijuana), is already legally available for prescription by physicians whose patients suffer from pain and chronic illness.
The right hand said (in a 2003 Patent application!)
<***Link removed see text below)
Cannabinoids have been found to have antioxidant properties, unrelated to NMDA receptor antagonism. This new found property makes cannabinoids useful in the treatment and prophylaxis of wide variety of oxidation
associated diseases, such as ischemic, age-related, inflammatory and autoimmune diseases. The cannabinoids are found to have particular application as neuroprotectants, for example in limiting neurological damage following ischemic insults, such as stroke and trauma, or in the treatment of neurodegenerative diseases, such as Alzheimer’s disease,
Setting aside the immorality of the United States government granting itself a patent on something - I find myself reminded of something asked of Senator McCarthy:
“You’ve done enough. Have you no sense of decency, sir? At long last, have you left no sense of decency?”
I think even Kafka would consider the U.S. government’s behavior with respect to marijuana too outlandish to write a story about.
**From Left Hand Says link
ONDCP
Medical Marijuana Reality Check
What’s Wrong With Permitting the Use of Smoked Marijuana?
Simply put, the smoked form of marijuana is not considered modern medicine. On April 20th, 2006, the FDA issued an advisory concluding that no sound scientific studies have supported medical use of smoked marijuana for treatment in the United States, and no animal or human data support the safety or efficacy of smoked marijuana for general medical use.
A number of states have passed voter referenda or legislative actions making smoked marijuana available for a variety of medical conditions upon a doctor's recommendation. According to the Food and Drug Administration (FDA), these measures are inconsistent with efforts to ensure medications undergo the rigorous scientific scrutiny of the FDA approval process and are proven safe and effective under the standards of the FD&C Act.
While smoking marijuana may allow patients to temporarily feel better, the medical community makes an important distinction between inebriation and the controlled delivery of pure pharmaceutical medication. The raw (leaf ) form of marijuana contains a complex mixture of compounds in uncertain concentrations, the majority of which have unknown pharmacological effects.
The Institute of Medicine (IOM) has concluded that smoking marijuana is not recommended for any long-term medical use, and a subsequent IOM report declared that, “marijuana is not modern medicine.” Additionally, the American Medical Association, the National Cancer Institute, the American Cancer Society, and the National Multiple Sclerosis Society do not support the smoked form of marijuana as medicine.
Smoking Marijuana May Unintentionally Cause Serious Harm to Patients
The delicate immune systems of seriously ill patients may become compromised by the smoking of marijuana. Additionally, the daily use of marijuana compromises lung function and increases the risk for respiratory diseases, similar to those associated with nicotine cigarettes.
Marijuana has a high potential for abuse and can incur addiction. Frequent use of marijuana leads to tolerance to the psychoactive effects and smokers compensate by smoking more often or seeking higher potency marijuana.
In people with psychotic or other problems, the use of marijuana can precipitate severe emotional disorders. Chronic use of marijuana may increase the risk of psychotic symptoms in people with a past history of schizophrenia. Marijuana smoking by young people may lead to severe impairment of higher brain function and neuropsychiatric disorders, as well as a higher risk for addiction and polydrug abuse problems.
Existing Legal Drugs Provide Superior Treatment for Serious Medical Conditions
The FDA has approved safe and effective medication for the treatment of glaucoma, nausea, wasting syndrome, cancer, and multiple sclerosis.
Marinol, the synthetic form of THC (the psychoactive ingredient contained in marijuana), is already legally available for prescription by physicians whose patients suffer from pain and chronic illness.
“Medical marijuana was supposed to be for the truly ill cancer victims and AIDS patients who could use the drug to relieve pain or restore their appetites. Yet the number of dispensaries has skyrocketed from five in 2005 to 143 by the end of 2006. In North Hollywood alone, there are more pot clinics than Starbucks.”
–Pasadena Star-News, January 21st, 2007
In Their Words: What the Experts Say:
The American Academy of Ophthalmology:
“Based on reviews by the National Eye Institute (NEI) and the Institute of Medicine and on available scientific evidence, the Task Force on Complementary Therapies believes that no scientific evidence has been found that demonstrates increased benefits and/or diminished risks of marijuana use to treat glaucoma compared with the wide variety of pharmaceutical agents now available.”
Complementary Therapy Assessment: Marijuana in the Treatment of Glaucoma, American Academy of Ophthalmology, May 2003
The American Medical Association:
“...AMA recommends that marijuana be retained in Schedule I of the Controlled Substances Act...AMA believes that the NIH should use its resources and influence to support the development of a smoke-free inhaled delivery system for marijuana or delta-9-tetrahydrocannabinol (THC) to reduce the health hazards associated with the combustion and inhalation of marijuana...”
Policy Statement H-95.952, American Medical Association, http://www.ama-assn.org
The National Multiple Sclerosis Society:
“Studies completed thus far have not provided convincing evidence that marijuana or its derivatives provide substantiated benefits for symptoms of MS.”
The MS Information Sourcebook, Marijuana (Cannabis), National Multiple Sclerosis Society, September 18th, 2006
The Institute of Medicine (IOM):
“Because of the health risks associated with smoking, smoked marijuana should generally not be recommended for long-term medical use.”
Marijuana and Medicine: Assessing the Science Base, Institute of Medicine, 1999
*** from Right Hand Said link
US Patent 6630507 - Cannabinoids as antioxidants and neuroprotectants
US Patent Issued on October 7, 2003Abstract
Cannabinoids have been found to have antioxidant properties, unrelated to
NMDA receptor antagonism. This new found property makes cannabinoids
useful in the treatment and prophylaxis of wide variety of oxidation
associated diseases, such as ischemic, age-related, inflammatory and
autoimmune diseases. The cannabinoids are found to have particular
application as neuroprotectants, for example in limiting neurological
damage following ischemic insults, such as stroke and trauma, or in the
treatment of neurodegenerative diseases, such as Alzheimer's disease,
Parkinson's disease and HIV dementia. Nonpsychoactive cannabinoids, such
as cannabidoil, are particularly advantageous to use because they avoid
toxicity that is encountered with psychoactive cannabinoids at high doses
useful in the method of the present invention. A particular disclosed
class of cannabinoids useful as neuroprotective antioxidants is formula
(I) wherein the R group is independently selected from the group
consisting of H, CH3, and COCH3.
##STR1##Claims
We claim:
1. A method of treating diseases caused by oxidative stress, comprising
administering a therapeutically effective amount of a cannabinoid that has
substantially no binding to the NMDA receptor to a subject who has a
disease caused by oxidative stress.
2. The method of claim 1, wherein the cannabinoid is nonpsychoactive.
3. The method of claim 2, wherein the cannabinoid has a volume of
distribution of 10 L/kg or more.
4. The method of claim 1, wherein the cannabinoid is not an antagonist at
the NMDA receptor.
5. The method of claim 1, wherein the cannabinoid is:
##STR22##
where R is H, substituted or unsubstituted alkyl, carboxyl, alkoxy, aryl,
aryloxy, arylalkyl, halo or amino.
6. The method of claim 5, wherein R is H, substituted or unsubstituted
alkyl, carboxyl or alkoxy.
7. The method of claim 2, wherein the cannabinoid is:
##STR23##
where
A is cyclohexyl, substituted or unsubstituted aryl, or
##STR24##
but not a pinene;
R1 is H, substituted or unsubstituted alkyl, or substituted or
unsubstituted carboxyl;
R2 is H, lower substituted or unsubstituted alkyl, or alkoxy;
R3 is of H, lower substituted or unsubstituted alkyl, or substituted
or unsubstituted carboxyl;
R4 is H, hydroxyl, or lower substituted or unsubstituted alkyl; and
R5 is H, hydroxyl, or lower substituted or unsubstituted alkyl.
8. The method of claim 7, wherein
R1 is lower alkyl, COOH or COCH3 ;
BR>R2 is unsubstituted C1 -C5 alkyl, hydroxyl, methoxy or
ethoxy;
R3 is H, unsubstituted C1 -C3 alkyl, or COCH3 ;
R4 is hydroxyl, pentyl, heptyl, or diemthylheptyl; and
R5 is hydroxyl or methyl.
9. The method of claim 1, wherein the cannabinoid is:
##STR25##
where R1, R2 and R3 are independently H, CH3, or
COCH3.
10. The method of claim 9, wherein the cannabinoid is:
##STR26##
where:
a) R1 =R2 =R3 =H;
b) R1 =R3 =H, R2 =CH3 ;
c) R1 =R2 =CH3, R3 =H;
d) R1 =R2 =COCH3, R3 =H; or
e) R1 =H, R2 =R3 =COCH3.
11. The method of claim 2, wherein the cannabinoid is:
##STR27##
where R19 is H, lower alkyl, lower alcohol, or carboxyl; R20 is H
or OH; and R21 -R25 are independently H or OH.
12. The method of claim 11, wherein R19 is H, CH3, CH2 OH,
or COOH, and R20 -R24 are independently H or OH.
13. The method of claim 2, wherein the cannabinoid is:
##STR28##
where R19 and R20 are H, and R26 is alkyl.
14. The method of claim 10, wherein the cannabinoid is cannabidiol.
15. A method of treating an ischemic or neurodegenerative disease in the
central nervous system of a subject, comprising administering to the
subject a therapeutically effective amount of a cannabinoid, where the
cannabinoid is
##STR29##
where R is H, substituted or unsubstituted alkyl, carboxyl, alkoxy, aryl,
aryloxy, arylalkyl, halo or amino.
16. The method of claim 15, wherein the cannabinoid is not a psychoactive
cannabinoid.
17. The method of claim 15 where the ischemic or neurodegenerative disease
is an ischemic infarct, Alzheimer's disease, Parkinson's disease, and
human immunodeficiency virus dementia, Down's syndrome, or heart disease.
18. A method of treating a disease with a cannabinoid that has
substantially no binding to the NMDA receptor, comprising determining
whether the disease is caused by oxidative stress, and if the disease is
caused by oxidative stress, administering the cannabinoid in a
therapeutically effective antioxidant amount.
19. The method of claim 18, wherein the cannabinoid has a volume of
distribution of at least 1.5 L/kg and substantially no activity at the
cannabinoid receptor.
20. The method of claim 19, wherein the cannabinoid has a volume of
distribution of at least 10 L/kg.
21. The method of claim 1, wherein the cannabinoid selectively inhibits an
enzyme activity of 5- and 15-lipoxygenase more than an enzyme activity of
12-lipoxygenase.
22. A method of treating a neurodegenerative or ischemic disease in the
central nervous system of a subject, comprising administering to the
subject a therapeutically effective amount of a compound selected from any
of the compounds of claims 9 through 13.
23. The method of claim 22 where the compound is cannabidiol.
24. The method of claim 22, wherein the ischemic or neurodegenerative
disease is an ischemic infarct, Alzheimer's disease, Parkinson's disease,
and human immunodeficiency virus dementia, Down's syndrome, or heart
disease.
25. The method of claim 24 wherein the disease is an ischemic infarct.
26. The method of claim 1, wherein the cannabinoid is not an antagonist at
the AMPA receptor.
Description
FIELD OF THE INVENTION
The present invention concerns pharmaceutical compounds and compositions
that are useful as tissue protectants, such as neuroprotectants and
cardioprotectants. The compounds and compositions may be used, for
example, in the treatment of acute ischemic neurological insults or
chronic neurodegenerative diseases.
BACKGROUND OF THE INVENTION
Permanent injury to the central nervous system (CNS) occurs in a variety of
medical conditions, and has been the subject of intense scientific
scrutiny in recent years. It is known that the brain has high metabolic
requirements, and that it can suffer permanent neurologic damage if
deprived of sufficient oxygen (hypoxia) for even a few minutes. In the
absence of oxygen (anoxia), mitochondrial production of ATP cannot meet
the metabolic requirements of the brain, and tissue damage occurs. This
process is exacerbated by neuronal release of the neurotransmitter
glutamate, which stimulates NMDA (N-methyl-D-aspartate), AMPA
(α-amino-3-hydroxy-5-methyl-4-isoxazole propionate) and kainate
receptors. Activation of these receptors initiates calcium influx into the
neurons, and production of reactive oxygen species, which are potent
toxins that damage important cellular structures such as membranes, DNA
and enzymes.
The brain has many redundant blood supplies, which means that its tissue is
seldom completely deprived of oxygen, even during acute ischemic events
caused by thromboembolic events or trauma. A combination of the injury of
hypoxia with the added insult of glutamate toxicity is therefore believed
to be ultimately responsible for cellular death. Hence if the additive
insult of glutamate toxicity can be alleviated, neurological damage could
also be lessened. Anti-oxidants and anti-inflammatory agents have been
proposed to reduce damage, but they often have poor access to structures
such as the brain (which are protected by the blood brain barrier).
Given the importance of the NMDA, AMPA and kainate receptors in the
mechanism of injury, research efforts have focused on using antagonists to
these receptors to interfere with the receptor mediated calcium influx
that ultimately leads to cellular death and tissue necrosis. In vitro
studies using cultured neurons have demonstrated that glutamate receptor
antagonists reduce neurotoxicity, but NMDA and AMPA/kainate receptor
antagonists have different effects. Antagonists to NMDAr prevent
neurotoxicity if present during the glutamate exposure period, but are
less effective if added after glutamate is removed. In contrast,
AMPA/kainate receptor antagonists are not as effective as NMDA antagonists
during the glutamate exposure period, but are more effective following
glutamate exposure.
Some of the research on these antagonists has focused on cannabinoids, a
subset of which have been found to be NMDA receptor antagonists. U.S. Pat.
No. 5,538,993 (3S,4S-delta-6-tetrahydrocannabinol-7-oic acids), U.S. Pat.
No. 5,521,215 (sterospecific (+) THC enantiomers), and U.S. Pat. No.
5,284,867 (dimethylheptyl benzopyrans) have reported that these
cannabinoids are effective NMDA receptor blockers. U.S. Pat. No. 5,434,295
discloses that the 1,1 dimethylheptyl (DMH) homolog of
[3R,4R]-7-hydroxy-Δ6 THC (known as HU-210) is a superpotent
cannabinoid receptor agonist with cannabinomimetic activity two orders of
magnitude greater than the natural Δ9 THC. The HU-210
dimethylheptyl cannabinoid, has severe side effects, including fatigue,
thirst, headache, and hypotension. J. Pharmacol. Sci. 60:1433-1457 (1971).
Subjects who received this synthetic cannabinoid with a dimethylheptyl
group experienced marked psychomotor retardation, and were unwilling or
incapable of assuming an erect position.
In contrast to HU-210, the (-)(3R,4R) THC-DMH enantiomer (known as HU-211)
displays low affinity to the cannabinoid receptors, but retains NMDA
receptor antagonist neuroprotective activity.
##STR2##
THC (tetrahydrocannabinol) is another of the cannabinoids that has been
shown to be neuroprotective in cell cultures, but this protection was
believed to be mediated by interaction at the cannabinoid receptor, and so
would be accompanied by undesired psychotropic side effects.
##STR3##
Although it has been unclear whether cannabimimetic activity plays a role
in neuroprotection against glutamate induced neurological injury, the
teaching in this field has clearly been that a cannabinoid must at least
be an antagonist at the NMDA receptor to have neuroprotective effect.
Hence cannabidiol
(2-[3-methyl-6-(1-methylethenyl)-2-cyclohexen-1-yl]-5-pentyl-1,3-benzenedi
ol or CBD), a cannabinoid devoid of psychoactive effect (Pharm. Rev.
38:21-43, 1986), has not been considered useful as a neuroprotectant.
Cannabidiol has been studied as an antiepileptic (Carlini et al., J. Clin.
Pharmacol. 21:417S-427S, 1981; Karler et al., J. Clin. Pharmacol.
21:437S-448S, 1981, Consroe et al., J. Clin Phannacol. 21:428S-436S,
1981), and has been found to lower intraocular pressure (Colasanti et al,
Exp. Eye Res. 39:251-259, 1984 and Gen. Pharmac. 15:479-484, 1984).
##STR4##
No signs of toxicity or serious side effects have been observed following
chronic administration of cannabidiol to healthy volunteers (Cunha et al.,
Pharmacology 21:175-185, 1980), even in large acute doses of 700 mg/day
(Consroe et al., Pharmacol. Biochem. Behav. 40:701-708, 1991) but
cannabidiol is inactive at the NMDA receptor. Hence in spite of its
potential use in treating glaucoma and seizures, cannabidiol has not been
considered a neuroprotective agent that could be used to prevent glutamate
induced damage in the central nervous system.
SUMMARY OF THE INVENTION
It is an object of this invention to provide a new class of antioxidant
drugs, that have particular application as neuroprotectants, although they
are generally useful in the treatment of many oxidation associated
diseases.
Yet another object of the invention is to provide a subset of such drugs
that can be substantially free of psychoactive or psychotoxic effects, are
substantially non-toxic even at very high doses, and have good tissue
penetration, for example crossing the blood brain barrier.
It has surprisingly been found that cannabidiol and other cannabinoids can
function as neuroprotectants, even though they lack NMDA receptor
antagonist activity. This discovery was made possible because of the
inventor's recognition of a previously unanticipated antioxidant property
of the cannabinoids in general (and cannabidiol in particular) that
functions completely independently of antagonism at the NMDA, AMPA and
kainate receptors. Hence the present invention includes methods of
preventing or treating diseases caused by oxidative stress, such as
neuronal hypoxia, by administering a prophylactic or therapeutically
effective amount of a cannabinoid to a subject who has a disease caused by
oxidative stress.
The cannabinoid may be a cannabinoid other than THC, HU-210, or other
potent cannabinoid receptor agonists. The cannabinoid may also be other
than HU-211 or any other NMDA receptor antagonist that has previously been
reported. A potent cannabinoid receptor agonist is one that has an
EC50 at the cannabinoid receptor of 50 nM or less, but in more
particular embodiments 190 nM or 250 nM or less. In disclosed embodiments
the cannabinoid is not psychoactive, and is not psychotoxic even at high
doses. In some particularly disclosed embodiments, the cannabinoid is
selected from the group:
##STR5##
where A is aryl, and particularly
##STR6##
but not a pinene such as:
##STR7##
and the R1 -R5 groups are each independently selected from the
groups of hydrogen, lower substituted or unsubstituted alkyl, substituted
or unsubstituted carboxyl, substituted or unsubstituted alkoxy,
substituted or unsubstituted alcohol, and substituted or unsubstituted
ethers, and R6 -R7 are H or methyl. In particular embodiments,
there are no nitrogens in the rings, and/or no amino substitutions on the
rings.
In other embodiments, the cannabinoid is one of the following:
##STR8##
where there can be 0 to 3 double bonds on the A ring, as indicated by the
optional double bonds indicated by dashed lines on the A ring. The C ring
is aromatic, and the B ring can be a pyran. Particular embodiments are
dibenzo pyrans and cyclohexenyl benzenediols. Particular embodiments of
the cannabinoids of the present invention may also be highly lipid
soluble, and in particular embodiments can be dissolved in an aqueous
solution only sparingly (for example 10 mg/ml or less). The octanol/water
partition ratio at neutral pH in useful embodiments is 5000 or greater,
for example 6000 or greater. This high lipid solubility enhances
penetration of the drug into the CNS, as reflected by its volume of
distribution (Vd) of 1.5 L/kg or more, for example 3.5 L/kg, 7 L/kg,
or ideally 10 L/kg or more, for example at least 20 L/kg. Particular
embodiments may also be highly water soluble derivatives that are able to
penetrate the CNS, for example carboxyl derivatives.
R7-18 are independently selected from the group of H, substituted or
unsubstituted alkyl, especially lower alkyl, for example unsubstituted
C1 -C3 alkyl, hydroxyl, alkoxy, especially lower alkoxy such as
methoxy or ethoxy, substituted or unsubstituted alcohol, and unsubstituted
or substituted carboxyl, for example COOH or COCH3. In other
embodiments R7-18 can also be substituted or unsubstituted amino, and
halogen.
The cannabinoid has substantially no binding to the NMDAr (for example an
IC50 greater than or equal to 5 μM or 10 μM), has substantially
no psychoactive activity mediated by the cannabinoid receptor (for example
an IC50 at the cannabinoid receptor of greater than or equal to 300
nM, for example greater than 1 μM and a Ki greater than 250 nM,
especially 500-1000 nM, for example greater than 1000 nM), and antioxidant
activity, as demonstratable by the Fenton reaction or cyclic voltametry.
In other particular embodiments, the cannabinoids are one of the following:
##STR9##
where R19 is substituted or unsubstituted alkyl, such as lower alkyl
(for example methyl), lower alcohol (such as methyl alcohol) or carboxyl
(such as carboxylic acid) and oxygen (as in .dbd.O); R20 is hydrogen
or hydroxy; R21 is hydrogen, hydroxy, or methoxy; R22 is
hydrogen or hydroxy; R23 is hydrogen or hydroxy; R24 is hydrogen
or hydroxy; R25 is hydrogen or hydroxy; and R26 is substituted
or unsubstituted alkyl (for example n-methyl alkyl), substituted or
unsubstituted alcohol, or substituted or unsubstituted carboxy.
In yet other embodiments of the invention, the cannabinoids are
##STR10##
wherein numbering conventions for each of the ring positions are shown, and
R27, R28 and R29 are independently selected from the group
consisting of H, unsubstituted lower alkyl such as CH3, and carboxyl
such as COCH3. Particular examples of nonpsychoactive cannabinoids
that fall within this definition are cannabidiol and
##STR11##
and other structural analogs of cannabidiol.
In more particular embodiments, the cannabinoid is used to prevent or treat
an ischemic or neurodegenerative disease in the central nervous system of
a subject, by administering to the subject a therapeutically effective
amount of a cannabinoid to protect against oxidative injury to the central
nervous system. The cannabinoid may be any of the compounds set forth
above, or more specifically
##STR12##
wherein R27, R28 and R29 are independently selected from the
group consisting of H, lower alkyl such as CH3, and carboxyl such as
COCH3, and particularly wherein
a) R27 =R28 =R29 =H
b) R27 =R29 =H; R28 =CH3
c) R27 =R28 =CH3 ; R29 =H
d) R27 =R28 =COCH3 ; R29 =H
e) R27 =H; R28 =R29 =COCH3
When R27 =R28 =R29 =H, then the compound is cannabidiol.
When R27 =R29 =H and R28 =CH3, the compound is CBD
monomethyl ether. When R27 =R28 =CH3 and R29 =H, the
compound is CBD dimethyl ether. When R27 =R28 =COCH3 and
R29 =H, the compound is CBD diacetate. When R27 =H and R28
=R29 =COCH3, the compound is CBD monoacetate. The ischemic or
neurodegenerative disease may be, for example, an ischemic infarct,
Alzheimer's disease, Parkinson's disease, Down's syndrome, human
immunodeficiency virus (HIV) dementia, myocardial infarction, or treatment
and prevention of intraoperative or perioperative hypoxic insults that can
leave persistent neurological deficits following open heart surgery
requiring heart/lung bypass machines, such as coronary artery bypass
grafts (CABG).
The invention also includes an assay for selecting a cannabinoid to use in
treating a neurological disease by determining whether the cannabinoid is
an antioxidant. Once it has been determined that the cannabinoid is an
antioxidant, an antioxidant effective amount of the cannabinoid is
administered to treat the neurological disease, such as a vascular
ischemic event in the central nervous system, for example the type caused
by a neurovascular thromboembolism. Similarly, the method of the present
invention includes determining whether a disease is caused by oxidative
stress, and if the disease is caused by oxidative stress, administering
the cannabinoid in a therapeutically effective antioxidant amount.
The invention also includes identifying and administering antioxidant and
neuroprotective compounds (such as cannabidiol) which selectively inhibit
the enzyme activity of both 5- and 15-lipoxygenase more than the enzyme
activity of 12-lipoxygenase. In addition, such compounds posses low NMDA
antagonist activity and low cannabinoid receptor activity. Assays for
selecting compounds with the desired effect on lipoxygenase enzymes, and
methods for using identified compounds to treat neurological or ischemic
diseases are also provided. Such diseases may include a vascular ischemic
event in the central nervous system, for example a thromboembolism in the
brain, or a vascular ischemic event in the myocardium. Useful
administration of the compounds involves administration both during and
after an ischemic injury.
These and other objects of the invention will be understood more clearly by
reference to the following detailed description and drawings.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1A is a graph showing NMDA induced cellular damage in a neuron (as
measured by LDH release) in cells that were exposed to glutamate for 10
minutes, which demonstrates that increasing concentrations of cannabidiol
in the cell culture protects against cellular damage.
FIG. 1B is a graph similar to FIG. 1A, but showing that AMPA/kainate
receptor mediated damage (induced by glutamate and the AMPA/kainate
receptor potentiating agents cyclothiazide or concanavalin A) is also
reduced in a concentration dependent manner by the presence of cannabidiol
in the culture medium.
FIG. 2A is a bar graph showing cellular damage (as measured by LDH release)
in the presence of glutamate alone (100 μM Glu), and in the presence of
glutamate and 5 μM cannabidiol (CBD) or 5 μM THC, and demonstrates
that CBD and THC were similarly protective.
FIG. 2B is a bar graph similar to FIG. 2A, but showing the cellular damage
assessed in the presence of the cannabinoid receptor antagonist SR 141716A
(SR), which was not found to alter the neuroprotective effect of CBD (5
μM) or THC (5 μM), indicating the effect is not a typical
cannabinoid effect mediated by the cannabinoid receptor.
FIG. 3 is a graph showing the reduction oxidation potentials determined by
cyclic voltametry for some natural and synthetic cannabinoids, the
antioxidant BHT, and the non-cannabinoid anandamide (arachidonyl
ethanolamide) which is a ligand for the cannabinoid receptor. The voltage
at which initial peaks occur is an indication of antioxidant activity.
FIG. 4 is a graph that demonstrates the antioxidant properties of BHT, CBD
and THC, by plotting the fluorescence of a fluorescent dye against
concentrations of these substances, where declining fluorescence is an
indication of greater antioxidant activity.
FIG. 5A is a graph illustrating decreased t-butyl peroxide induced toxicity
(as measured by LDH release) in the presence of increasing concentrations
of cannabidiol, demonstrating that cannabidiol is an effective antioxidant
in living cells.
FIG. 5B is a bar graph comparing the antioxidant activity of several
antioxidants against glutamate induced toxicity in neurons, showing that
CBD has superior antioxidant activity.
FIG. 6A is a graph showing the effect of CBD (as measured by the change in
absorbance at 234 nm) on the enzymatic activity of two lipoxygenase
enzymes, rabbit 15-LO and porcine 12-LO, which demonstrates that CBD
inhibits 15-LO, but not 12-LO enzyme.
FIG. 6B is a graph demonstrating that inhibitory effect of CBD on 15-LO is
competitive.
FIG. 7A is a graph similar to FIG. 6A, but was performed in whole cells
rather than purified enzyme preparations, and shows the effect of CBD (as
measured by the change in absorbance at 236 nm) on the enzymatic activity
of 5-LO from cultured rat basophillic leukemia cells (RBL-2H3), which
demonstrates that CBD inhibits 5-LO.
FIG. 7B is a graph showing the effect of CBD (as measured by the change in
absorbance at 236 nm) on the formation of 12-HETE (the product of 12-LO)
by human leukocytes (12-LO type 1).
FIG. 7C is a graph similar to FIG. 7B, showing the effect of CBD (as
measured by the change in absorbance at 236 nm) on the formation of
12-HETE by human platelets (12-LO type 2).
FIG. 8 is a bar graph demonstrating that 12-HETE can protect cortical
neurons from NMDAr toxicity most effectively when administered during and
post ischemia.
DETAILED DESCRIPTION OF SOME SPECIFIC EMBODIMENTS
This invention provides antioxidant compounds and compositions, such as
pharmaceutical compositions, that include cannabinoids that act as free
radical scavengers for use in prophylaxis and treatment of disease. The
invention also includes methods for using the antioxidants in prevention
and treatment of pathological conditions such as ischemia (tissue
hypoxia), and in subjects who have been exposed to oxidant inducing agents
such as cancer chemotherapy, toxins, radiation, or other sources of
oxidative stress. The compositions and methods described herein are also
used for preventing oxidative damage in transplanted organs, for
inhibiting reoxygenation injury following reperfusion of ischemic tissues
(for example in heart disease), and for any other condition that is
mediated by oxidative or free radical mechanisms of injury. In particular
embodiments of the invention, the compounds and compositions are used in
the treatment of ischemic cardiovascular and neurovascular conditions, and
neurodegenerative diseases. However the present invention can also be used
as an antioxidant treatment in non-neurological diseases.
Molecular oxygen is essential for aerobic organisms, where it participates
in many biochemical reactions, including its role as the terminal electron
acceptor in oxidative phosphorylation. However excessive concentrations of
various forms of reactive oxygen species and other free radicals can have
serious adverse biological consequences, including the peroxidation of
membrane lipids, hydroxylation of nucleic acid bases, and the oxidation of
sulfhydryl groups and other protein moieties. Biological antioxidants
include tocopherols and tocotrieneols, carotenoids, quinones, bilirubin,
ascorbic acid, uric acid, and metal binding proteins. However these
endogenous antioxidant systems are often overwhelmed by pathological
processes that allow permanent oxidative damage to occur to tissue.
Free radicals are atoms, ions or molecules that contain an unpaired
electron, are usually unstable, and exhibit short half-lives. Reactive
oxygen species (ROS) is a collective term, designating the oxygen radicals
(e.g. .O2- superoxide radical), which by sequential univalent
reduction produces hydrogen peroxide (H2 O2) and hydroxyl
radical (.OH). The hydroxyl radical sets off chain reactions and can
interact with nucleic acids. Other ROS include nitric oxide (NO.) and
peroxy nitrite (NOO.), and other peroxyl (RO2.) and alkoxyl (RO.)
radicals. Increased production of these poisonous metabolites in certain
pathological conditions is believed to cause cellular damage through the
action of the highly reactive molecules on proteins, lipids and DNA. In
particular, ROS are believed to accumulate when tissues are subjected to
ischemia, particularly when followed by reperfusion.
The pharmaceutical compositions of the present invention have potent
antioxidant and/or free radical scavenging properties, that prevent or
reduce oxidative damage in biological systems, such as occurs in
ischemic/reperfusion injury, or in chronic neurodegenerative diseases such
as Alzheimer's disease, HIV dementia, and many other oxidation associated
diseases.
DEFINITIONS
"Oxidative associated diseases" refers to pathological conditions that
result at least in part from the production of or exposure to free
radicals, particularly oxyradicals, or reactive oxygen species. It is
evident to those of skill in the art that most pathological conditions are
multifactorial, and that assigning or identifying the predominant causal
factors for any particular condition is frequently difficult. For these
reasons, the term "free radical associated disease" encompasses
pathological states that are recognized as conditions in which free
radicals or ROS contribute to the pathology of the disease, or wherein
administration of a free radical inhibitor (e.g. desferroxamine),
scavenger (e.g. tocopherol, glutathione) or catalyst (e.g. superoxide
dismutase, catalase) is shown to produce detectable benefit by decreasing
symptoms, increasing survival, or providing other detectable clinical
benefits in treating or preventing the pathological state.
Oxidative associated diseases include, without limitation, free radical
associated diseases, such as ischemia, ischemic reperfusion injury,
inflammatory diseases, systemic lupus erythematosis, myocardial ischemia
or infarction, cerebrovascular accidents (such as a thromboembolic or
hemorrhagic stroke) that can lead to ischemia or an infarct in the brain,
operative ischemia, traumatic hemorrhage (for example a hypovolemic stroke
that can lead to CNS hypoxia or anoxia), spinal cord trauma, Down's
syndrome, Crohn's disease, autoimmune diseases (e.g. rheumatoid arthritis
or diabetes), cataract formation, uveitis, emphysema, gastric ulcers,
oxygen toxicity, neoplasia, undesired cellular apoptosis, radiation
sickness, and others. The present invention is believed to be particularly
beneficial in the treatment of oxidative associated diseases of the CNS,
because of the ability of the cannabinoids to cross the blood brain
barrier and exert their antioxidant effects in the brain. In particular
embodiments, the pharmaceutical composition of the present invention is
used for preventing, arresting, or treating neurological damage in
Parkinson's disease, Alzheimer's disease and HIV dementia; autoimmune
neurodegeneration of the type that can occur in encephalitis, and hypoxic
or anoxic neuronal damage that can result from apnea, respiratory arrest
or cardiac arrest, and anoxia caused by drowning, brain surgery or trauma
(such as concussion or spinal cord shock).
As used herein, an "antioxidant" is a substance that, when present in a
mixture containing an oxidizable substrate biological molecule,
significantly delays or prevents oxidation of the substrate biological
molecule. Antioxidants can act by scavenging biologically important
reactive free radicals or other reactive oxygen species (.O2-,
H2 O2, .OH, HOCl, ferryl, peroxyl, peroxynitrite, and alkoxyl),
or by preventing their formation, or by catalytically converting the free
radical or other reactive oxygen species to a less reactive species.
Relative antioxidant activity can be measured by cyclic voltametry studies
of the type disclosed in Example 5 (and FIG. 3), where the voltage
(x-axis) is an index of relative antioxidant activity. The voltage at
which the first peak occurs is an indication of the voltage at which an
electron is donated, which in turn is an index of antioxidant activity.
"Therapeutically effective antioxidant doses" can be determined by various
methods, including generating an empirical dose-response curve, predicting
potency and efficacy of a congener by using quantitative structure
activity relationships (QSAR) methods or molecular modeling, and other
methods used in the pharmaceutical sciences. Since oxidative damage is
generally cumulative, there is no minimum threshold level (or dose) with
respect to efficacy. However, minimum doses for producing a detectable
therapeutic or prophylactic effect for particular disease states can be
established.
As used herein, a "cannabinoid" is a chemical compound (such as cannabinol,
THC or cannabidiol) that is found in the plant species Cannabis saliva
(marijuana), and metabolites and synthetic analogues thereof that may or
may not have psychoactive properties. Cannabinoids therefore include
(without limitation) compounds (such as THC) that have high affinity for
the cannabinoid receptor (for example Ki <250 nM), and compounds
that do not have significant affinity for the cannabinoid receptor (such
as cannabidiol, CBD). Cannabinoids also include compounds that have a
characteristic dibenzopyran ring structure (of the type seen in THC) and
cannabinoids which do not possess a pyran ring (such as cannabidiol).
Hence a partial list of cannabinoids includes THC, CBD, dimethyl
heptylpentyl cannabidiol (DMHP-CBD), 6,12-dihydro-6-hydroxy-cannabidiol
(described in U.S. Pat. No. 5,227,537, incorporated by reference);
(3S,4R)-7-hydroxy-Δ6 -tetrahydrocannabinol homologs and
derivatives described in U.S. Pat. No. 4,876,276, incorporated by
reference;
(+)-4-[4-DMH-2,6-diacetoxy-phenyl]-2-carboxy-6,6-dimethylbicyclo[3.1.
1]hept-2-en, and other 4-phenylpinene derivatives disclosed in U.S. Pat.
No. 5,434,295, which is incorporated by reference; and cannabidiol
(-)(CBD) analogs such as (-)CBD-monomethylether, (-)CBD dimethyl ether;
(-)CBD diacetate; (-)3'-acetyl-CBD monoacetate; and ±AF11, all of which
are disclosed in Consroe et al., J. Clin. Phannacol. 21:428S-436S, 1981,
which is also incorporated by reference. Many other cannabinoids are
similarly disclosed in Agurell et al., Pharmacol. Rev. 38:31-43, 1986,
which is also incorporated by reference.
As referred to herein, the term "psychoactivity" means "cannabinoid
receptor mediated psychoactivity." Such effects include, euphoria,
lightheadedness, reduced motor coordination, and memory impairment.
Psychoactivity is not meant to include non-cannabinoid receptor mediated
effects such as the anxiolytic effect of CBD.
The "lipoxygenase enzyme activity" refers to the relative level of
lipoxygenase enzyme activity for a particular lipoxgenase, such as 5-, 15-
or 12-lipoxygenase, as measured in Example 8. A compound would be said to
"selectively inhibit a lipoxgenase enzyme" if the concentration of
inhibitor required to reduce enzyme activity by 50% was at least about 5
times less than the amount required to reduce activity of a second
lipoxgenase enzyme by the same degree (under the same conditions, i.e.
temperature, substrate concentration, etc.)
An "antagonist" is a compound that binds and occupies a receptor without
activating it. In the presence of a sufficient concentration of
antagonist, an agonist cannot activate its receptor. Therefore,
antagonists may decrease the neurotoxicity mediated by NMDA (as described
in Example 3) or AMPA and Kainate (as described in Example 4).
An "agonist" is a compound that activates a receptor. When the receptor is
activated for a longer than normal period of time, this may cause
neurotoxicity, as in the case of NMDA, AMPA and kainate receptors (see
Examples 3 and 4).
The term "alkyl" refers to a cyclic, branched, or straight chain alkyl
group containing only carbon and hydrogen, and unless otherwise mentioned
contains one to twelve carbon atoms. This term is further exemplified by
groups such as methyl, ethyl, n-propyl, isobutyl, t-butyl, pentyl,
pivalyl, heptyl, adamantyl, and cyclopentyl. Alkyl groups can either be
unsubstituted or substituted with one or more substituents, e.g. halogen,
alkyl, alkoxy, alkylthio, trifluoromethyl, acyloxy, hydroxy, mercapto,
carboxy, aryloxy, aryloxy, aryl, arylalkyl, heteroaryl, amino, alkylamino,
dialkylamino, morpholino, piperidino, pyrrolidin-1-yl, piperazin-1-yl, or
other functionality.
The term "lower alkyl" refers to a cyclic, branched or straight chain
monovalent alkyl radical of one to seven carbon atoms. This term is
further exemplified by such radicals as methyl, ethyl, n-propyl, i-propyl,
n-butyl, t-butyl, i-butyl (or 2-methylpropyl), cyclopropylmethyl, i-amyl,
n-amyl, hexyl and heptyl. Lower alkyl groups can also be unsubstituted or
substituted, where a specific example of a substituted alkyl is
1,1-dimethyl heptyl.
"Hydroxyl" refers to --OH.
"Alcohol" refers to R--OH, wherein R is alkyl, especially lower alkyl (for
example in methyl, ethyl or propyl alcohol). An alcohol may be either
linear or branched, such as isopropyl alcohol.
"Carboxyl" refers to the radical --COOH, and substituted carboxyl refers to
--COR where R is alkyl, lower alkyl or a carboxylic acid or ester.
The term "aryl" or "Ar" refers to a monovalent unsaturated aromatic
carbocyclic group having a single ring (e.g. phenyl) or multiple condensed
rings (e.g. naphthyl or anthryl), which can optionally be unsubstituted or
substituted with, e.g., halogen, alkyl, alkoxy, alkylthio,
trifluoromethyl, acyloxy, hydroxy, mercapto, carboxy, aryloxy, aryl,
arylalkyl, heteroaryl, amino, alkylamino, dialkylamino, morpholino,
piperidino, pyrrolidin-1-yl, piperazin-1-yl, or other functionality.
The term "alkoxy" refers to a substituted or unsubstituted alkoxy, where an
alkoxy has the structure --O--R, where R is substituted or unsubstituted
alkyl. In an unsubstituted alkoxy, the R is an unsubstituted alkyl. The
term "substituted alkoxy" refers to a group having the structure --O--R,
where R is alkyl which is substituted with a non-interfering substituent.
The term "arylalkoxy" refers to a group having the structure --O--R--Ar,
where R is alkyl and Ar is an aromatic substituent. Arylalkoxys are a
subset of substituted alkoxys. Examples of useful substituted alkoxy
groups are: benzyloxy, naphthyloxy, and chlorobenzyloxy.
The term "aryloxy" refers to a group having the structure --O--Ar, where Ar
is an aromatic group. A particular aryloxy group is phenoxy.
The term "heterocycle" refers to a monovalent saturated, unsaturated, or
aromatic carbocyclic group having a single ring (e.g. morpholino, pyridyl
or faryl) or multiple condensed rings (e.g. indolizinyl or
benzothienyl) and having at least one heteroatom, defined as N, O, P,
or S, within the ring, which can optionally be unsubstituted or
substituted with, e.g. halogen, alkyl, alkoxy, alkylthio, trifluoromethyl,
acyloxy, hydroxy, mercapto, carboxy, aryloxy, aryl, arylakyl, heteroaryl,
amino, alkylamino, dialkylamino, morpholino, piperidino, pyrrolidin-1-yl,
piperazin-1-yl, or other functionality.
"Arylalkyl" refers to the groups --R--Ar and --R--HetAr, where Ar is an
aryl group. HetAr is a heteroaryl group, and R is a straight-chain or
branched chain aliphatic group. Example of arylaklyl groups include benzyl
and furfuryl. Arylalkyl groups can optionally be unsubstituted or
substituted with, e.g., halogen, alkyl, alkoxy, alkylthio,
trifluoromethyl, acyloxy, hydroxy, mercapto, carboxy, aryloxy, aryl,
arylalkyl, heteroaryl, amino, alkylamino, dialkylamino, morpholino,
peperidino, pyrrolidin-1-yl, piperazin-1-yl, or other functionality.
The term "halo" or "halide" refers to fluoro, bromo, chloro and iodo
substituents.
The term "amino" refers to a chemical functionality --NR'R" where R' and R"
are independently hydrogen, alkyl, or aryl. The term "quaternary amine"
refers to the positively charged group --N+ R'R", where R'R" and R"
are independently selected and are alkyl or aryl. A particular amino group
is --NH2.
A "pharmaceutical agent" or "drug" refers to a chemical compound or
composition capable of inducing a desired therapeutic or prophylactic
effect when properly administered to a subject.
All chemical compounds include both the (+) and (-) stereoisomers, as well
as either the (+) or (-) stereoisomer.
Other chemistry terms herein are used according to conventional usage in
the art, as exemplified by The McGraw-Hill Dictionary of Chemical Terms
(1985) and The Condensed Chemical Dictionary (1981).
The following examples show that both nonpsychoactive cannabidiol, and
psychoactive cannabinoids such as THC, can protect neurons from glutamate
induced death, by a mechanism independent of cannabinoid receptors.
Cannabinoids are also be shown to be potent antioxidants capable of
preventing ROS toxicity in neurons.
EXAMPLE 1
Preparation of Cannabinoids and Neuronal Cultures
Cannabidiol, THC and reactants other than those specifically listed below
were purchased from Sigma Chemical, Co. (St. Louis, Mo.). Cyclothiazide,
glutamatergic ligands and MK-801 were obtained from Tocris Cookson (UK).
Dihydrorhodamine was supplied by Molecular Probes (Eugene, Oreg.). T-butyl
hydroperoxide, tetraethylammonium chloride, ferric citrate and sodium
dithionite were all purchased from Aldrich (WI). All culture media were
Gibco/BRL (MD) products.
Solutions of cannabinoids, cyclothiazide and other lipophiles were prepared
by evaporating a 10 mM ethanolic solution (under a stream of nitrogen) in
a siliconized microcentrifuge tube. Dimethyl sulfoxide (DMSO, less than
0.05% of final volume) was added to ethanol to prevent the lipophile
completely drying onto the tube wall. After evaporation, 1 ml of culture
media was added and the drug was dispersed using a high power sonic probe.
Special attention was used to ensure the solution did not overheat or
generate foam. Following dispersal, all solutions were made up to their
final volume in siliconized glass tubes by mixing with an appropriate
quantity of culture media.
Primary neuronal cultures were prepared according to the method of Ventra
et al. (J. Neurochem. 66:1752-1761, 1996). Fetuses were extracted by
Cesarian section from a 17 day pregnant Wistar rat, and the feral brains
were placed into phosphate buffered saline. The cortices were then
dissected out, cut into small pieces and incubated with papain for nine
minutes at 37° C. After this time the tissue was dissociated by
passage through a fire polished Pasteur pipette, and the resultant cell
suspension separated by centrifugation over a gradient consisting of 10
mg/ml bovine serum albumin and 10 mg/ml ovomucoid (a trypsin inhibitor) in
Earls buffered salt solution. The pellet was then re-suspended in high
glucose, phenol red free Dulbeco's modified Eagles medium containing 10%
fetal bovine serum, 2 mM glutamine, 100 IU penicillin, and 100 μg/ml
streptomycin (DMEM). Cells were counted, tested for vitality using the
trypan blue exclusion test and seeded onto poly-D-lysine coated 24
multiwell plates. After 96 hours, 10 μM fluoro-deoxyuridine and 10
μM uridine were added to block glial cell growth. This protocol
resulted in a highly neuron-enriched culture.
EXAMPLE 2
Preparation of Astrocytes and Conditioned Media
Astrocyte conditioned DMEM was used throughout the AMPA/kainate toxicity
procedure and following glutamate exposure in the NMDAr mediated toxicity
protocol. Media was conditioned by 24 hour treatment over a confluent
layer of type I astrocytes, prepared from two day old Wistar rat pups.
Cortices were dissected, cut into small pieces, and enzymatically digested
with 0.25% trypsin. Tissue was then dissociated by passage through a fire
polished Pasteur pipette and the cell suspension plated into untreated 75
cm2 T-flasks. After 24 hours the media was replaced and unattached
cells removed. Once astrocytes achieved confluence, cells were divided
into four flasks. Media for experiments was conditioned by a 24 hour
exposure to these astrocytes, after which time it was frozen at
-20° C. until use. Astrocyte cultures were used to condition DMEM
for no longer than two months.
EXAMPLE 3
NMDA Mediated Toxicity Studies
Glutamate neurotoxicity can be mediated by NMDA, AMPA or kainate receptors.
To examine NMDAr mediated toxicity, cultured neurons (cultured for 14-18
days) were exposed to 250 μM glutamate for 10 minutes in a magnesium
free saline solution. The saline was composed of 125 mM NaCl, 25 mM
glucose, 10 mM HEPES (pH 7.4), 5 mM KCl, 1.8 mM calcium chloride and 5%
bovine serum albumin. Following exposure, cells were washed twice with
saline, and incubated for 18 hours in conditioned DMEM. The level of
lactate dehydrogenase (LDH) in the media was used as an index of cell
injury.
Toxicity was completely prevented by addition of the NMDAr antagonist,
MK-801 (500 nM, data not shown). However, FIG. 1A shows that cannabidiol
also prevented neurotoxicity (maximum protection 88±9%) with an
EC50 of 2-4 μM (specifically about 3.5 μM).
EXAMPLE 4
AMPA and Kainate Receptor Mediated Toxicity Studies
Unlike NMDA receptors, which are regulated by magnesium ions, AMPA/kainate
receptors rapidly desensitize following ligand binding. To examine AMPA
and kainate receptor mediated toxicity, neurons were cultured for 7-13
days, then exposed to 100 μM glutamate and 50 μM cyclothiazide (used
to prevent AMPA receptor desensitization). Cells were incubated with
glutamate in the presence of 500 nM MK-801 (an NMDAr antagonist) for 18-20
hours prior to analysis. Specific AMPA and kainate receptor ligands were
also used to separately examine the effects of cannabinoids on AMPA and
kainate receptor mediated events. Fluorowillardiine (1.5 μM) was the
AMPA agonist and 4-methyl glutamate (10 μM) was the kainate agonist
used to investigate receptor mediated toxicity. When specifically
examining kainate receptor activity, cyclothiazide was replaced with 0.15
mg/ml Concanavalin-A.
Cannabidiol protection against AMPA/kainate mediated neurotoxicity is
illustrated in FIG. 1B, where LDH in the media was used as an index of
cell injury. The neuroprotective effect of cannabidiol was similar to that
observed in the NMDA mediated toxicity model (FIG. 1A). Cannabidiol
prevented neurotoxicity (maximum protection 80±17%) with an EC50
of 2-4 μM (specifically about 3.3 μM). Comparable results were
obtained with either the AMPA receptor ligand, fluorowillardiine or the
kainate receptor specific ligand, 4-methyl-glutamate (data not shown).
Hence cannabidiol protects similarly against toxicity mediated by NMDA,
AMPA or kainate receptors.
Unlike cannabidiol, THC is a ligand (and agonist) for the brain cannabinoid
receptor. The action of THC at the cannabinoid receptor has been proposed
to explain the ability of THC to protect neurons from NMDAr toxicity in
vitro. However in AMPA/kainate receptor toxicity assays, THC and
cannabidiol were similarly protective (FIG. 2A), indicating that
cannabinoid neuroprotection is independent of cannabinoid receptor
activation. This was confirmed by inclusion of cannabinoid receptor
antagonist SR-141716A in the culture media (SR in FIG. 2B). See Mansbach
et al., Psychopharmacology 124:315-22, 1996, for a description of
SR-141716A. Neither THC nor cannabidiol neuroprotection was affected by
cannabinoid receptor antagonist (FIG. 2B).
EXAMPLE 5
Cyclic Voltametery Studies or ReDox Potentials
To investigate whether cannabinoids protect neurons against glutamate
damage by reacting with ROS, the antioxidant properties of cannabidiol and
other cannabinoids were assessed. Cyclic voltametry, a procedure that
measures the ability of a compound to accept or donate electrons under a
variable voltage potential, was used to measure the oxidation potentials
of several natural and synthetic cannabinoids. These studies were
performed with an EG&G Princeton Applied Research potentiostat/galvanostat
(Model 273/PAR 270 software, NJ). The working electrode was a glassy
carbon disk with a platinum counter electrode and silver/silver chloride
reference. Tetraethylammonium chloride in acetonitrile (0.1 M) was used as
an electrolyte. Cyclic voltametry scans were done from +0 to 1.8 V at scan
rate of 100 mV per second. The reducing ability of cannabidiol (CBD), THC,
HU-211, and BHT were measured in this fashion. Anandamide, a cannabinoid
receptor ligand without a cannabinoid like structure, was used as a
non-responsive control. Each experiment was repeated twice with
essentially the same results.
Cannabidiol, THC and the synthetic cannabinoid HU-211 all donated electrons
at a similar potential as the antioxidant BHT. Anandamide (arachidonyl
ethanolamide) did not undergo oxidation at these potentials (FIG. 3).
Several other natural and synthetic cannabinoids, including cannabidiol,
nabilone, and levanantrodol were also tested, and they too exhibited
oxidation profiles similar to cannabidiol and THC (data not shown).
EXAMPLE 6
Iron Catalyzed Dihydrorhodamine Oxidation (Fenton Reaction)
The ability of cannabinoids to be readily oxidized, as illustrated in
Example 5, indicated they possess antioxidant properties comparable to
BHT. The antioxidant activity of BHT was examined in a Fenton reaction, in
which iron is catalyzed to produce ROS. Cannabidiol (CBD) and
tetrahydrocannabinol (THC) were evaluated for their ability to prevent
oxidation of dihydrorhodamine to the fluorescent compound rhodamine.
Oxidant was generated by ferrous catalysis (diothionite reduced ferric
citrate) of t-butyl hydroperoxide in a 50:50 water:acetonitrile (v/v)
solution. Dihydrorhodamine (50 μM) was incubated with 300 μM t-butyl
hydroperoxide and 0.5 μM iron for 5 minutes. After this time, oxidation
was assessed by spectrofluorimetry (Excit=500 nm, Emiss=570 nm). Various
concentrations of cannabinoids and BHT were included to examine their
ability to prevent dihydrorhodiamine oxidation.
Cannabidiol, THC and BHT all prevented dihydrorhodamine oxidation in a
similar, concentration dependent manner (FIG. 4), indicating that
cannabinoids have antioxidant potency comparable to BHT.
To confirm that cannabinoids act as antioxidants in the intact cell,
neurons were also incubated with the oxidant t-butyl hydroperoxide and
varying concentrations of cannabidiol (FIG. 5A). The t-butyl hydroperoxide
oxidant was chosen for its solubility in both aqueous and organic
solvents, which facilitates oxidation in both cytosolic and membrane cell
compartments. Cell toxicity was assessed 18-20 hours after insult by
measuring lactate dehydrogenase (LDH) release into the culture media. All
experiments were conducted with triple or quadruple values at each point
and all plates contained positive (glutamate alone) and baseline controls.
The assay was validated by comparison with an XTT based metabolic activity
assay. As shown in FIG. 5A, cannabidiol protected neurons against ROS
toxicity in a dose related manner, with an EC50 of about 6 μM. The
maximum protection observed was 88±9%.
Cannabidiol was also compared with known antioxidants in an AMPA/kainate
toxicity protocol. Neurons were exposed to 100 μM glutamate and
equimolar (5 μM) cannabidiol, α-tocopherol, BHT or ascorbate
(FIG. 5B). Although all of the antioxidants attenuated glutamate toxicity,
cannabidiol was significantly more protective than either
α-tocopherol or ascorbate. The similar antioxidant abilities of
cannabidiol and BHT in this chemical system (FIG. 4), and their comparable
protection in neuronal cultures (FIG. 5B), implies that cannabidiol
neuroprotection is due to an antioxidant effect.
EXAMPLE 7
In vivo Rat Studies
The middle cerebral artery of chloral hydrate anesthetized rats was
occluded by insertion of suture thread into it. The animals were allowed
to recover from the anesthetic and move freely for a period of two hours.
After this time the suture was removed under mild anesthetic and the
animals allowed to recover for 48 hours. Then the animals were tested for
neurological deficits, sacrificed, and the infarct volume calculated. To
examine the infarct volume, animals were anesthetized, ex-sanguinated, and
a metabolically active dye (3-phenyl tetrazolium chloride) was pumped
throughout the body. All living tissues were stained pink by the dye,
while morbid regions of infarcted tissue remained white. Brains were then
fixed for 24 hours in formaldehyde, sliced and the infarct volumes
measured.
One hour prior to induction of ischemia 20 mg/kg of cannabidiol was
administered by intra-peritoneal injection (ip) in a 90% saline:5%
emulphor 620 (emulsifier):5% ethanol vehicle. A second ip 10 mg/kg dose of
cannabidiol was administered 8 hours later using the same vehicle. Control
animals received injections of vehicle without drug. IV doses would be
expected to be 3-5 times less because of reduction of first pass
metabolism.
The infarct size and neurological assessment of the test animals is shown
Table 1.
TABLE 1
Cannabidiol protects rat brains from ischemia damage
Volume of Infarct Behavioral Deficit
(mm3) Score
Animal Drug Control Drug Control
1 108.2 110.5 3 2
2 83.85 119.6 4 4
3 8.41 118.9 3 4
4 75.5 177.7 1 4
5 60.53 33.89 1 3
6 27.52 255.5 1 5
7 23.16 143 1 4
Mean 55.3 137.0 2.0 3.7
SEM 13.8 25.7 0.5 0.4
p = 0.016 significant p = 0.015 significant
*Neurological scoring is performed on a subjective 1-5 scale of impairment.
0 = no impairment, 5 = severe (paralysis)
This data shows that infarct size was approximately halved in the animals
treated with cannabidiol, which was also accompanied by a substantial
improvement in the neurological status of the animal.
These studies with the nonpsychotropic marijuana constituent, cannabidiol,
demonstrate that protection can be achieved against both glutamate
neurotoxicity and free radical induced cell death. THC, the psychoactive
principle of cannabis, also blocked glutamate neurotoxicity with a potency
similar to cannabidiol. In both cases, neuroprotection is unaffected by
the presence of a cannabinoid receptor antagonist. These results therefore
surprisingly demonstrate that cannabinoids can have useful therapeutic
effects that are not mediated by cannabinoid receptors, and therefore are
not necessarily accompanied by psychoactive side effects. Cannabidiol also
acts as an anti-epileptic and anxiolytic, which makes it particularly
useful in the treatment of neurological diseases in which neuroanatomic
defects can predispose to seizures (e.g. subarachnoid hemorrhage).
A particular advantage of the cannabinoid compounds of the present
invention is that they are highly lipophilic, and have good penetration
into the central nervous system. The volume of distribution of some of
these compounds is at least 100 L in a 70 kg person (1.4 L/kg), more
particularly at least 250 L, and most particularly 500 L or even 700 L in
a 70 kg person (10 L/kg). The lipophilicity of particular compounds is
also about as great as that of THC, cannabidiol or other compounds that
have excellent penetration into the brain and other portions of the CNS.
Cannabinoids that lack psychoactivity or psychotoxicity are particularly
useful embodiments of the present invention, because the absence of such
side effects allows very high doses of the drug to be used without
encountering unpleasant side effects (such as dysphoria) or dangerous
complications (such as obtundation in a patient who may already have an
altered mental status). For example, therapeutic antioxidant blood levels
of cannabidiol can be 5-20 mg/kg, without significant toxicity, while
blood levels of psychoactive cannabinoids at this level would produce
obtundation, headache, conjunctival irritation, and other problems.
Particular examples of the compounds of the present invention have low
affinity to the cannabinoid receptor, for example a Ki of greater
than 250 nM, for example Ki≥500-1000 nM. A compound with a
Ki≥1000 nM is particularly useful, which compound has
essentially no psychoactivity mediated by the cannabinoid receptor.
Cannabidiol blocks glutamate toxicity with equal potency regardless of
whether the insult is mediated by NMDA, AMPA or kainate receptors.
Cannabidiol and THC have been shown to be comparable to the antioxidant
BHT, both in their ability to prevent dihydrorhodamine oxidation and in
their cyclic voltametric profiles. Several synthetic cannabinoids also
exhibited profiles similar to the BHT, although anandamide, which is not
structurally related to cannabinoids, did not. These findings indicate
that cannabinoids act as antioxidants in a non-biological situation, which
was confirmed in living cells by showing that cannabidiol attenuates
hydroperoxide induced neurotoxicity. The potency of cannabidiol as an
antioxidant was examined by comparing it on an equimolar basis with three
other commonly used compounds.
In the AMPA/kainate receptor dependent neurotoxicity model, cannabidiol
neuroprotection was comparable to the potent antioxidant, BHT, but
significantly greater than that observed with either α-tocopherol or
ascorbate. This unexpected superior antioxidant activity (in the absence
of BHT tumor promoting activity) shows for the first time that
cannabidiol, and other cannabinoids, can be used as antioxidant drugs in
the treatment (including prophylaxis) of oxidation associated diseases,
and is particularly useful as a neuroprotectant. The therapeutic potential
of nonpsychoactive cannabinoids is particularly promising, because of the
absence of psychotoxicity, and the ability to administer higher doses than
with psychotropic cannabinoids, such as THC. Previous studies have also
indicated that cannabidiol is not toxic, even when chronically
administered to humans or given in large acute doses (700 mg/day).
EXAMPLE 8
Effect of Cannabidiol on Lipoxygenase Enzymes
This example describes in vitro and in vivo assays to examine the effect of
cannabidiol (CBD) on three lipoxygenase (LO) enzymes: 5-LO, 12-LO and
15-LO.
In vitro Enzyme Assay
The ability of CBD to inhibit lipoxygenase was examined by measuring the
time dependent change in absorption at 234 nM following addition of 5 U of
each lipoxygenase (rabbit 15-LO purchased from Biomol (PA), porcine 12-LO
purchased from Cayman chemicals (MI)) to a solution containing 10 μM
(final concentration) linoleic acid.
Enzyme studies were performed using a u.v. spectrophotometer and a 3 ml
quartz cuvette containing 2.5 ml of a stirred solution of 12.5 μM
sodium linoleic acid (sodium salt) in solution A (25 mM Tris (pH 8.1), 1
mM EDTA 0.1% methyl cellulose). The reaction was initiated by addition of
0.5 ml enzyme solution (10 U/ml enzyme in solution A) and recorded for 60
seconds. Lipoxygenase exhibits non-Michaelis-Menten kinetics, an initial
"lag" (priming) phase followed by a linear phase which is terminated by
product inhibition. These complications were reduced by assessing enzyme
activity (change in absorption) over the "steepest" 20 second period in a
60 second run time. Recordings examined the absorption at 234 nm minus the
value at a reference wavelength of 280 nm. Linoleic acid was used as the
substrate rather than arachidonic acid, because the products are less
inhibitory to the enzyme, thereby providing a longer "linear phase".
Cell Purification and Separation
Human platelets and leukocytes were purified from buffy coat preparations
(NIH Blood Bank) using a standard Ficoll based centrifugation method used
in blood banks. Prior to use, cells were washed three times to eliminate
contaminating cell types. Cultured rat basophillic leukemia cells
(RBL-2H3) were used as a source of 5-lipoxygenase.
In vivo Determination of Lipoxygenase Activity
Cells were incubated with arachidonic acid and stimulated with the calcium
ionophore A23187. Lipids were extracted and separated by reverse phase
HPLC. Product formation was assessed as the area of a peak that co-eluted
with an authentic standard, had a greater absorbance at 236 nm than at
either 210 or 280 nm, and the formation of which was inhibited by a
lipoxygenase inhibitor.
Cell pellets were triturated in DMEM culture media, aliquoted and
pre-incubated for 15 minutes with 20 μM arachidonic acid and varying
concentrations of cannabidiol and/or 40 μM nordihydroguaiaretic acid (a
lipxygenase inhibitor). Platelets and leukocytes were also pre-incubated
with 80 μM manoalide (Biomol) to prevent phospholipase A2 activation.
Product formation was initiated by addition of 5 μM A23187 and
incubation for 10 minutes at 37° C. At the end of the incubation,
the reaction was stopped by addition of 15% 1M HCl and 10 ng/ml
prostaglandin B2 (internal standard). Lipids were extracted with 1 volume
of ethyl ether, which was dried under a stream of nitrogen. Samples were
reconstituted in 50% acetonitrile:50% H2 O and separated by reverse
phase HPLC using a gradient running from 63% acetonitrile: 37% H2
O:0.2% acetic acid to 90% acetonitrile (0.2% acetic acid) over 13 minutes.
Measurement of NMDAr Toxicity
The ability of 12-HETE (12-(s)-hydroxy-eicosatetraenoic acid, the product
of the action of 12-lipoxygenase on arachidonic (eicosatetraenoic) acid)
to protect cortical neurons from NMDAr toxicity was measured as described
in Example 3. The 12-HETE (0.5 μg/ml) was added either during ischemia
(co-incubated with the glutamate), during post-ischemia (co-incubated with
the DMEM after washing the cells), or during both ischemia and
post-ischemia.
Results
Using semi-purified enzyme preparations, the effect of CBD on rabbit 15-LO
and porcine 12-LO was compared. As shown in FIGS. 6A and B, CBD is a
potent competitive inhibitor of 15-LO with an EC50 of 598 nM.
However, CBD had no effect on the 12-LO enzyme.
Using whole cell preparations, the effect of CBD on 5- and 12-LO enzymes
was investigated. As shown in FIG. 7A, CBD inhibited 5-LO in cultured rat
basophillic leukemia cells (RBL-2H3) with an EC50 of 1.92 μM.
However, CBD had no effect on 12-LO, as monitored by the production of
12-HETE (the product of 12-LO), in either human leukocytes or platelets
(FIGS. 7B and C). The leukocyte 12-LO is similar, while the platelet 12-LO
is structurally and functionally different, from the porcine 12-LO used in
the in vitro enzyme study.
The ability of 12-HETE to protect cortical neurons from NMDAr toxicity is
shown in FIG. 8. To achieve best protection from NMDAr toxicity, 12-HETE
was administered both during and post ischemia.
Therefore, CBD serves as a selective inhibitor of at least two lipoxygenase
enzymes, 5-LO and 15-LO, but had no effect on 12-LO. Importantly, this is
the first demonstration (FIG. 8) that the 12-LO product 12-HETE can play a
significant role in protecting neurons from NMDAr mediated toxicity.
Although the mechanism of this protection is unknown at the present time,
12-HETE is known to be an important neuromodulator, due to its ability to
influence potassium channel activity.
EXAMPLE 9
Methods of Treatment
The present invention includes a treatment that inhibits oxidation
associated diseases in a subject such as an animal, for example a rat or
human. The method includes administering the antioxidant drugs of the
present invention, or a combination of the antioxidant drug and one or
more other pharmaceutical agents, to the subject in a pharmaceutically
compatible carrier and in an effective amount to inhibit the development
or progression of oxidation associated diseases. Although the treatment
can be used prophylactically in any patient in a demographic group at
significant risk for such diseases, subjects can also be selected using
more specific criteria, such as a definitive diagnosis of the condition.
The administration of any exogenous antioxidant cannabinoid would inhibit
the progression of the oxidation associated disease as compared to a
subject to whom the cannabinoid was not administered. The antioxidant
effect, however, increases with the dose of the cannabinoid.
The vehicle in which the drug is delivered can include pharmaceutically
acceptable compositions of the drugs of the present invention using
methods well known to those with skill in the art. Any of the common
carriers, such as sterile saline or glucose solution, can be utilized with
the drugs provided by the invention. Routes of administration include but
are not limited to oral, intracranial ventricular (icv), intrathecal (it),
intravenous (iv), parenteral, rectal, topical ophthalmic, subconjunctival,
nasal, aural, sub-lingual (under the tongue) and transdermal. The
antioxidant drugs of the invention may be administered intravenously in
any conventional medium for intravenous injection such as an aqueous
saline medium, or in blood plasma medium. Such medium may also contain
conventional pharmaceutical adjunct materials such as, for example,
pharmaceutically acceptable salts to adjust the osmotic pressure, lipid
carriers such as cyclodextrins, proteins such as serum albumin,
hydrophilic agents such as methyl cellulose, detergents, buffers,
preservatives and the like. Given the low solubility of many cannabinoids,
they may be suspended in sesame oil.
Given the excellent absorption of the compounds of the present invention
via an inhaled route, the compounds may also be administered as inhalants,
for example in pharmaceutical aerosols utilizing solutions, suspensions,
emulsions, powders and semisolid preparations of the type more fully
described in Remington: The Science and Practice of Pharmacy (19th
Edition, 1995) in chapter 95. A particular inhalant form is a metered dose
inhalant containing the active ingredient, in a suspension or a dispersing
agent (such as sorbitan trioleate, oleyl alcohol, oleic acid, or lecithin,
and a propellant such as 12/11 or 12/114).
Embodiments of the invention comprising pharmaceutical compositions can be
prepared with conventional pharmaceutically acceptable carriers, adjuvants
and counterions as would be known to those of skill in the art. The
compositions are preferably in the form of a unit dose in solid,
semi-solid and liquid dosage forms such as tablets, pills, powders, liquid
solutions or suspensions, injectable and infusible solutions, for example
a unit dose vial, or a metered dose inhaler. Effective oral human dosage
ranges for cannabidiol are contemplated to vary from about 1-40 mg/kg, for
example 5-20 mg/kg, and in particular a dose of about 20 mg/kg of body
weight.
If the antioxidant drugs are to be used in the prevention of cataracts,
they may be administered in the form of eye drops formulated in a
pharmaceutically inert, biologically acceptable carrier, such as isotonic
saline or an ointment. Conventional preservatives, such as benzalkonium
chloride, can also be added to the formulation. In ophthalmic ointments,
the active ingredient is admixed with a suitable base, such as white
petrolatum and mineral oil, along with antimicrobial preservatives.
Specific methods of compounding these dosage forms, as well as appropriate
pharmaceutical carriers, are known in the art. Remington: The Science and
Practice of Pharmacy, 19th Ed., Mack Publishing Co. (1995), particularly
Part 7.
The compounds of the present invention are ideally administered as soon as
a diagnosis is made of an ischemic event, or other oxidative insult. For
example, once a myocardial infarction has been confirmed by
electrocardiograph, or an elevation in enzymes characteristic of cardiac
injury (e.g. CKMB), a therapeutically effective amount of the cannabinoid
drug is administered. A dose can also be given following symptoms
characteristic of a stroke (motor or sensory abnormalities), or
radiographic confirmation of a cerebral infarct in a distribution
characteristic of a neurovascular thromboembolic event. The dose can be
given by frequent bolus administration, or as a continuous IV dose. In the
case of cannabidiol, for example, the drug could be given in a dose of 5
mg/kg active ingredient as a continuous intravenous infusion; or hourly
intramuscular injections of that dose.
EXAMPLE 10
The following table lists examples of some dibenzopyran cannabinoids that
may be useful as antioxidants in the method of the present invention.
##STR13##
##STR14##
Compound R19 R20 R21
R22 R23 R24 R25 R26
H 5 7-OH-Δ1 -THC CH2 OH H H H
H H H C5 H11
H 6 6α-OH-Δ1 -THC CH3 α-OH
H 7 6β-OH-Δ1 -THC CH3 β-OH
8 1"-OH-Δ1 -THC CH3 OH
H 9 2"-OH-Δ1 -THC CH3 OH
10 3"-OH-Δ1 -THC CH3
OH
11 4"-OH-Δ1 -THC CH3
OH
H 12 6α,7-diOH-Δ1 -THC CH2 OH α-OH
H 13 6v,7-diOH-Δ1 -THC CH2 OH β-OH
14 1",7-diOH-Δ1 -THC CH2 OH OH
H 15 2",7-diOH-Δ1 -THC CH2 OH
OH
H 16 3",7-diOH-Δ1 -THC CH2 OH
OH
H 17 4",7-diOH-Δ1 -THC CH2 OH
OH
18 1",6β-diOH-Δ1 -THC CH3 β-OH
OH
19 1",3"-diOH-Δ1 -THC CH3 OH
OH
20 1",6α,7-triOH-Δ1 -THC CH2 OH
α-OH OH
H 21 Δ1 -THC-6-one CH3 .dbd.O
22 Epoxyhexahydrocannabinol CH3
(EHHC)*
23 7-oxo-Δ1 -THC CHO
H 24 Δ1 -THC-7"-oic acid COOH
H 25 Δ1 -THC-3"-oic acid CH3
C2 H4 COOH
H 26 1"-OH-Δ1 -THC-7"-oic acid COOH
OH
H 27 2"-OH-Δ1 -THC-7"-oic acid COOH
OH
H 28 3"-OH-Δ1 -THC-7"-oic acid COOH
OH
H 29 4"-OH-Δ1 -THC-7"-oic acid COOH
OH
H 30 3",4",5"-trisnor-2"-OH-Δ1 - COOH
C2 H4 OH
THC-7-oic acid
H 31 7-OH-Δ1 -THC-2"-oic acid CH2 OH
CH2 COOH
H 32 6β-OH-Δ1 -THC-2"-oic acid CH3
β-OH CH2 COOH
H 33 7-OH-Δ1 -THC-3"-oic acid CH2 OH
C2 H4 COOH
H 34 6β-OH-Δ1 -THC-3"-oic acid CH3
β-OH C2 H4 COOH
H 35 6α-OH-Δ1 -THC-4"-oic acid CH3
α-OH C3 H6 COOH
H 36 2",3"-dehydro-6U-OH-Δ1 - CH3 α-OH
C3 H4 COOH
THC-4"-oic acid
H 37 Δ1 -THC-1",7-dioic acid COOH
COOH
H 38 Δ1 -THC-2",7-dioic acid COOH
CH2 COOH
H 39 Δ1 -THC-3",7-dioic acid COOH
C2 H4 COOH
H 40 Δ1 -THC-4",7-dioic acid COOH
C3 H6 COOH
H 41 1",2"-dehydro-Δ1 -THC-3",7- COOH
C2 H2 COOH
dioic acid
H 42 Δ1 -THC-glucuronic acid CH3
gluc.dagger.
H 43 Δ1 -THC-7-oic acid COO gluc.dagger.
glucuronide
*Epoxy group in C-1 and C-2 positions
.dagger. Glucuronide
Note: R-group substituents are H if not indicated otherwise.
Chemical structures of some of the dibenzopyran cannabinoids are shown
below.
##STR15##
##STR16##
##STR17##
EXAMPLE 11
Examples of Structural Analogs of Cannabidiol
The following table lists examples of some cannabinoids which are
structural analogs of cannabidiol and that may be useful as antioxidants
in the method of the present invention. A particularly useful example is
compound CBD, cannabidiol.
Compound R19 R20 R21 R22 R23
R24 R25 R26
##STR18## ##STR19##
44 CBD CH3 H H H H H H C5
H11
45 7-OH--CBD CH2 OH
46 6α- CH3 α-OH
47 6β- CH3 β-OH
48 1"- CH3 OH
49 2"- CH3 OH
50 3"- CH3 OH
51 4"- CH3 OH
52 5"- CH3 C4
H8 CH2 OH
53 6,7-diOH--CBD CH2 OH OH
54 3",7-diOH--CBD CH2 OH OH
55 4",7-diOH--CBD CH2 OH OH
56 CBD-7-oic acid COOH
57 CBD-3"-oic acid CH3 C2
H4 COOH
##STR20## ##STR21##
58 CBN CH3 H H H H H H C5
H11
59 7-OH--CBN CH2 OH
60 1"-OH--CBN CH3 OH
61 2"-OH--CBN CH3 OH
62 3"-OH--CBN CH3 OH
63 4"-OH--CBN CH3 OH
64 5"-OH--CBN CH3 C4
H8 CH2 OH
65 2"-7-diOH--CBN CH2 OH OH
66 CBN-7-oic acid COOH
67 CBN-1"-oic acid CH3 COOH
68 CBN-3"-oic acid CH3 C2
H4 COOH
Note: R-group substituents are H if not indicated otherwise.
The invention being thus described, variation in the materials and methods
for practicing the invention will be apparent to one of ordinary skill in
the art. Such variations are to be considered within the scope of the
invention, which is set forth in the claims below.
* * * * *
[B]Other References
Windholz et al., The Merck Index, Tenth Edition (1983) p. 241, abstract No. 1723.
Mechoulam et al., "A Total Synthesis of d1-Ɗ1 -Tetrahydrocannabinol, the Active Constituent of Hashish1," Journal of the American Chemical Society, 87:14:3273-3275 (1965)
Mechoulam et al., "Chemical Basis of Hashish Activity," Science, 18:611-612 (1970)
Ottersen et al., "The Crystal and Molecular Structure of Cannabidiol," Acta Chem. Scand. B 31, 9:807-812 (1977)
Cunha et al., "Chronic Administration of Cannabidiol to Healthy Volunteers and Epileptic Patients1," Pharmacology, 21:175-185 (1980)
Consroe et al., "Acute and Chronic Antiepileptic Drug Effects in Audiogenic Seizure-Susceptible Rats," Experimental Neurology, Academic Press Inc., 70:626-637 (1980)
Turkanis et al., "Electrophysiologic Properties of the Cannabinoids," J. Clin. Pharmacol., 21:449S-463S (1981)
Carlini et al., "Hypnotic and Antielpileptic Effects of Cannabidiol," J. Clin. Pharmacol., 21:417S-427S (1981)
Karler et al., "The Cannabinoids as Potential Antiepileptics," J. Clin. Pharmacol., 21:437S-448S (1981)
Consroe et al., "Antiepileptic Potential of Cannabidiol Analgos," J. Clin. Pharmacol., 21:428S-436S (1981)
Colasanti et al., "Ocular Hypotension, Ocular Toxicity,a nd Neurotoxicity in Response to Marihuana Extract and Cannabidiol," Gen Pharm., Pergamon Press Ltd., 15(6):479-484 (1984)
Colasanti et al., "Intraocular Pressure, Ocular Toxicity and Neurotoxicity after Administration of Cannabinol or Cannabigerol," Exp. Eye Res., Academic Press Inc., 39:251-259 (1984)
Volfe et al., "Cannabinoids Block Release of Serotonin from Platelets Induced by Plasma frm Migraine Patients," Int. J. Clin. Pharm. Res., Bioscience Ediprint Inc., 4:243-246 (1985)
Agurell et al., "Pharmacokinetics and Metabolism of Ɗ1 -Tetrahydrocannabinol and Other Cannabinoids with Emphasis on Man*," Pharmacological Reviews, 38(1):21-43 (1986)
Karler et al., "Different Cannabinoids Exhibit Different Pharmacological and Toxicological Properties,"NIDA Res. Monogr., 79:96-107 (1987)
Samara et al., "Pharmacokinetics of Cannabidiol in Dogs," Drug Metabolism and Disposition, 16(3):469-472 (1988)
Choi, "Glutamate Neurotoxicity and Diseases of the Nervous System," Neuron, Cell Press, 1:623-634 (1988)
Eshhar et al., "Neuroprotective and Antioxidant Activities of HU-211, A Novel NMDA Receptor Antagonist," European Journal of Pharmacology, 283:19-29 (1995)
Skaper et al., "The ALIAmide Palmitoylethanolamide and Cannabinoids, but not Anandamide, are Protective in a Delayed Postglutamate Paradigm of Excitotoxic Death in Cerebellar Granule Neurons," Neurobiology, Proc. Natl. Acad. Sci. USA, 93:3984-3989 (1996)
Alonso et al., "Simple Synthesis of 5-Substituted Resorcinols: A Revisited Family of Interesting Bioactive Molecules," J. Org. Chem., American Chemical Society, 62(2):417-421 (1997)
Combes et al. "A Simple Synthesis of the Natural 2,5-Dialkylresorcinol Free Radical Scavenger Antioxidant: Resorstation," Synthetic Communications, Marcel Dekker, Inc., 27(21):3769-3778 (1997)
Shohami et al., "Oxidative Stress in Closed-Head Injury: Brain Antioxidant Capacity as an Indicator of Functional Outcome," Journal of Cerebral Blood Flow and Metabolism, Lippincott-Raven Publishers, 17(10):1007-1019 (1997)
Zurier et al., "Dimethylheptyl-THC-11 OIC Acid," Arthritis & Rheumatism, 41(1):163-170 (1998)
Hampson et al., "Dual Effects of Anandamide on NMDA Receptor-Mediated Responses and Neurotransmission," Journal of Neurochemistry, Lippincott-Raven Publishers, 70(2):671-676 (1998)
Hampson et al., "Cannabidiol and (-)Ɗ9 -tetrahydrocannabiono are Neuroprotective Antioxidants," Medical Sciences, Proc. Natl. Acad. Sci. USA, 8268-8273 (1998)
July 3, 2008
The left hand says <**link removed, see text below
Existing Legal Drugs Provide Superior Treatment for Serious Medical Conditions
The FDA has approved safe and effective medication for the treatment of glaucoma, nausea, wasting syndrome, cancer, and multiple sclerosis.
Marinol, the synthetic form of THC (the psychoactive ingredient contained in marijuana), is already legally available for prescription by physicians whose patients suffer from pain and chronic illness.
The right hand said (in a 2003 Patent application!)
<***Link removed see text below)
Cannabinoids have been found to have antioxidant properties, unrelated to NMDA receptor antagonism. This new found property makes cannabinoids useful in the treatment and prophylaxis of wide variety of oxidation
associated diseases, such as ischemic, age-related, inflammatory and autoimmune diseases. The cannabinoids are found to have particular application as neuroprotectants, for example in limiting neurological damage following ischemic insults, such as stroke and trauma, or in the treatment of neurodegenerative diseases, such as Alzheimer’s disease,
Setting aside the immorality of the United States government granting itself a patent on something - I find myself reminded of something asked of Senator McCarthy:
“You’ve done enough. Have you no sense of decency, sir? At long last, have you left no sense of decency?”
I think even Kafka would consider the U.S. government’s behavior with respect to marijuana too outlandish to write a story about.
**From Left Hand Says link
ONDCP
Medical Marijuana Reality Check
What’s Wrong With Permitting the Use of Smoked Marijuana?
Simply put, the smoked form of marijuana is not considered modern medicine. On April 20th, 2006, the FDA issued an advisory concluding that no sound scientific studies have supported medical use of smoked marijuana for treatment in the United States, and no animal or human data support the safety or efficacy of smoked marijuana for general medical use.
A number of states have passed voter referenda or legislative actions making smoked marijuana available for a variety of medical conditions upon a doctor's recommendation. According to the Food and Drug Administration (FDA), these measures are inconsistent with efforts to ensure medications undergo the rigorous scientific scrutiny of the FDA approval process and are proven safe and effective under the standards of the FD&C Act.
While smoking marijuana may allow patients to temporarily feel better, the medical community makes an important distinction between inebriation and the controlled delivery of pure pharmaceutical medication. The raw (leaf ) form of marijuana contains a complex mixture of compounds in uncertain concentrations, the majority of which have unknown pharmacological effects.
The Institute of Medicine (IOM) has concluded that smoking marijuana is not recommended for any long-term medical use, and a subsequent IOM report declared that, “marijuana is not modern medicine.” Additionally, the American Medical Association, the National Cancer Institute, the American Cancer Society, and the National Multiple Sclerosis Society do not support the smoked form of marijuana as medicine.
Smoking Marijuana May Unintentionally Cause Serious Harm to Patients
The delicate immune systems of seriously ill patients may become compromised by the smoking of marijuana. Additionally, the daily use of marijuana compromises lung function and increases the risk for respiratory diseases, similar to those associated with nicotine cigarettes.
Marijuana has a high potential for abuse and can incur addiction. Frequent use of marijuana leads to tolerance to the psychoactive effects and smokers compensate by smoking more often or seeking higher potency marijuana.
In people with psychotic or other problems, the use of marijuana can precipitate severe emotional disorders. Chronic use of marijuana may increase the risk of psychotic symptoms in people with a past history of schizophrenia. Marijuana smoking by young people may lead to severe impairment of higher brain function and neuropsychiatric disorders, as well as a higher risk for addiction and polydrug abuse problems.
Existing Legal Drugs Provide Superior Treatment for Serious Medical Conditions
The FDA has approved safe and effective medication for the treatment of glaucoma, nausea, wasting syndrome, cancer, and multiple sclerosis.
Marinol, the synthetic form of THC (the psychoactive ingredient contained in marijuana), is already legally available for prescription by physicians whose patients suffer from pain and chronic illness.
“Medical marijuana was supposed to be for the truly ill cancer victims and AIDS patients who could use the drug to relieve pain or restore their appetites. Yet the number of dispensaries has skyrocketed from five in 2005 to 143 by the end of 2006. In North Hollywood alone, there are more pot clinics than Starbucks.”
–Pasadena Star-News, January 21st, 2007
In Their Words: What the Experts Say:
The American Academy of Ophthalmology:
“Based on reviews by the National Eye Institute (NEI) and the Institute of Medicine and on available scientific evidence, the Task Force on Complementary Therapies believes that no scientific evidence has been found that demonstrates increased benefits and/or diminished risks of marijuana use to treat glaucoma compared with the wide variety of pharmaceutical agents now available.”
Complementary Therapy Assessment: Marijuana in the Treatment of Glaucoma, American Academy of Ophthalmology, May 2003
The American Medical Association:
“...AMA recommends that marijuana be retained in Schedule I of the Controlled Substances Act...AMA believes that the NIH should use its resources and influence to support the development of a smoke-free inhaled delivery system for marijuana or delta-9-tetrahydrocannabinol (THC) to reduce the health hazards associated with the combustion and inhalation of marijuana...”
Policy Statement H-95.952, American Medical Association, http://www.ama-assn.org
The National Multiple Sclerosis Society:
“Studies completed thus far have not provided convincing evidence that marijuana or its derivatives provide substantiated benefits for symptoms of MS.”
The MS Information Sourcebook, Marijuana (Cannabis), National Multiple Sclerosis Society, September 18th, 2006
The Institute of Medicine (IOM):
“Because of the health risks associated with smoking, smoked marijuana should generally not be recommended for long-term medical use.”
Marijuana and Medicine: Assessing the Science Base, Institute of Medicine, 1999
*** from Right Hand Said link
US Patent 6630507 - Cannabinoids as antioxidants and neuroprotectants
US Patent Issued on October 7, 2003Abstract
Cannabinoids have been found to have antioxidant properties, unrelated to
NMDA receptor antagonism. This new found property makes cannabinoids
useful in the treatment and prophylaxis of wide variety of oxidation
associated diseases, such as ischemic, age-related, inflammatory and
autoimmune diseases. The cannabinoids are found to have particular
application as neuroprotectants, for example in limiting neurological
damage following ischemic insults, such as stroke and trauma, or in the
treatment of neurodegenerative diseases, such as Alzheimer's disease,
Parkinson's disease and HIV dementia. Nonpsychoactive cannabinoids, such
as cannabidoil, are particularly advantageous to use because they avoid
toxicity that is encountered with psychoactive cannabinoids at high doses
useful in the method of the present invention. A particular disclosed
class of cannabinoids useful as neuroprotective antioxidants is formula
(I) wherein the R group is independently selected from the group
consisting of H, CH3, and COCH3.
##STR1##Claims
We claim:
1. A method of treating diseases caused by oxidative stress, comprising
administering a therapeutically effective amount of a cannabinoid that has
substantially no binding to the NMDA receptor to a subject who has a
disease caused by oxidative stress.
2. The method of claim 1, wherein the cannabinoid is nonpsychoactive.
3. The method of claim 2, wherein the cannabinoid has a volume of
distribution of 10 L/kg or more.
4. The method of claim 1, wherein the cannabinoid is not an antagonist at
the NMDA receptor.
5. The method of claim 1, wherein the cannabinoid is:
##STR22##
where R is H, substituted or unsubstituted alkyl, carboxyl, alkoxy, aryl,
aryloxy, arylalkyl, halo or amino.
6. The method of claim 5, wherein R is H, substituted or unsubstituted
alkyl, carboxyl or alkoxy.
7. The method of claim 2, wherein the cannabinoid is:
##STR23##
where
A is cyclohexyl, substituted or unsubstituted aryl, or
##STR24##
but not a pinene;
R1 is H, substituted or unsubstituted alkyl, or substituted or
unsubstituted carboxyl;
R2 is H, lower substituted or unsubstituted alkyl, or alkoxy;
R3 is of H, lower substituted or unsubstituted alkyl, or substituted
or unsubstituted carboxyl;
R4 is H, hydroxyl, or lower substituted or unsubstituted alkyl; and
R5 is H, hydroxyl, or lower substituted or unsubstituted alkyl.
8. The method of claim 7, wherein
R1 is lower alkyl, COOH or COCH3 ;
BR>R2 is unsubstituted C1 -C5 alkyl, hydroxyl, methoxy or
ethoxy;
R3 is H, unsubstituted C1 -C3 alkyl, or COCH3 ;
R4 is hydroxyl, pentyl, heptyl, or diemthylheptyl; and
R5 is hydroxyl or methyl.
9. The method of claim 1, wherein the cannabinoid is:
##STR25##
where R1, R2 and R3 are independently H, CH3, or
COCH3.
10. The method of claim 9, wherein the cannabinoid is:
##STR26##
where:
a) R1 =R2 =R3 =H;
b) R1 =R3 =H, R2 =CH3 ;
c) R1 =R2 =CH3, R3 =H;
d) R1 =R2 =COCH3, R3 =H; or
e) R1 =H, R2 =R3 =COCH3.
11. The method of claim 2, wherein the cannabinoid is:
##STR27##
where R19 is H, lower alkyl, lower alcohol, or carboxyl; R20 is H
or OH; and R21 -R25 are independently H or OH.
12. The method of claim 11, wherein R19 is H, CH3, CH2 OH,
or COOH, and R20 -R24 are independently H or OH.
13. The method of claim 2, wherein the cannabinoid is:
##STR28##
where R19 and R20 are H, and R26 is alkyl.
14. The method of claim 10, wherein the cannabinoid is cannabidiol.
15. A method of treating an ischemic or neurodegenerative disease in the
central nervous system of a subject, comprising administering to the
subject a therapeutically effective amount of a cannabinoid, where the
cannabinoid is
##STR29##
where R is H, substituted or unsubstituted alkyl, carboxyl, alkoxy, aryl,
aryloxy, arylalkyl, halo or amino.
16. The method of claim 15, wherein the cannabinoid is not a psychoactive
cannabinoid.
17. The method of claim 15 where the ischemic or neurodegenerative disease
is an ischemic infarct, Alzheimer's disease, Parkinson's disease, and
human immunodeficiency virus dementia, Down's syndrome, or heart disease.
18. A method of treating a disease with a cannabinoid that has
substantially no binding to the NMDA receptor, comprising determining
whether the disease is caused by oxidative stress, and if the disease is
caused by oxidative stress, administering the cannabinoid in a
therapeutically effective antioxidant amount.
19. The method of claim 18, wherein the cannabinoid has a volume of
distribution of at least 1.5 L/kg and substantially no activity at the
cannabinoid receptor.
20. The method of claim 19, wherein the cannabinoid has a volume of
distribution of at least 10 L/kg.
21. The method of claim 1, wherein the cannabinoid selectively inhibits an
enzyme activity of 5- and 15-lipoxygenase more than an enzyme activity of
12-lipoxygenase.
22. A method of treating a neurodegenerative or ischemic disease in the
central nervous system of a subject, comprising administering to the
subject a therapeutically effective amount of a compound selected from any
of the compounds of claims 9 through 13.
23. The method of claim 22 where the compound is cannabidiol.
24. The method of claim 22, wherein the ischemic or neurodegenerative
disease is an ischemic infarct, Alzheimer's disease, Parkinson's disease,
and human immunodeficiency virus dementia, Down's syndrome, or heart
disease.
25. The method of claim 24 wherein the disease is an ischemic infarct.
26. The method of claim 1, wherein the cannabinoid is not an antagonist at
the AMPA receptor.
Description
FIELD OF THE INVENTION
The present invention concerns pharmaceutical compounds and compositions
that are useful as tissue protectants, such as neuroprotectants and
cardioprotectants. The compounds and compositions may be used, for
example, in the treatment of acute ischemic neurological insults or
chronic neurodegenerative diseases.
BACKGROUND OF THE INVENTION
Permanent injury to the central nervous system (CNS) occurs in a variety of
medical conditions, and has been the subject of intense scientific
scrutiny in recent years. It is known that the brain has high metabolic
requirements, and that it can suffer permanent neurologic damage if
deprived of sufficient oxygen (hypoxia) for even a few minutes. In the
absence of oxygen (anoxia), mitochondrial production of ATP cannot meet
the metabolic requirements of the brain, and tissue damage occurs. This
process is exacerbated by neuronal release of the neurotransmitter
glutamate, which stimulates NMDA (N-methyl-D-aspartate), AMPA
(α-amino-3-hydroxy-5-methyl-4-isoxazole propionate) and kainate
receptors. Activation of these receptors initiates calcium influx into the
neurons, and production of reactive oxygen species, which are potent
toxins that damage important cellular structures such as membranes, DNA
and enzymes.
The brain has many redundant blood supplies, which means that its tissue is
seldom completely deprived of oxygen, even during acute ischemic events
caused by thromboembolic events or trauma. A combination of the injury of
hypoxia with the added insult of glutamate toxicity is therefore believed
to be ultimately responsible for cellular death. Hence if the additive
insult of glutamate toxicity can be alleviated, neurological damage could
also be lessened. Anti-oxidants and anti-inflammatory agents have been
proposed to reduce damage, but they often have poor access to structures
such as the brain (which are protected by the blood brain barrier).
Given the importance of the NMDA, AMPA and kainate receptors in the
mechanism of injury, research efforts have focused on using antagonists to
these receptors to interfere with the receptor mediated calcium influx
that ultimately leads to cellular death and tissue necrosis. In vitro
studies using cultured neurons have demonstrated that glutamate receptor
antagonists reduce neurotoxicity, but NMDA and AMPA/kainate receptor
antagonists have different effects. Antagonists to NMDAr prevent
neurotoxicity if present during the glutamate exposure period, but are
less effective if added after glutamate is removed. In contrast,
AMPA/kainate receptor antagonists are not as effective as NMDA antagonists
during the glutamate exposure period, but are more effective following
glutamate exposure.
Some of the research on these antagonists has focused on cannabinoids, a
subset of which have been found to be NMDA receptor antagonists. U.S. Pat.
No. 5,538,993 (3S,4S-delta-6-tetrahydrocannabinol-7-oic acids), U.S. Pat.
No. 5,521,215 (sterospecific (+) THC enantiomers), and U.S. Pat. No.
5,284,867 (dimethylheptyl benzopyrans) have reported that these
cannabinoids are effective NMDA receptor blockers. U.S. Pat. No. 5,434,295
discloses that the 1,1 dimethylheptyl (DMH) homolog of
[3R,4R]-7-hydroxy-Δ6 THC (known as HU-210) is a superpotent
cannabinoid receptor agonist with cannabinomimetic activity two orders of
magnitude greater than the natural Δ9 THC. The HU-210
dimethylheptyl cannabinoid, has severe side effects, including fatigue,
thirst, headache, and hypotension. J. Pharmacol. Sci. 60:1433-1457 (1971).
Subjects who received this synthetic cannabinoid with a dimethylheptyl
group experienced marked psychomotor retardation, and were unwilling or
incapable of assuming an erect position.
In contrast to HU-210, the (-)(3R,4R) THC-DMH enantiomer (known as HU-211)
displays low affinity to the cannabinoid receptors, but retains NMDA
receptor antagonist neuroprotective activity.
##STR2##
THC (tetrahydrocannabinol) is another of the cannabinoids that has been
shown to be neuroprotective in cell cultures, but this protection was
believed to be mediated by interaction at the cannabinoid receptor, and so
would be accompanied by undesired psychotropic side effects.
##STR3##
Although it has been unclear whether cannabimimetic activity plays a role
in neuroprotection against glutamate induced neurological injury, the
teaching in this field has clearly been that a cannabinoid must at least
be an antagonist at the NMDA receptor to have neuroprotective effect.
Hence cannabidiol
(2-[3-methyl-6-(1-methylethenyl)-2-cyclohexen-1-yl]-5-pentyl-1,3-benzenedi
ol or CBD), a cannabinoid devoid of psychoactive effect (Pharm. Rev.
38:21-43, 1986), has not been considered useful as a neuroprotectant.
Cannabidiol has been studied as an antiepileptic (Carlini et al., J. Clin.
Pharmacol. 21:417S-427S, 1981; Karler et al., J. Clin. Pharmacol.
21:437S-448S, 1981, Consroe et al., J. Clin Phannacol. 21:428S-436S,
1981), and has been found to lower intraocular pressure (Colasanti et al,
Exp. Eye Res. 39:251-259, 1984 and Gen. Pharmac. 15:479-484, 1984).
##STR4##
No signs of toxicity or serious side effects have been observed following
chronic administration of cannabidiol to healthy volunteers (Cunha et al.,
Pharmacology 21:175-185, 1980), even in large acute doses of 700 mg/day
(Consroe et al., Pharmacol. Biochem. Behav. 40:701-708, 1991) but
cannabidiol is inactive at the NMDA receptor. Hence in spite of its
potential use in treating glaucoma and seizures, cannabidiol has not been
considered a neuroprotective agent that could be used to prevent glutamate
induced damage in the central nervous system.
SUMMARY OF THE INVENTION
It is an object of this invention to provide a new class of antioxidant
drugs, that have particular application as neuroprotectants, although they
are generally useful in the treatment of many oxidation associated
diseases.
Yet another object of the invention is to provide a subset of such drugs
that can be substantially free of psychoactive or psychotoxic effects, are
substantially non-toxic even at very high doses, and have good tissue
penetration, for example crossing the blood brain barrier.
It has surprisingly been found that cannabidiol and other cannabinoids can
function as neuroprotectants, even though they lack NMDA receptor
antagonist activity. This discovery was made possible because of the
inventor's recognition of a previously unanticipated antioxidant property
of the cannabinoids in general (and cannabidiol in particular) that
functions completely independently of antagonism at the NMDA, AMPA and
kainate receptors. Hence the present invention includes methods of
preventing or treating diseases caused by oxidative stress, such as
neuronal hypoxia, by administering a prophylactic or therapeutically
effective amount of a cannabinoid to a subject who has a disease caused by
oxidative stress.
The cannabinoid may be a cannabinoid other than THC, HU-210, or other
potent cannabinoid receptor agonists. The cannabinoid may also be other
than HU-211 or any other NMDA receptor antagonist that has previously been
reported. A potent cannabinoid receptor agonist is one that has an
EC50 at the cannabinoid receptor of 50 nM or less, but in more
particular embodiments 190 nM or 250 nM or less. In disclosed embodiments
the cannabinoid is not psychoactive, and is not psychotoxic even at high
doses. In some particularly disclosed embodiments, the cannabinoid is
selected from the group:
##STR5##
where A is aryl, and particularly
##STR6##
but not a pinene such as:
##STR7##
and the R1 -R5 groups are each independently selected from the
groups of hydrogen, lower substituted or unsubstituted alkyl, substituted
or unsubstituted carboxyl, substituted or unsubstituted alkoxy,
substituted or unsubstituted alcohol, and substituted or unsubstituted
ethers, and R6 -R7 are H or methyl. In particular embodiments,
there are no nitrogens in the rings, and/or no amino substitutions on the
rings.
In other embodiments, the cannabinoid is one of the following:
##STR8##
where there can be 0 to 3 double bonds on the A ring, as indicated by the
optional double bonds indicated by dashed lines on the A ring. The C ring
is aromatic, and the B ring can be a pyran. Particular embodiments are
dibenzo pyrans and cyclohexenyl benzenediols. Particular embodiments of
the cannabinoids of the present invention may also be highly lipid
soluble, and in particular embodiments can be dissolved in an aqueous
solution only sparingly (for example 10 mg/ml or less). The octanol/water
partition ratio at neutral pH in useful embodiments is 5000 or greater,
for example 6000 or greater. This high lipid solubility enhances
penetration of the drug into the CNS, as reflected by its volume of
distribution (Vd) of 1.5 L/kg or more, for example 3.5 L/kg, 7 L/kg,
or ideally 10 L/kg or more, for example at least 20 L/kg. Particular
embodiments may also be highly water soluble derivatives that are able to
penetrate the CNS, for example carboxyl derivatives.
R7-18 are independently selected from the group of H, substituted or
unsubstituted alkyl, especially lower alkyl, for example unsubstituted
C1 -C3 alkyl, hydroxyl, alkoxy, especially lower alkoxy such as
methoxy or ethoxy, substituted or unsubstituted alcohol, and unsubstituted
or substituted carboxyl, for example COOH or COCH3. In other
embodiments R7-18 can also be substituted or unsubstituted amino, and
halogen.
The cannabinoid has substantially no binding to the NMDAr (for example an
IC50 greater than or equal to 5 μM or 10 μM), has substantially
no psychoactive activity mediated by the cannabinoid receptor (for example
an IC50 at the cannabinoid receptor of greater than or equal to 300
nM, for example greater than 1 μM and a Ki greater than 250 nM,
especially 500-1000 nM, for example greater than 1000 nM), and antioxidant
activity, as demonstratable by the Fenton reaction or cyclic voltametry.
In other particular embodiments, the cannabinoids are one of the following:
##STR9##
where R19 is substituted or unsubstituted alkyl, such as lower alkyl
(for example methyl), lower alcohol (such as methyl alcohol) or carboxyl
(such as carboxylic acid) and oxygen (as in .dbd.O); R20 is hydrogen
or hydroxy; R21 is hydrogen, hydroxy, or methoxy; R22 is
hydrogen or hydroxy; R23 is hydrogen or hydroxy; R24 is hydrogen
or hydroxy; R25 is hydrogen or hydroxy; and R26 is substituted
or unsubstituted alkyl (for example n-methyl alkyl), substituted or
unsubstituted alcohol, or substituted or unsubstituted carboxy.
In yet other embodiments of the invention, the cannabinoids are
##STR10##
wherein numbering conventions for each of the ring positions are shown, and
R27, R28 and R29 are independently selected from the group
consisting of H, unsubstituted lower alkyl such as CH3, and carboxyl
such as COCH3. Particular examples of nonpsychoactive cannabinoids
that fall within this definition are cannabidiol and
##STR11##
and other structural analogs of cannabidiol.
In more particular embodiments, the cannabinoid is used to prevent or treat
an ischemic or neurodegenerative disease in the central nervous system of
a subject, by administering to the subject a therapeutically effective
amount of a cannabinoid to protect against oxidative injury to the central
nervous system. The cannabinoid may be any of the compounds set forth
above, or more specifically
##STR12##
wherein R27, R28 and R29 are independently selected from the
group consisting of H, lower alkyl such as CH3, and carboxyl such as
COCH3, and particularly wherein
a) R27 =R28 =R29 =H
b) R27 =R29 =H; R28 =CH3
c) R27 =R28 =CH3 ; R29 =H
d) R27 =R28 =COCH3 ; R29 =H
e) R27 =H; R28 =R29 =COCH3
When R27 =R28 =R29 =H, then the compound is cannabidiol.
When R27 =R29 =H and R28 =CH3, the compound is CBD
monomethyl ether. When R27 =R28 =CH3 and R29 =H, the
compound is CBD dimethyl ether. When R27 =R28 =COCH3 and
R29 =H, the compound is CBD diacetate. When R27 =H and R28
=R29 =COCH3, the compound is CBD monoacetate. The ischemic or
neurodegenerative disease may be, for example, an ischemic infarct,
Alzheimer's disease, Parkinson's disease, Down's syndrome, human
immunodeficiency virus (HIV) dementia, myocardial infarction, or treatment
and prevention of intraoperative or perioperative hypoxic insults that can
leave persistent neurological deficits following open heart surgery
requiring heart/lung bypass machines, such as coronary artery bypass
grafts (CABG).
The invention also includes an assay for selecting a cannabinoid to use in
treating a neurological disease by determining whether the cannabinoid is
an antioxidant. Once it has been determined that the cannabinoid is an
antioxidant, an antioxidant effective amount of the cannabinoid is
administered to treat the neurological disease, such as a vascular
ischemic event in the central nervous system, for example the type caused
by a neurovascular thromboembolism. Similarly, the method of the present
invention includes determining whether a disease is caused by oxidative
stress, and if the disease is caused by oxidative stress, administering
the cannabinoid in a therapeutically effective antioxidant amount.
The invention also includes identifying and administering antioxidant and
neuroprotective compounds (such as cannabidiol) which selectively inhibit
the enzyme activity of both 5- and 15-lipoxygenase more than the enzyme
activity of 12-lipoxygenase. In addition, such compounds posses low NMDA
antagonist activity and low cannabinoid receptor activity. Assays for
selecting compounds with the desired effect on lipoxygenase enzymes, and
methods for using identified compounds to treat neurological or ischemic
diseases are also provided. Such diseases may include a vascular ischemic
event in the central nervous system, for example a thromboembolism in the
brain, or a vascular ischemic event in the myocardium. Useful
administration of the compounds involves administration both during and
after an ischemic injury.
These and other objects of the invention will be understood more clearly by
reference to the following detailed description and drawings.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1A is a graph showing NMDA induced cellular damage in a neuron (as
measured by LDH release) in cells that were exposed to glutamate for 10
minutes, which demonstrates that increasing concentrations of cannabidiol
in the cell culture protects against cellular damage.
FIG. 1B is a graph similar to FIG. 1A, but showing that AMPA/kainate
receptor mediated damage (induced by glutamate and the AMPA/kainate
receptor potentiating agents cyclothiazide or concanavalin A) is also
reduced in a concentration dependent manner by the presence of cannabidiol
in the culture medium.
FIG. 2A is a bar graph showing cellular damage (as measured by LDH release)
in the presence of glutamate alone (100 μM Glu), and in the presence of
glutamate and 5 μM cannabidiol (CBD) or 5 μM THC, and demonstrates
that CBD and THC were similarly protective.
FIG. 2B is a bar graph similar to FIG. 2A, but showing the cellular damage
assessed in the presence of the cannabinoid receptor antagonist SR 141716A
(SR), which was not found to alter the neuroprotective effect of CBD (5
μM) or THC (5 μM), indicating the effect is not a typical
cannabinoid effect mediated by the cannabinoid receptor.
FIG. 3 is a graph showing the reduction oxidation potentials determined by
cyclic voltametry for some natural and synthetic cannabinoids, the
antioxidant BHT, and the non-cannabinoid anandamide (arachidonyl
ethanolamide) which is a ligand for the cannabinoid receptor. The voltage
at which initial peaks occur is an indication of antioxidant activity.
FIG. 4 is a graph that demonstrates the antioxidant properties of BHT, CBD
and THC, by plotting the fluorescence of a fluorescent dye against
concentrations of these substances, where declining fluorescence is an
indication of greater antioxidant activity.
FIG. 5A is a graph illustrating decreased t-butyl peroxide induced toxicity
(as measured by LDH release) in the presence of increasing concentrations
of cannabidiol, demonstrating that cannabidiol is an effective antioxidant
in living cells.
FIG. 5B is a bar graph comparing the antioxidant activity of several
antioxidants against glutamate induced toxicity in neurons, showing that
CBD has superior antioxidant activity.
FIG. 6A is a graph showing the effect of CBD (as measured by the change in
absorbance at 234 nm) on the enzymatic activity of two lipoxygenase
enzymes, rabbit 15-LO and porcine 12-LO, which demonstrates that CBD
inhibits 15-LO, but not 12-LO enzyme.
FIG. 6B is a graph demonstrating that inhibitory effect of CBD on 15-LO is
competitive.
FIG. 7A is a graph similar to FIG. 6A, but was performed in whole cells
rather than purified enzyme preparations, and shows the effect of CBD (as
measured by the change in absorbance at 236 nm) on the enzymatic activity
of 5-LO from cultured rat basophillic leukemia cells (RBL-2H3), which
demonstrates that CBD inhibits 5-LO.
FIG. 7B is a graph showing the effect of CBD (as measured by the change in
absorbance at 236 nm) on the formation of 12-HETE (the product of 12-LO)
by human leukocytes (12-LO type 1).
FIG. 7C is a graph similar to FIG. 7B, showing the effect of CBD (as
measured by the change in absorbance at 236 nm) on the formation of
12-HETE by human platelets (12-LO type 2).
FIG. 8 is a bar graph demonstrating that 12-HETE can protect cortical
neurons from NMDAr toxicity most effectively when administered during and
post ischemia.
DETAILED DESCRIPTION OF SOME SPECIFIC EMBODIMENTS
This invention provides antioxidant compounds and compositions, such as
pharmaceutical compositions, that include cannabinoids that act as free
radical scavengers for use in prophylaxis and treatment of disease. The
invention also includes methods for using the antioxidants in prevention
and treatment of pathological conditions such as ischemia (tissue
hypoxia), and in subjects who have been exposed to oxidant inducing agents
such as cancer chemotherapy, toxins, radiation, or other sources of
oxidative stress. The compositions and methods described herein are also
used for preventing oxidative damage in transplanted organs, for
inhibiting reoxygenation injury following reperfusion of ischemic tissues
(for example in heart disease), and for any other condition that is
mediated by oxidative or free radical mechanisms of injury. In particular
embodiments of the invention, the compounds and compositions are used in
the treatment of ischemic cardiovascular and neurovascular conditions, and
neurodegenerative diseases. However the present invention can also be used
as an antioxidant treatment in non-neurological diseases.
Molecular oxygen is essential for aerobic organisms, where it participates
in many biochemical reactions, including its role as the terminal electron
acceptor in oxidative phosphorylation. However excessive concentrations of
various forms of reactive oxygen species and other free radicals can have
serious adverse biological consequences, including the peroxidation of
membrane lipids, hydroxylation of nucleic acid bases, and the oxidation of
sulfhydryl groups and other protein moieties. Biological antioxidants
include tocopherols and tocotrieneols, carotenoids, quinones, bilirubin,
ascorbic acid, uric acid, and metal binding proteins. However these
endogenous antioxidant systems are often overwhelmed by pathological
processes that allow permanent oxidative damage to occur to tissue.
Free radicals are atoms, ions or molecules that contain an unpaired
electron, are usually unstable, and exhibit short half-lives. Reactive
oxygen species (ROS) is a collective term, designating the oxygen radicals
(e.g. .O2- superoxide radical), which by sequential univalent
reduction produces hydrogen peroxide (H2 O2) and hydroxyl
radical (.OH). The hydroxyl radical sets off chain reactions and can
interact with nucleic acids. Other ROS include nitric oxide (NO.) and
peroxy nitrite (NOO.), and other peroxyl (RO2.) and alkoxyl (RO.)
radicals. Increased production of these poisonous metabolites in certain
pathological conditions is believed to cause cellular damage through the
action of the highly reactive molecules on proteins, lipids and DNA. In
particular, ROS are believed to accumulate when tissues are subjected to
ischemia, particularly when followed by reperfusion.
The pharmaceutical compositions of the present invention have potent
antioxidant and/or free radical scavenging properties, that prevent or
reduce oxidative damage in biological systems, such as occurs in
ischemic/reperfusion injury, or in chronic neurodegenerative diseases such
as Alzheimer's disease, HIV dementia, and many other oxidation associated
diseases.
DEFINITIONS
"Oxidative associated diseases" refers to pathological conditions that
result at least in part from the production of or exposure to free
radicals, particularly oxyradicals, or reactive oxygen species. It is
evident to those of skill in the art that most pathological conditions are
multifactorial, and that assigning or identifying the predominant causal
factors for any particular condition is frequently difficult. For these
reasons, the term "free radical associated disease" encompasses
pathological states that are recognized as conditions in which free
radicals or ROS contribute to the pathology of the disease, or wherein
administration of a free radical inhibitor (e.g. desferroxamine),
scavenger (e.g. tocopherol, glutathione) or catalyst (e.g. superoxide
dismutase, catalase) is shown to produce detectable benefit by decreasing
symptoms, increasing survival, or providing other detectable clinical
benefits in treating or preventing the pathological state.
Oxidative associated diseases include, without limitation, free radical
associated diseases, such as ischemia, ischemic reperfusion injury,
inflammatory diseases, systemic lupus erythematosis, myocardial ischemia
or infarction, cerebrovascular accidents (such as a thromboembolic or
hemorrhagic stroke) that can lead to ischemia or an infarct in the brain,
operative ischemia, traumatic hemorrhage (for example a hypovolemic stroke
that can lead to CNS hypoxia or anoxia), spinal cord trauma, Down's
syndrome, Crohn's disease, autoimmune diseases (e.g. rheumatoid arthritis
or diabetes), cataract formation, uveitis, emphysema, gastric ulcers,
oxygen toxicity, neoplasia, undesired cellular apoptosis, radiation
sickness, and others. The present invention is believed to be particularly
beneficial in the treatment of oxidative associated diseases of the CNS,
because of the ability of the cannabinoids to cross the blood brain
barrier and exert their antioxidant effects in the brain. In particular
embodiments, the pharmaceutical composition of the present invention is
used for preventing, arresting, or treating neurological damage in
Parkinson's disease, Alzheimer's disease and HIV dementia; autoimmune
neurodegeneration of the type that can occur in encephalitis, and hypoxic
or anoxic neuronal damage that can result from apnea, respiratory arrest
or cardiac arrest, and anoxia caused by drowning, brain surgery or trauma
(such as concussion or spinal cord shock).
As used herein, an "antioxidant" is a substance that, when present in a
mixture containing an oxidizable substrate biological molecule,
significantly delays or prevents oxidation of the substrate biological
molecule. Antioxidants can act by scavenging biologically important
reactive free radicals or other reactive oxygen species (.O2-,
H2 O2, .OH, HOCl, ferryl, peroxyl, peroxynitrite, and alkoxyl),
or by preventing their formation, or by catalytically converting the free
radical or other reactive oxygen species to a less reactive species.
Relative antioxidant activity can be measured by cyclic voltametry studies
of the type disclosed in Example 5 (and FIG. 3), where the voltage
(x-axis) is an index of relative antioxidant activity. The voltage at
which the first peak occurs is an indication of the voltage at which an
electron is donated, which in turn is an index of antioxidant activity.
"Therapeutically effective antioxidant doses" can be determined by various
methods, including generating an empirical dose-response curve, predicting
potency and efficacy of a congener by using quantitative structure
activity relationships (QSAR) methods or molecular modeling, and other
methods used in the pharmaceutical sciences. Since oxidative damage is
generally cumulative, there is no minimum threshold level (or dose) with
respect to efficacy. However, minimum doses for producing a detectable
therapeutic or prophylactic effect for particular disease states can be
established.
As used herein, a "cannabinoid" is a chemical compound (such as cannabinol,
THC or cannabidiol) that is found in the plant species Cannabis saliva
(marijuana), and metabolites and synthetic analogues thereof that may or
may not have psychoactive properties. Cannabinoids therefore include
(without limitation) compounds (such as THC) that have high affinity for
the cannabinoid receptor (for example Ki <250 nM), and compounds
that do not have significant affinity for the cannabinoid receptor (such
as cannabidiol, CBD). Cannabinoids also include compounds that have a
characteristic dibenzopyran ring structure (of the type seen in THC) and
cannabinoids which do not possess a pyran ring (such as cannabidiol).
Hence a partial list of cannabinoids includes THC, CBD, dimethyl
heptylpentyl cannabidiol (DMHP-CBD), 6,12-dihydro-6-hydroxy-cannabidiol
(described in U.S. Pat. No. 5,227,537, incorporated by reference);
(3S,4R)-7-hydroxy-Δ6 -tetrahydrocannabinol homologs and
derivatives described in U.S. Pat. No. 4,876,276, incorporated by
reference;
(+)-4-[4-DMH-2,6-diacetoxy-phenyl]-2-carboxy-6,6-dimethylbicyclo[3.1.
1]hept-2-en, and other 4-phenylpinene derivatives disclosed in U.S. Pat.
No. 5,434,295, which is incorporated by reference; and cannabidiol
(-)(CBD) analogs such as (-)CBD-monomethylether, (-)CBD dimethyl ether;
(-)CBD diacetate; (-)3'-acetyl-CBD monoacetate; and ±AF11, all of which
are disclosed in Consroe et al., J. Clin. Phannacol. 21:428S-436S, 1981,
which is also incorporated by reference. Many other cannabinoids are
similarly disclosed in Agurell et al., Pharmacol. Rev. 38:31-43, 1986,
which is also incorporated by reference.
As referred to herein, the term "psychoactivity" means "cannabinoid
receptor mediated psychoactivity." Such effects include, euphoria,
lightheadedness, reduced motor coordination, and memory impairment.
Psychoactivity is not meant to include non-cannabinoid receptor mediated
effects such as the anxiolytic effect of CBD.
The "lipoxygenase enzyme activity" refers to the relative level of
lipoxygenase enzyme activity for a particular lipoxgenase, such as 5-, 15-
or 12-lipoxygenase, as measured in Example 8. A compound would be said to
"selectively inhibit a lipoxgenase enzyme" if the concentration of
inhibitor required to reduce enzyme activity by 50% was at least about 5
times less than the amount required to reduce activity of a second
lipoxgenase enzyme by the same degree (under the same conditions, i.e.
temperature, substrate concentration, etc.)
An "antagonist" is a compound that binds and occupies a receptor without
activating it. In the presence of a sufficient concentration of
antagonist, an agonist cannot activate its receptor. Therefore,
antagonists may decrease the neurotoxicity mediated by NMDA (as described
in Example 3) or AMPA and Kainate (as described in Example 4).
An "agonist" is a compound that activates a receptor. When the receptor is
activated for a longer than normal period of time, this may cause
neurotoxicity, as in the case of NMDA, AMPA and kainate receptors (see
Examples 3 and 4).
The term "alkyl" refers to a cyclic, branched, or straight chain alkyl
group containing only carbon and hydrogen, and unless otherwise mentioned
contains one to twelve carbon atoms. This term is further exemplified by
groups such as methyl, ethyl, n-propyl, isobutyl, t-butyl, pentyl,
pivalyl, heptyl, adamantyl, and cyclopentyl. Alkyl groups can either be
unsubstituted or substituted with one or more substituents, e.g. halogen,
alkyl, alkoxy, alkylthio, trifluoromethyl, acyloxy, hydroxy, mercapto,
carboxy, aryloxy, aryloxy, aryl, arylalkyl, heteroaryl, amino, alkylamino,
dialkylamino, morpholino, piperidino, pyrrolidin-1-yl, piperazin-1-yl, or
other functionality.
The term "lower alkyl" refers to a cyclic, branched or straight chain
monovalent alkyl radical of one to seven carbon atoms. This term is
further exemplified by such radicals as methyl, ethyl, n-propyl, i-propyl,
n-butyl, t-butyl, i-butyl (or 2-methylpropyl), cyclopropylmethyl, i-amyl,
n-amyl, hexyl and heptyl. Lower alkyl groups can also be unsubstituted or
substituted, where a specific example of a substituted alkyl is
1,1-dimethyl heptyl.
"Hydroxyl" refers to --OH.
"Alcohol" refers to R--OH, wherein R is alkyl, especially lower alkyl (for
example in methyl, ethyl or propyl alcohol). An alcohol may be either
linear or branched, such as isopropyl alcohol.
"Carboxyl" refers to the radical --COOH, and substituted carboxyl refers to
--COR where R is alkyl, lower alkyl or a carboxylic acid or ester.
The term "aryl" or "Ar" refers to a monovalent unsaturated aromatic
carbocyclic group having a single ring (e.g. phenyl) or multiple condensed
rings (e.g. naphthyl or anthryl), which can optionally be unsubstituted or
substituted with, e.g., halogen, alkyl, alkoxy, alkylthio,
trifluoromethyl, acyloxy, hydroxy, mercapto, carboxy, aryloxy, aryl,
arylalkyl, heteroaryl, amino, alkylamino, dialkylamino, morpholino,
piperidino, pyrrolidin-1-yl, piperazin-1-yl, or other functionality.
The term "alkoxy" refers to a substituted or unsubstituted alkoxy, where an
alkoxy has the structure --O--R, where R is substituted or unsubstituted
alkyl. In an unsubstituted alkoxy, the R is an unsubstituted alkyl. The
term "substituted alkoxy" refers to a group having the structure --O--R,
where R is alkyl which is substituted with a non-interfering substituent.
The term "arylalkoxy" refers to a group having the structure --O--R--Ar,
where R is alkyl and Ar is an aromatic substituent. Arylalkoxys are a
subset of substituted alkoxys. Examples of useful substituted alkoxy
groups are: benzyloxy, naphthyloxy, and chlorobenzyloxy.
The term "aryloxy" refers to a group having the structure --O--Ar, where Ar
is an aromatic group. A particular aryloxy group is phenoxy.
The term "heterocycle" refers to a monovalent saturated, unsaturated, or
aromatic carbocyclic group having a single ring (e.g. morpholino, pyridyl
or faryl) or multiple condensed rings (e.g. indolizinyl or
benzothienyl) and having at least one heteroatom, defined as N, O, P,
or S, within the ring, which can optionally be unsubstituted or
substituted with, e.g. halogen, alkyl, alkoxy, alkylthio, trifluoromethyl,
acyloxy, hydroxy, mercapto, carboxy, aryloxy, aryl, arylakyl, heteroaryl,
amino, alkylamino, dialkylamino, morpholino, piperidino, pyrrolidin-1-yl,
piperazin-1-yl, or other functionality.
"Arylalkyl" refers to the groups --R--Ar and --R--HetAr, where Ar is an
aryl group. HetAr is a heteroaryl group, and R is a straight-chain or
branched chain aliphatic group. Example of arylaklyl groups include benzyl
and furfuryl. Arylalkyl groups can optionally be unsubstituted or
substituted with, e.g., halogen, alkyl, alkoxy, alkylthio,
trifluoromethyl, acyloxy, hydroxy, mercapto, carboxy, aryloxy, aryl,
arylalkyl, heteroaryl, amino, alkylamino, dialkylamino, morpholino,
peperidino, pyrrolidin-1-yl, piperazin-1-yl, or other functionality.
The term "halo" or "halide" refers to fluoro, bromo, chloro and iodo
substituents.
The term "amino" refers to a chemical functionality --NR'R" where R' and R"
are independently hydrogen, alkyl, or aryl. The term "quaternary amine"
refers to the positively charged group --N+ R'R", where R'R" and R"
are independently selected and are alkyl or aryl. A particular amino group
is --NH2.
A "pharmaceutical agent" or "drug" refers to a chemical compound or
composition capable of inducing a desired therapeutic or prophylactic
effect when properly administered to a subject.
All chemical compounds include both the (+) and (-) stereoisomers, as well
as either the (+) or (-) stereoisomer.
Other chemistry terms herein are used according to conventional usage in
the art, as exemplified by The McGraw-Hill Dictionary of Chemical Terms
(1985) and The Condensed Chemical Dictionary (1981).
The following examples show that both nonpsychoactive cannabidiol, and
psychoactive cannabinoids such as THC, can protect neurons from glutamate
induced death, by a mechanism independent of cannabinoid receptors.
Cannabinoids are also be shown to be potent antioxidants capable of
preventing ROS toxicity in neurons.
EXAMPLE 1
Preparation of Cannabinoids and Neuronal Cultures
Cannabidiol, THC and reactants other than those specifically listed below
were purchased from Sigma Chemical, Co. (St. Louis, Mo.). Cyclothiazide,
glutamatergic ligands and MK-801 were obtained from Tocris Cookson (UK).
Dihydrorhodamine was supplied by Molecular Probes (Eugene, Oreg.). T-butyl
hydroperoxide, tetraethylammonium chloride, ferric citrate and sodium
dithionite were all purchased from Aldrich (WI). All culture media were
Gibco/BRL (MD) products.
Solutions of cannabinoids, cyclothiazide and other lipophiles were prepared
by evaporating a 10 mM ethanolic solution (under a stream of nitrogen) in
a siliconized microcentrifuge tube. Dimethyl sulfoxide (DMSO, less than
0.05% of final volume) was added to ethanol to prevent the lipophile
completely drying onto the tube wall. After evaporation, 1 ml of culture
media was added and the drug was dispersed using a high power sonic probe.
Special attention was used to ensure the solution did not overheat or
generate foam. Following dispersal, all solutions were made up to their
final volume in siliconized glass tubes by mixing with an appropriate
quantity of culture media.
Primary neuronal cultures were prepared according to the method of Ventra
et al. (J. Neurochem. 66:1752-1761, 1996). Fetuses were extracted by
Cesarian section from a 17 day pregnant Wistar rat, and the feral brains
were placed into phosphate buffered saline. The cortices were then
dissected out, cut into small pieces and incubated with papain for nine
minutes at 37° C. After this time the tissue was dissociated by
passage through a fire polished Pasteur pipette, and the resultant cell
suspension separated by centrifugation over a gradient consisting of 10
mg/ml bovine serum albumin and 10 mg/ml ovomucoid (a trypsin inhibitor) in
Earls buffered salt solution. The pellet was then re-suspended in high
glucose, phenol red free Dulbeco's modified Eagles medium containing 10%
fetal bovine serum, 2 mM glutamine, 100 IU penicillin, and 100 μg/ml
streptomycin (DMEM). Cells were counted, tested for vitality using the
trypan blue exclusion test and seeded onto poly-D-lysine coated 24
multiwell plates. After 96 hours, 10 μM fluoro-deoxyuridine and 10
μM uridine were added to block glial cell growth. This protocol
resulted in a highly neuron-enriched culture.
EXAMPLE 2
Preparation of Astrocytes and Conditioned Media
Astrocyte conditioned DMEM was used throughout the AMPA/kainate toxicity
procedure and following glutamate exposure in the NMDAr mediated toxicity
protocol. Media was conditioned by 24 hour treatment over a confluent
layer of type I astrocytes, prepared from two day old Wistar rat pups.
Cortices were dissected, cut into small pieces, and enzymatically digested
with 0.25% trypsin. Tissue was then dissociated by passage through a fire
polished Pasteur pipette and the cell suspension plated into untreated 75
cm2 T-flasks. After 24 hours the media was replaced and unattached
cells removed. Once astrocytes achieved confluence, cells were divided
into four flasks. Media for experiments was conditioned by a 24 hour
exposure to these astrocytes, after which time it was frozen at
-20° C. until use. Astrocyte cultures were used to condition DMEM
for no longer than two months.
EXAMPLE 3
NMDA Mediated Toxicity Studies
Glutamate neurotoxicity can be mediated by NMDA, AMPA or kainate receptors.
To examine NMDAr mediated toxicity, cultured neurons (cultured for 14-18
days) were exposed to 250 μM glutamate for 10 minutes in a magnesium
free saline solution. The saline was composed of 125 mM NaCl, 25 mM
glucose, 10 mM HEPES (pH 7.4), 5 mM KCl, 1.8 mM calcium chloride and 5%
bovine serum albumin. Following exposure, cells were washed twice with
saline, and incubated for 18 hours in conditioned DMEM. The level of
lactate dehydrogenase (LDH) in the media was used as an index of cell
injury.
Toxicity was completely prevented by addition of the NMDAr antagonist,
MK-801 (500 nM, data not shown). However, FIG. 1A shows that cannabidiol
also prevented neurotoxicity (maximum protection 88±9%) with an
EC50 of 2-4 μM (specifically about 3.5 μM).
EXAMPLE 4
AMPA and Kainate Receptor Mediated Toxicity Studies
Unlike NMDA receptors, which are regulated by magnesium ions, AMPA/kainate
receptors rapidly desensitize following ligand binding. To examine AMPA
and kainate receptor mediated toxicity, neurons were cultured for 7-13
days, then exposed to 100 μM glutamate and 50 μM cyclothiazide (used
to prevent AMPA receptor desensitization). Cells were incubated with
glutamate in the presence of 500 nM MK-801 (an NMDAr antagonist) for 18-20
hours prior to analysis. Specific AMPA and kainate receptor ligands were
also used to separately examine the effects of cannabinoids on AMPA and
kainate receptor mediated events. Fluorowillardiine (1.5 μM) was the
AMPA agonist and 4-methyl glutamate (10 μM) was the kainate agonist
used to investigate receptor mediated toxicity. When specifically
examining kainate receptor activity, cyclothiazide was replaced with 0.15
mg/ml Concanavalin-A.
Cannabidiol protection against AMPA/kainate mediated neurotoxicity is
illustrated in FIG. 1B, where LDH in the media was used as an index of
cell injury. The neuroprotective effect of cannabidiol was similar to that
observed in the NMDA mediated toxicity model (FIG. 1A). Cannabidiol
prevented neurotoxicity (maximum protection 80±17%) with an EC50
of 2-4 μM (specifically about 3.3 μM). Comparable results were
obtained with either the AMPA receptor ligand, fluorowillardiine or the
kainate receptor specific ligand, 4-methyl-glutamate (data not shown).
Hence cannabidiol protects similarly against toxicity mediated by NMDA,
AMPA or kainate receptors.
Unlike cannabidiol, THC is a ligand (and agonist) for the brain cannabinoid
receptor. The action of THC at the cannabinoid receptor has been proposed
to explain the ability of THC to protect neurons from NMDAr toxicity in
vitro. However in AMPA/kainate receptor toxicity assays, THC and
cannabidiol were similarly protective (FIG. 2A), indicating that
cannabinoid neuroprotection is independent of cannabinoid receptor
activation. This was confirmed by inclusion of cannabinoid receptor
antagonist SR-141716A in the culture media (SR in FIG. 2B). See Mansbach
et al., Psychopharmacology 124:315-22, 1996, for a description of
SR-141716A. Neither THC nor cannabidiol neuroprotection was affected by
cannabinoid receptor antagonist (FIG. 2B).
EXAMPLE 5
Cyclic Voltametery Studies or ReDox Potentials
To investigate whether cannabinoids protect neurons against glutamate
damage by reacting with ROS, the antioxidant properties of cannabidiol and
other cannabinoids were assessed. Cyclic voltametry, a procedure that
measures the ability of a compound to accept or donate electrons under a
variable voltage potential, was used to measure the oxidation potentials
of several natural and synthetic cannabinoids. These studies were
performed with an EG&G Princeton Applied Research potentiostat/galvanostat
(Model 273/PAR 270 software, NJ). The working electrode was a glassy
carbon disk with a platinum counter electrode and silver/silver chloride
reference. Tetraethylammonium chloride in acetonitrile (0.1 M) was used as
an electrolyte. Cyclic voltametry scans were done from +0 to 1.8 V at scan
rate of 100 mV per second. The reducing ability of cannabidiol (CBD), THC,
HU-211, and BHT were measured in this fashion. Anandamide, a cannabinoid
receptor ligand without a cannabinoid like structure, was used as a
non-responsive control. Each experiment was repeated twice with
essentially the same results.
Cannabidiol, THC and the synthetic cannabinoid HU-211 all donated electrons
at a similar potential as the antioxidant BHT. Anandamide (arachidonyl
ethanolamide) did not undergo oxidation at these potentials (FIG. 3).
Several other natural and synthetic cannabinoids, including cannabidiol,
nabilone, and levanantrodol were also tested, and they too exhibited
oxidation profiles similar to cannabidiol and THC (data not shown).
EXAMPLE 6
Iron Catalyzed Dihydrorhodamine Oxidation (Fenton Reaction)
The ability of cannabinoids to be readily oxidized, as illustrated in
Example 5, indicated they possess antioxidant properties comparable to
BHT. The antioxidant activity of BHT was examined in a Fenton reaction, in
which iron is catalyzed to produce ROS. Cannabidiol (CBD) and
tetrahydrocannabinol (THC) were evaluated for their ability to prevent
oxidation of dihydrorhodamine to the fluorescent compound rhodamine.
Oxidant was generated by ferrous catalysis (diothionite reduced ferric
citrate) of t-butyl hydroperoxide in a 50:50 water:acetonitrile (v/v)
solution. Dihydrorhodamine (50 μM) was incubated with 300 μM t-butyl
hydroperoxide and 0.5 μM iron for 5 minutes. After this time, oxidation
was assessed by spectrofluorimetry (Excit=500 nm, Emiss=570 nm). Various
concentrations of cannabinoids and BHT were included to examine their
ability to prevent dihydrorhodiamine oxidation.
Cannabidiol, THC and BHT all prevented dihydrorhodamine oxidation in a
similar, concentration dependent manner (FIG. 4), indicating that
cannabinoids have antioxidant potency comparable to BHT.
To confirm that cannabinoids act as antioxidants in the intact cell,
neurons were also incubated with the oxidant t-butyl hydroperoxide and
varying concentrations of cannabidiol (FIG. 5A). The t-butyl hydroperoxide
oxidant was chosen for its solubility in both aqueous and organic
solvents, which facilitates oxidation in both cytosolic and membrane cell
compartments. Cell toxicity was assessed 18-20 hours after insult by
measuring lactate dehydrogenase (LDH) release into the culture media. All
experiments were conducted with triple or quadruple values at each point
and all plates contained positive (glutamate alone) and baseline controls.
The assay was validated by comparison with an XTT based metabolic activity
assay. As shown in FIG. 5A, cannabidiol protected neurons against ROS
toxicity in a dose related manner, with an EC50 of about 6 μM. The
maximum protection observed was 88±9%.
Cannabidiol was also compared with known antioxidants in an AMPA/kainate
toxicity protocol. Neurons were exposed to 100 μM glutamate and
equimolar (5 μM) cannabidiol, α-tocopherol, BHT or ascorbate
(FIG. 5B). Although all of the antioxidants attenuated glutamate toxicity,
cannabidiol was significantly more protective than either
α-tocopherol or ascorbate. The similar antioxidant abilities of
cannabidiol and BHT in this chemical system (FIG. 4), and their comparable
protection in neuronal cultures (FIG. 5B), implies that cannabidiol
neuroprotection is due to an antioxidant effect.
EXAMPLE 7
In vivo Rat Studies
The middle cerebral artery of chloral hydrate anesthetized rats was
occluded by insertion of suture thread into it. The animals were allowed
to recover from the anesthetic and move freely for a period of two hours.
After this time the suture was removed under mild anesthetic and the
animals allowed to recover for 48 hours. Then the animals were tested for
neurological deficits, sacrificed, and the infarct volume calculated. To
examine the infarct volume, animals were anesthetized, ex-sanguinated, and
a metabolically active dye (3-phenyl tetrazolium chloride) was pumped
throughout the body. All living tissues were stained pink by the dye,
while morbid regions of infarcted tissue remained white. Brains were then
fixed for 24 hours in formaldehyde, sliced and the infarct volumes
measured.
One hour prior to induction of ischemia 20 mg/kg of cannabidiol was
administered by intra-peritoneal injection (ip) in a 90% saline:5%
emulphor 620 (emulsifier):5% ethanol vehicle. A second ip 10 mg/kg dose of
cannabidiol was administered 8 hours later using the same vehicle. Control
animals received injections of vehicle without drug. IV doses would be
expected to be 3-5 times less because of reduction of first pass
metabolism.
The infarct size and neurological assessment of the test animals is shown
Table 1.
TABLE 1
Cannabidiol protects rat brains from ischemia damage
Volume of Infarct Behavioral Deficit
(mm3) Score
Animal Drug Control Drug Control
1 108.2 110.5 3 2
2 83.85 119.6 4 4
3 8.41 118.9 3 4
4 75.5 177.7 1 4
5 60.53 33.89 1 3
6 27.52 255.5 1 5
7 23.16 143 1 4
Mean 55.3 137.0 2.0 3.7
SEM 13.8 25.7 0.5 0.4
p = 0.016 significant p = 0.015 significant
*Neurological scoring is performed on a subjective 1-5 scale of impairment.
0 = no impairment, 5 = severe (paralysis)
This data shows that infarct size was approximately halved in the animals
treated with cannabidiol, which was also accompanied by a substantial
improvement in the neurological status of the animal.
These studies with the nonpsychotropic marijuana constituent, cannabidiol,
demonstrate that protection can be achieved against both glutamate
neurotoxicity and free radical induced cell death. THC, the psychoactive
principle of cannabis, also blocked glutamate neurotoxicity with a potency
similar to cannabidiol. In both cases, neuroprotection is unaffected by
the presence of a cannabinoid receptor antagonist. These results therefore
surprisingly demonstrate that cannabinoids can have useful therapeutic
effects that are not mediated by cannabinoid receptors, and therefore are
not necessarily accompanied by psychoactive side effects. Cannabidiol also
acts as an anti-epileptic and anxiolytic, which makes it particularly
useful in the treatment of neurological diseases in which neuroanatomic
defects can predispose to seizures (e.g. subarachnoid hemorrhage).
A particular advantage of the cannabinoid compounds of the present
invention is that they are highly lipophilic, and have good penetration
into the central nervous system. The volume of distribution of some of
these compounds is at least 100 L in a 70 kg person (1.4 L/kg), more
particularly at least 250 L, and most particularly 500 L or even 700 L in
a 70 kg person (10 L/kg). The lipophilicity of particular compounds is
also about as great as that of THC, cannabidiol or other compounds that
have excellent penetration into the brain and other portions of the CNS.
Cannabinoids that lack psychoactivity or psychotoxicity are particularly
useful embodiments of the present invention, because the absence of such
side effects allows very high doses of the drug to be used without
encountering unpleasant side effects (such as dysphoria) or dangerous
complications (such as obtundation in a patient who may already have an
altered mental status). For example, therapeutic antioxidant blood levels
of cannabidiol can be 5-20 mg/kg, without significant toxicity, while
blood levels of psychoactive cannabinoids at this level would produce
obtundation, headache, conjunctival irritation, and other problems.
Particular examples of the compounds of the present invention have low
affinity to the cannabinoid receptor, for example a Ki of greater
than 250 nM, for example Ki≥500-1000 nM. A compound with a
Ki≥1000 nM is particularly useful, which compound has
essentially no psychoactivity mediated by the cannabinoid receptor.
Cannabidiol blocks glutamate toxicity with equal potency regardless of
whether the insult is mediated by NMDA, AMPA or kainate receptors.
Cannabidiol and THC have been shown to be comparable to the antioxidant
BHT, both in their ability to prevent dihydrorhodamine oxidation and in
their cyclic voltametric profiles. Several synthetic cannabinoids also
exhibited profiles similar to the BHT, although anandamide, which is not
structurally related to cannabinoids, did not. These findings indicate
that cannabinoids act as antioxidants in a non-biological situation, which
was confirmed in living cells by showing that cannabidiol attenuates
hydroperoxide induced neurotoxicity. The potency of cannabidiol as an
antioxidant was examined by comparing it on an equimolar basis with three
other commonly used compounds.
In the AMPA/kainate receptor dependent neurotoxicity model, cannabidiol
neuroprotection was comparable to the potent antioxidant, BHT, but
significantly greater than that observed with either α-tocopherol or
ascorbate. This unexpected superior antioxidant activity (in the absence
of BHT tumor promoting activity) shows for the first time that
cannabidiol, and other cannabinoids, can be used as antioxidant drugs in
the treatment (including prophylaxis) of oxidation associated diseases,
and is particularly useful as a neuroprotectant. The therapeutic potential
of nonpsychoactive cannabinoids is particularly promising, because of the
absence of psychotoxicity, and the ability to administer higher doses than
with psychotropic cannabinoids, such as THC. Previous studies have also
indicated that cannabidiol is not toxic, even when chronically
administered to humans or given in large acute doses (700 mg/day).
EXAMPLE 8
Effect of Cannabidiol on Lipoxygenase Enzymes
This example describes in vitro and in vivo assays to examine the effect of
cannabidiol (CBD) on three lipoxygenase (LO) enzymes: 5-LO, 12-LO and
15-LO.
In vitro Enzyme Assay
The ability of CBD to inhibit lipoxygenase was examined by measuring the
time dependent change in absorption at 234 nM following addition of 5 U of
each lipoxygenase (rabbit 15-LO purchased from Biomol (PA), porcine 12-LO
purchased from Cayman chemicals (MI)) to a solution containing 10 μM
(final concentration) linoleic acid.
Enzyme studies were performed using a u.v. spectrophotometer and a 3 ml
quartz cuvette containing 2.5 ml of a stirred solution of 12.5 μM
sodium linoleic acid (sodium salt) in solution A (25 mM Tris (pH 8.1), 1
mM EDTA 0.1% methyl cellulose). The reaction was initiated by addition of
0.5 ml enzyme solution (10 U/ml enzyme in solution A) and recorded for 60
seconds. Lipoxygenase exhibits non-Michaelis-Menten kinetics, an initial
"lag" (priming) phase followed by a linear phase which is terminated by
product inhibition. These complications were reduced by assessing enzyme
activity (change in absorption) over the "steepest" 20 second period in a
60 second run time. Recordings examined the absorption at 234 nm minus the
value at a reference wavelength of 280 nm. Linoleic acid was used as the
substrate rather than arachidonic acid, because the products are less
inhibitory to the enzyme, thereby providing a longer "linear phase".
Cell Purification and Separation
Human platelets and leukocytes were purified from buffy coat preparations
(NIH Blood Bank) using a standard Ficoll based centrifugation method used
in blood banks. Prior to use, cells were washed three times to eliminate
contaminating cell types. Cultured rat basophillic leukemia cells
(RBL-2H3) were used as a source of 5-lipoxygenase.
In vivo Determination of Lipoxygenase Activity
Cells were incubated with arachidonic acid and stimulated with the calcium
ionophore A23187. Lipids were extracted and separated by reverse phase
HPLC. Product formation was assessed as the area of a peak that co-eluted
with an authentic standard, had a greater absorbance at 236 nm than at
either 210 or 280 nm, and the formation of which was inhibited by a
lipoxygenase inhibitor.
Cell pellets were triturated in DMEM culture media, aliquoted and
pre-incubated for 15 minutes with 20 μM arachidonic acid and varying
concentrations of cannabidiol and/or 40 μM nordihydroguaiaretic acid (a
lipxygenase inhibitor). Platelets and leukocytes were also pre-incubated
with 80 μM manoalide (Biomol) to prevent phospholipase A2 activation.
Product formation was initiated by addition of 5 μM A23187 and
incubation for 10 minutes at 37° C. At the end of the incubation,
the reaction was stopped by addition of 15% 1M HCl and 10 ng/ml
prostaglandin B2 (internal standard). Lipids were extracted with 1 volume
of ethyl ether, which was dried under a stream of nitrogen. Samples were
reconstituted in 50% acetonitrile:50% H2 O and separated by reverse
phase HPLC using a gradient running from 63% acetonitrile: 37% H2
O:0.2% acetic acid to 90% acetonitrile (0.2% acetic acid) over 13 minutes.
Measurement of NMDAr Toxicity
The ability of 12-HETE (12-(s)-hydroxy-eicosatetraenoic acid, the product
of the action of 12-lipoxygenase on arachidonic (eicosatetraenoic) acid)
to protect cortical neurons from NMDAr toxicity was measured as described
in Example 3. The 12-HETE (0.5 μg/ml) was added either during ischemia
(co-incubated with the glutamate), during post-ischemia (co-incubated with
the DMEM after washing the cells), or during both ischemia and
post-ischemia.
Results
Using semi-purified enzyme preparations, the effect of CBD on rabbit 15-LO
and porcine 12-LO was compared. As shown in FIGS. 6A and B, CBD is a
potent competitive inhibitor of 15-LO with an EC50 of 598 nM.
However, CBD had no effect on the 12-LO enzyme.
Using whole cell preparations, the effect of CBD on 5- and 12-LO enzymes
was investigated. As shown in FIG. 7A, CBD inhibited 5-LO in cultured rat
basophillic leukemia cells (RBL-2H3) with an EC50 of 1.92 μM.
However, CBD had no effect on 12-LO, as monitored by the production of
12-HETE (the product of 12-LO), in either human leukocytes or platelets
(FIGS. 7B and C). The leukocyte 12-LO is similar, while the platelet 12-LO
is structurally and functionally different, from the porcine 12-LO used in
the in vitro enzyme study.
The ability of 12-HETE to protect cortical neurons from NMDAr toxicity is
shown in FIG. 8. To achieve best protection from NMDAr toxicity, 12-HETE
was administered both during and post ischemia.
Therefore, CBD serves as a selective inhibitor of at least two lipoxygenase
enzymes, 5-LO and 15-LO, but had no effect on 12-LO. Importantly, this is
the first demonstration (FIG. 8) that the 12-LO product 12-HETE can play a
significant role in protecting neurons from NMDAr mediated toxicity.
Although the mechanism of this protection is unknown at the present time,
12-HETE is known to be an important neuromodulator, due to its ability to
influence potassium channel activity.
EXAMPLE 9
Methods of Treatment
The present invention includes a treatment that inhibits oxidation
associated diseases in a subject such as an animal, for example a rat or
human. The method includes administering the antioxidant drugs of the
present invention, or a combination of the antioxidant drug and one or
more other pharmaceutical agents, to the subject in a pharmaceutically
compatible carrier and in an effective amount to inhibit the development
or progression of oxidation associated diseases. Although the treatment
can be used prophylactically in any patient in a demographic group at
significant risk for such diseases, subjects can also be selected using
more specific criteria, such as a definitive diagnosis of the condition.
The administration of any exogenous antioxidant cannabinoid would inhibit
the progression of the oxidation associated disease as compared to a
subject to whom the cannabinoid was not administered. The antioxidant
effect, however, increases with the dose of the cannabinoid.
The vehicle in which the drug is delivered can include pharmaceutically
acceptable compositions of the drugs of the present invention using
methods well known to those with skill in the art. Any of the common
carriers, such as sterile saline or glucose solution, can be utilized with
the drugs provided by the invention. Routes of administration include but
are not limited to oral, intracranial ventricular (icv), intrathecal (it),
intravenous (iv), parenteral, rectal, topical ophthalmic, subconjunctival,
nasal, aural, sub-lingual (under the tongue) and transdermal. The
antioxidant drugs of the invention may be administered intravenously in
any conventional medium for intravenous injection such as an aqueous
saline medium, or in blood plasma medium. Such medium may also contain
conventional pharmaceutical adjunct materials such as, for example,
pharmaceutically acceptable salts to adjust the osmotic pressure, lipid
carriers such as cyclodextrins, proteins such as serum albumin,
hydrophilic agents such as methyl cellulose, detergents, buffers,
preservatives and the like. Given the low solubility of many cannabinoids,
they may be suspended in sesame oil.
Given the excellent absorption of the compounds of the present invention
via an inhaled route, the compounds may also be administered as inhalants,
for example in pharmaceutical aerosols utilizing solutions, suspensions,
emulsions, powders and semisolid preparations of the type more fully
described in Remington: The Science and Practice of Pharmacy (19th
Edition, 1995) in chapter 95. A particular inhalant form is a metered dose
inhalant containing the active ingredient, in a suspension or a dispersing
agent (such as sorbitan trioleate, oleyl alcohol, oleic acid, or lecithin,
and a propellant such as 12/11 or 12/114).
Embodiments of the invention comprising pharmaceutical compositions can be
prepared with conventional pharmaceutically acceptable carriers, adjuvants
and counterions as would be known to those of skill in the art. The
compositions are preferably in the form of a unit dose in solid,
semi-solid and liquid dosage forms such as tablets, pills, powders, liquid
solutions or suspensions, injectable and infusible solutions, for example
a unit dose vial, or a metered dose inhaler. Effective oral human dosage
ranges for cannabidiol are contemplated to vary from about 1-40 mg/kg, for
example 5-20 mg/kg, and in particular a dose of about 20 mg/kg of body
weight.
If the antioxidant drugs are to be used in the prevention of cataracts,
they may be administered in the form of eye drops formulated in a
pharmaceutically inert, biologically acceptable carrier, such as isotonic
saline or an ointment. Conventional preservatives, such as benzalkonium
chloride, can also be added to the formulation. In ophthalmic ointments,
the active ingredient is admixed with a suitable base, such as white
petrolatum and mineral oil, along with antimicrobial preservatives.
Specific methods of compounding these dosage forms, as well as appropriate
pharmaceutical carriers, are known in the art. Remington: The Science and
Practice of Pharmacy, 19th Ed., Mack Publishing Co. (1995), particularly
Part 7.
The compounds of the present invention are ideally administered as soon as
a diagnosis is made of an ischemic event, or other oxidative insult. For
example, once a myocardial infarction has been confirmed by
electrocardiograph, or an elevation in enzymes characteristic of cardiac
injury (e.g. CKMB), a therapeutically effective amount of the cannabinoid
drug is administered. A dose can also be given following symptoms
characteristic of a stroke (motor or sensory abnormalities), or
radiographic confirmation of a cerebral infarct in a distribution
characteristic of a neurovascular thromboembolic event. The dose can be
given by frequent bolus administration, or as a continuous IV dose. In the
case of cannabidiol, for example, the drug could be given in a dose of 5
mg/kg active ingredient as a continuous intravenous infusion; or hourly
intramuscular injections of that dose.
EXAMPLE 10
The following table lists examples of some dibenzopyran cannabinoids that
may be useful as antioxidants in the method of the present invention.
##STR13##
##STR14##
Compound R19 R20 R21
R22 R23 R24 R25 R26
H 5 7-OH-Δ1 -THC CH2 OH H H H
H H H C5 H11
H 6 6α-OH-Δ1 -THC CH3 α-OH
H 7 6β-OH-Δ1 -THC CH3 β-OH
8 1"-OH-Δ1 -THC CH3 OH
H 9 2"-OH-Δ1 -THC CH3 OH
10 3"-OH-Δ1 -THC CH3
OH
11 4"-OH-Δ1 -THC CH3
OH
H 12 6α,7-diOH-Δ1 -THC CH2 OH α-OH
H 13 6v,7-diOH-Δ1 -THC CH2 OH β-OH
14 1",7-diOH-Δ1 -THC CH2 OH OH
H 15 2",7-diOH-Δ1 -THC CH2 OH
OH
H 16 3",7-diOH-Δ1 -THC CH2 OH
OH
H 17 4",7-diOH-Δ1 -THC CH2 OH
OH
18 1",6β-diOH-Δ1 -THC CH3 β-OH
OH
19 1",3"-diOH-Δ1 -THC CH3 OH
OH
20 1",6α,7-triOH-Δ1 -THC CH2 OH
α-OH OH
H 21 Δ1 -THC-6-one CH3 .dbd.O
22 Epoxyhexahydrocannabinol CH3
(EHHC)*
23 7-oxo-Δ1 -THC CHO
H 24 Δ1 -THC-7"-oic acid COOH
H 25 Δ1 -THC-3"-oic acid CH3
C2 H4 COOH
H 26 1"-OH-Δ1 -THC-7"-oic acid COOH
OH
H 27 2"-OH-Δ1 -THC-7"-oic acid COOH
OH
H 28 3"-OH-Δ1 -THC-7"-oic acid COOH
OH
H 29 4"-OH-Δ1 -THC-7"-oic acid COOH
OH
H 30 3",4",5"-trisnor-2"-OH-Δ1 - COOH
C2 H4 OH
THC-7-oic acid
H 31 7-OH-Δ1 -THC-2"-oic acid CH2 OH
CH2 COOH
H 32 6β-OH-Δ1 -THC-2"-oic acid CH3
β-OH CH2 COOH
H 33 7-OH-Δ1 -THC-3"-oic acid CH2 OH
C2 H4 COOH
H 34 6β-OH-Δ1 -THC-3"-oic acid CH3
β-OH C2 H4 COOH
H 35 6α-OH-Δ1 -THC-4"-oic acid CH3
α-OH C3 H6 COOH
H 36 2",3"-dehydro-6U-OH-Δ1 - CH3 α-OH
C3 H4 COOH
THC-4"-oic acid
H 37 Δ1 -THC-1",7-dioic acid COOH
COOH
H 38 Δ1 -THC-2",7-dioic acid COOH
CH2 COOH
H 39 Δ1 -THC-3",7-dioic acid COOH
C2 H4 COOH
H 40 Δ1 -THC-4",7-dioic acid COOH
C3 H6 COOH
H 41 1",2"-dehydro-Δ1 -THC-3",7- COOH
C2 H2 COOH
dioic acid
H 42 Δ1 -THC-glucuronic acid CH3
gluc.dagger.
H 43 Δ1 -THC-7-oic acid COO gluc.dagger.
glucuronide
*Epoxy group in C-1 and C-2 positions
.dagger. Glucuronide
Note: R-group substituents are H if not indicated otherwise.
Chemical structures of some of the dibenzopyran cannabinoids are shown
below.
##STR15##
##STR16##
##STR17##
EXAMPLE 11
Examples of Structural Analogs of Cannabidiol
The following table lists examples of some cannabinoids which are
structural analogs of cannabidiol and that may be useful as antioxidants
in the method of the present invention. A particularly useful example is
compound CBD, cannabidiol.
Compound R19 R20 R21 R22 R23
R24 R25 R26
##STR18## ##STR19##
44 CBD CH3 H H H H H H C5
H11
45 7-OH--CBD CH2 OH
46 6α- CH3 α-OH
47 6β- CH3 β-OH
48 1"- CH3 OH
49 2"- CH3 OH
50 3"- CH3 OH
51 4"- CH3 OH
52 5"- CH3 C4
H8 CH2 OH
53 6,7-diOH--CBD CH2 OH OH
54 3",7-diOH--CBD CH2 OH OH
55 4",7-diOH--CBD CH2 OH OH
56 CBD-7-oic acid COOH
57 CBD-3"-oic acid CH3 C2
H4 COOH
##STR20## ##STR21##
58 CBN CH3 H H H H H H C5
H11
59 7-OH--CBN CH2 OH
60 1"-OH--CBN CH3 OH
61 2"-OH--CBN CH3 OH
62 3"-OH--CBN CH3 OH
63 4"-OH--CBN CH3 OH
64 5"-OH--CBN CH3 C4
H8 CH2 OH
65 2"-7-diOH--CBN CH2 OH OH
66 CBN-7-oic acid COOH
67 CBN-1"-oic acid CH3 COOH
68 CBN-3"-oic acid CH3 C2
H4 COOH
Note: R-group substituents are H if not indicated otherwise.
The invention being thus described, variation in the materials and methods
for practicing the invention will be apparent to one of ordinary skill in
the art. Such variations are to be considered within the scope of the
invention, which is set forth in the claims below.
* * * * *
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