US patents on cannbinoids, WOW!

Clearly another fine example how how the Government & big pharm could give two shits about us. :321fu:


The list of patents on Cannabinoids.


http://patft.uspto.gov/netacgi/nph-Parser?Sect1=PTO1&Sect2=HITOFF&d=PALL&p=1&u=/netahtml/PTO/srchnum.htm&r=1&f=G&l=50&s1=6630507.PN.&OS=PN/6630507&RS=PN/6630507





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United States Patent 6,630,507Hampson , et al. October 7, 2003


Cannabinoids as antioxidants and neuroprotectants


AbstractCannabinoids 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, CH.sub.3, and COCH.sub.3. ##STR1##


Inventors: Hampson; Aidan J. (Irvine, CA), Axelrod; Julius (Rockville, MD), Grimaldi; Maurizio (Bethesda, MD) Assignee:The United States of America as represented by the Department of Health and Human Services (Washington, DC)


Appl. No.: 09/674,028Filed: February 2, 2001PCT Filed: April 21, 1999 PCT No.: PCT/US99/08769 PCT Pub. No.: WO99/53917 PCT Pub. Date: October 28, 1999


Current U.S. Class:514/454Current International Class: A61K 31/35 (20060101); A61K 031/35 ()Field of Search: 514/454 References Cited [Referenced By]U.S. Patent Documents2304669December 1942Adams4876276October 1989Mechoulam et al.5227537July 1993Stoss et al.5284867February 1994Kloog et al.5434295July 1995Mechoulam et al.5462946October 1995Mitchell et al.5512270April 1996Ghio et al.5521215May 1996Mechoulam et al.5538993July 1996Mechoulam et al.5635530June 1997Mechoulam et al.5696109December 1997Malfroy-Camine et al.6410588June 2002Feldmann et al.


Foreign Patent Documents427518May., 1991EP576357Dec., 1993EP656354Jun., 1995EP658546Jun., 1995EPWO9305031Mar., 1993WOWO9412667Jun., 1994WOWO9612485May., 1996WOWO9618600Jun., 1996WOWO9719063May., 1997WO99/53917Oct., 1999WO


Other References


Windholz et al., The Merck Index, th Edition (1983) p. 241, abstract No. 1723.* .


Mechoulam et al., "A Total Synthesis of d1-.DELTA..sup.1 -Tetrahydrocannabinol, the Active Constituent of Hashish.sup.1," 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 Patients.sup.1," 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 .DELTA..sup.1 -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 (-).DELTA..sup.9 -tetrahydrocannabiono are Neuroprotective Antioxidants," Medical Sciences, Proc. Natl. Acad. Sci. USA, 8268-8273 (1998)..


Primary Examiner: Weddington; Kevin E.


Attorney, Agent or Firm: Klarquist Sparkman, LLP


Parent Case Text


This application is a 371 of PCT/US99/08769 filed Apr. 21, 1999, which claims benefit of No. 60/082,589 filed Apr. 21, 1998, which claims benefit of No. 60/095,993 filed Aug. 10, 1998.


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; R.sub.1 is H, substituted or unsubstituted alkyl, or substituted or unsubstituted carboxyl; R.sub.2 is H, lower substituted or unsubstituted alkyl, or alkoxy; R.sub.3 is of H, lower substituted or unsubstituted alkyl, or substituted or unsubstituted carboxyl; R.sub.4 is H, hydroxyl, or lower substituted or unsubstituted alkyl; and R.sub.5 is H, hydroxyl, or lower substituted or unsubstituted alkyl.


8. The method of claim 7, wherein R.sub.1 is lower alkyl, COOH or COCH.sub.3 ; R.sub.2 is unsubstituted C.sub.1 -C.sub.5 alkyl, hydroxyl, methoxy or ethoxy; R.sub.3 is H, unsubstituted C.sub.1 -C.sub.3 alkyl, or COCH.sub.3 ; R.sub.4 is hydroxyl, pentyl, heptyl, or diemthylheptyl; and R.sub.5 is hydroxyl or methyl.


9. The method of claim 1, wherein the cannabinoid is: ##STR25##


where R.sub.1, R.sub.2 and R.sub.3 are independently H, CH.sub.3, or COCH.sub.3.


10. The method of claim 9, wherein the cannabinoid is: ##STR26##


where: a) R.sub.1 =R.sub.2 =R.sub.3 =H; b) R.sub.1 =R.sub.3 =H, R.sub.2 =CH.sub.3 ; c) R.sub.1 =R.sub.2 =CH.sub.3, R.sub.3 =H; d) R.sub.1 =R.sub.2 =COCH.sub.3, R.sub.3 =H; or e) R.sub.1 =H, R.sub.2 =R.sub.3 =COCH.sub.3.


11. The method of claim 2, wherein the cannabinoid is: ##STR27##


where R.sub.19 is H, lower alkyl, lower alcohol, or carboxyl; R.sub.20 is H or OH; and R.sub.21 -R.sub.25 are independently H or OH.


12. The method of claim 11, wherein R.sub.19 is H, CH.sub.3, CH.sub.2 OH, or COOH, and R.sub.20 -R.sub.24 are independently H or OH.


13. The method of claim 2, wherein the cannabinoid is: ##STR28##


where R.sub.19 and R.sub.20 are H, and R.sub.26 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


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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.degree. 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 .mu.g/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 .mu.M fluoro-deoxyuridine and 10 .mu.M 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 cm.sup.2 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.degree. 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 .mu.M 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 EC.sub.50 of 2-4 .mu.M (specifically about 3.5 .mu.M).


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 .mu.M glutamate and 50 .mu.M 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 .mu.M) was the AMPA agonist and 4-methyl glutamate (10 .mu.M) 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 EC.sub.50 of 2-4 .mu.M (specifically about 3.3 .mu.M). 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 .mu.M) was incubated with 300 .mu.M t-butyl hydroperoxide and 0.5 .mu.M 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 EC.sub.50 of about 6 .mu.M. 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 .mu.M glutamate and equimolar (5 .mu.M) cannabidiol, .alpha.-tocopherol, BHT or ascorbate (FIG. 5B). Although all of the antioxidants attenuated glutamate toxicity, cannabidiol was significantly more protective than either .alpha.-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 K.sub.i of greater than 250 nM, for example K.sub.i.gtoreq.500-1000 nM. A compound with a K.sub.i.gtoreq.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 .alpha.-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 .mu.M (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 .mu.M 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 .mu.M arachidonic acid and varying concentrations of cannabidiol and/or 40 .mu.M nordihydroguaiaretic acid (a lipxygenase inhibitor). Platelets and leukocytes were also pre-incubated with 80 .mu.M manoalide (Biomol) to prevent phospholipase A2 activation. Product formation was initiated by addition of 5 .mu.M A23187 and incubation for 10 minutes at 37.degree. 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% H.sub.2 O and separated by reverse phase HPLC using a gradient running from 63% acetonitrile: 37% H.sub.2 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 .mu.g/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 EC.sub.50 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 EC.sub.50 of 1.92 .mu.M. 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 (19.sup.th 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 R.sub.19 R.sub.20 R.sub.21 R.sub.22 R.sub.23 R.sub.24 R.sub.25 R.sub.26 H 5 7-OH-.DELTA..sup.1 -THC CH.sub.2 OH H H H H H H C.sub.5 H.sub.11 H 6 6.alpha.-OH-.DELTA..sup.1 -THC CH.sub.3 .alpha.-OH H 7 6.beta.-OH-.DELTA..sup.1 -THC CH.sub.3 .beta.-OH 8 1"-OH-.DELTA..sup.1 -THC CH.sub.3 OH H 9 2"-OH-.DELTA..sup.1 -THC CH.sub.3 OH 10 3"-OH-.DELTA..sup.1 -THC CH.sub.3 OH 11 4"-OH-.DELTA..sup.1 -THC CH.sub.3 OH H 12 6.alpha.,7-diOH-.DELTA..sup.1 -THC CH.sub.2 OH .alpha.-OH H 13 6v,7-diOH-.DELTA..sup.1 -THC CH.sub.2 OH .beta.-OH 14 1",7-diOH-.DELTA..sup.1 -THC CH.sub.2 OH OH H 15 2",7-diOH-.DELTA..sup.1 -THC CH.sub.2 OH OH H 16 3",7-diOH-.DELTA..sup.1 -THC CH.sub.2 OH OH H 17 4",7-diOH-.DELTA..sup.1 -THC CH.sub.2 OH OH 18 1",6.beta.-diOH-.DELTA..sup.1 -THC CH.sub.3 .beta.-OH OH 19 1",3"-diOH-.DELTA..sup.1 -THC CH.sub.3 OH OH 20 1",6.alpha.,7-triOH-.DELTA..sup.1 -THC CH.sub.2 OH .alpha.-OH OH H 21 .DELTA..sup.1 -THC-6-one CH.sub.3 .dbd.O 22 Epoxyhexahydrocannabinol CH.sub.3 (EHHC)* 23 7-oxo-.DELTA..sup.1 -THC CHO H 24 .DELTA..sup.1 -THC-7"-oic acid COOH H 25 .DELTA..sup.1 -THC-3"-oic acid CH.sub.3 C.sub.2 H.sub.4 COOH H 26 1"-OH-.DELTA..sup.1 -THC-7"-oic acid COOH OH H 27 2"-OH-.DELTA..sup.1 -THC-7"-oic acid COOH OH H 28 3"-OH-.DELTA..sup.1 -THC-7"-oic acid COOH OH H 29 4"-OH-.DELTA..sup.1 -THC-7"-oic acid COOH OH H 30 3",4",5"-trisnor-2"-OH-.DELTA..sup.1 - COOH C.sub.2 H.sub.4 OH THC-7-oic acid H 31 7-OH-.DELTA..sup.1 -THC-2"-oic acid CH.sub.2 OH CH.sub.2 COOH H 32 6.beta.-OH-.DELTA..sup.1 -THC-2"-oic acid CH.sub.3 .beta.-OH CH.sub.2 COOH H 33 7-OH-.DELTA..sup.1 -THC-3"-oic acid CH.sub.2 OH C.sub.2 H.sub.4 COOH H 34 6.beta.-OH-.DELTA..sup.1 -THC-3"-oic acid CH.sub.3 .beta.-OH C.sub.2 H.sub.4 COOH H 35 6.alpha.-OH-.DELTA..sup.1 -THC-4"-oic acid CH.sub.3 .alpha.-OH C.sub.3 H.sub.6 COOH H 36 2",3"-dehydro-6U-OH-.DELTA..sup.1 - CH.sub.3 .alpha.-OH C.sub.3 H.sub.4 COOH THC-4"-oic acid H 37 .DELTA..sup.1 -THC-1",7-dioic acid COOH COOH H 38 .DELTA..sup.1 -THC-2",7-dioic acid COOH CH.sub.2 COOH H 39 .DELTA..sup.1 -THC-3",7-dioic acid COOH C.sub.2 H.sub.4 COOH H 40 .DELTA..sup.1 -THC-4",7-dioic acid COOH C.sub.3 H.sub.6 COOH H 41 1",2"-dehydro-.DELTA..sup.1 -THC-3",7- COOH C.sub.2 H.sub.2 COOH dioic acid H 42 .DELTA..sup.1 -THC-glucuronic acid CH.sub.3 gluc.sup..dagger. H 43 .DELTA..sup.1 -THC-7-oic acid COO gluc.sup..dagger. glucuronide *Epoxy group in C-1 and C-2 positions .sup..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 R.sub.19 R.sub.20 R.sub.21 R.sub.22 R.sub.23 R.sub.24 R.sub.25 R.sub.26 ##STR18## ##STR19## 44 CBD CH.sub.3 H H H H H H C.sub.5 H.sub.11 45 7-OH--CBD CH.sub.2 OH 46 6.alpha.- CH.sub.3 .alpha.-OH 47 6.beta.- CH.sub.3 .beta.-OH 48 1"- CH.sub.3 OH 49 2"- CH.sub.3 OH 50 3"- CH.sub.3 OH 51 4"- CH.sub.3 OH 52 5"- CH.sub.3 C.sub.4 H.sub.8 CH.sub.2 OH 53 6,7-diOH--CBD CH.sub.2 OH OH 54 3",7-diOH--CBD CH.sub.2 OH OH 55 4",7-diOH--CBD CH.sub.2 OH OH 56 CBD-7-oic acid COOH 57 CBD-3"-oic acid CH.sub.3 C.sub.2 H.sub.4 COOH ##STR20## ##STR21## 58 CBN CH.sub.3 H H H H H H C.sub.5 H.sub.11 59 7-OH--CBN CH.sub.2 OH 60 1"-OH--CBN CH.sub.3 OH 61 2"-OH--CBN CH.sub.3 OH 62 3"-OH--CBN CH.sub.3 OH 63 4"-OH--CBN CH.sub.3 OH 64 5"-OH--CBN CH.sub.3 C.sub.4 H.sub.8 CH.sub.2 OH 65 2"-7-diOH--CBN CH.sub.2 OH OH 66 CBN-7-oic acid COOH 67 CBN-1"-oic acid CH.sub.3 COOH 68 CBN-3"-oic acid CH.sub.3 C.sub.2 H.sub.4 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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Yeah I couldn't find it either, sorry for the double post.


I agree that this does give the government a graceful way to back of the classification of marijuana but with that comes more regulations. I don't really think it will do much for flat out legalization though.

 

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