NON PSYCHOACTIVE CANNABINOIDS AND USES THEREOF

FIELD OF THE INVENTION

This invention relates to non-psychoactive cannabinoid derivatives and uses thereof for the manufacture of medicaments for the treatment of inflammation and/or inflammatory relates diseases, disorders and /or symptoms. BACKGROUND OF THE INVENTION

The non-psychoactive cannabis constituent cannabidiol (CBD) is a potent antiinflammatory agent [1-3]. It was shown to have anti-arthritic properties in murine collagen- induced arthritis [4]. CBD is essentially non toxic and has been administered to human patients without any side effects [5]. Another cannabinoid anti-inflammatory compound is ajulemic acid (a derivative of the psychoactive constituent of cannabis, THC) [6, 7]. However ajulemic acid binds to the CB1 receptor and exhibits psychoactivity [8, 9].

The following publications are relevant for describing the state of the art in the field of the invention:

1. R. Mechoulam, L.A. Parker and R. Gallily. Cannabidiol: An overview of some pharmacological aspects. J. Clin. Pharmacol. 42, 11S-19S (2002).

2. R. Mechoulam, P.F.Sumariwalla, M.Feldmann and R.Gallily. Cannabinoids in models of chronic inflammatory conditions. Phytochemistry Reviews 4, 11-18 (2005).

3. R. Mechoulam, M. Peters, E. Murillo-Rodriguez, L.O. Hanus. Cannabidiol - recent advances. Chemistry & Biodiversity 4, 1678-1692 (2007).

4. A.M. Malfait, R. Gallily, P.F. Sumariwalla, A.S. Malik, E. Andreakos, R.

Mechoulam, M. Feldmann. The non-psychoactive cannabis-constituent cannabidiol is an oral anti-arthritic therapeutic in murine collagen-induced arthritis. Proc. Natl. Acad. Sci (USA) 97, 9561-9566 (2000). 5. J.M. Cunha, E.A. Carlini, A.E. Pereira, O.L. Ramos, G. Pimentel, R. Gagliardi, E.L. Sanvito, N. Lander and R. Mechoulam. Chronic administration of CBD to healthy volunteers and epileptic patients. Pharmacologia, 21, 175-185 (1980).

6. S.H. Burstein, C.A. Audette, A. Breuer, W.A. Devane, S. Colodner, S.A. Doyle and R. Mechoulam. Synthetic non-psychotropic cannabinoids with potent antiinflammatory, analgesic and leukocyte antiadhesion activities. J. Med. Chem. 35, 3135- 3141 (1992).

7. R.B. Zurier, Y.P. Sun, K.L. George, J.A. Stebulis, R.G. Rossetti, A. Skulas, E.

Judge, C. N. Serhan. Ajulemic acid, a synthetic cannabinoid, increases formation of the endogenous proresolving and anti-inflammatory eicosanoid, lipoxin A4. FASEB J. 23, 1503-9 (2009).

8. Ajulemic acid. Drugs. 8, 1002-11 (2005).

9. P.F. Sumariwalla, R. Gallily, S. Tchilibon, E. Fride, R. Mechoulam and M.

Feldmann. A novel synthetic, nonpsychoactive cannabinoid acid (HU-320) with anti-inflammatory properties in murine collagen-induced arthritis. Arthritis & Rheumatism 50, 985-998 (2004).

10. S.E. O'Sullivan, D.A. Kendall DA. Cannabinoid activation of peroxisome proliferator-activated receptors: Potential for modulation of inflammatory disease. Immunobiology. 215, 611-616 (2010).

U . S. Ben-Shabat, L.O. Hanus, G. Katzavian, R. Gallily. New Cannabidiol Derivative: Synthesis, Binding to Cannabinoid Receptor, and Evaluation of Their Antiinflammatory Activity. J. Med. Chem. 49, 1113 - 1117 (2006).

SUMMARY OF THE INVENTION

In the first aspect, the invention provides a compound having the general formula (I):

wherein

is a single or double bond; provided that between 0 to 3 of is a double bond;

Ri is selected from -ReOR^ -C(=0)R ₈ , -OC(=0)R ₉ ;

R ₂ is a straight or branched Q-Cs alkyl;

R ₃ and R ₄ are each independently selected from H, straight or branched C1-C5 alkyl, -OR ₁₀ , -C(=0)R _n , -OC(=0)R ₁₂ ; provided that at least one of R ₃ and R4 is different than H;

R ₅ is selected from a straight or branched C ₆ -C ₁₂ alkyl, a straight or branched C ₅ - C ₉ alkoxy, a straight or branched C!-C ₇ ether, each being optionally substituted by at least one substituent selected from -OH, -NH ₃ , straight or branched C -Cs amine, halogen, phenyl, aryl, heteroaryl, cycloalkyl and heterocycloalkyl;

R^ is a straight or branched C _! -C ₅ alkylene;

R ₇ is selected from H, -C(=0)OH, -C(=0)NH ₃ , -C(=0)H, straight or branched C C ₅ alkyl, straight or branched Q-C5 acyl, straight or branched Q-Cs amide;

R ₈ , and R ₉ are independently selected from H, OH, straight or branched Q-Cs alkyl, straight or branched Cj-Csalkoxy, -NH ₃ , straight or branched C C ₅ amine;

R ₁₀ is selected from H, a straight or branched C C alkyl; and

Rn and R ₁₂ are independently selected from H, OH, straight or branched C1-C5 alkyl, straight or branched C _\ -Cs alkoxy, -NH ₃ , straight or branched C Cs amine.

It should be noted that the chemical bonding of atoms of the compounds of the present invention involve only the allowed bonding of each atom as reflected by the valence properties of each atom. Thus, in the case where two carbon atoms in the cyclohexene ring are connected via a double bond (i.e. one of is a double bond) the adjacent carbon atoms will not be able to be connected via a double bond as well. On the other hand in cases where two carbon atoms in the cyclohexene ring are connected via a single sigma bond (i.e. one of is a single bond), in order to fulfill the four valencies of each carbon atom, a hydrogen atom is taught to be added where appropriate.

In a further aspect, the invention provides a compound having the general formula (II):

wherein

is a single or double bond; provided that at least one of is a double bond;

Ri is selected from -R60R ₇ , -C(=0)R ₈ , -OC(=0)R ₉ ;

R ₂ is a straight or branched Q-Cs alkyl;

R ₃ and R ₄ are each independently selected from H, straight or branched Q-C5 alkyl, -OR ₁₀ , -C(=0)R _n , -OC(=0)R ₁₂ ; provided that at least one of R ₃ and R ₄ is different than H;

R ₅ is selected from a straight or branched C ₆ -C ₁₂ alkyl, a straight or branched C ₅ - C ₉ alkoxy, a straight or branched Q-O7 ether, each being optionally substituted by at least one substituent selected from -OH, -NH ₃ , straight or branched Cj-Cs amine, halogen, phenyl, aryl, heteroaryl, cycloalkyl and heterocycloalkyl;

is a straight or branched Ci-C ₅ alkylene;

R ₇ is selected from H, -C(=0)OH, -C(=0)NH ₃ , -C(=0)H, straight or branched C!-C ₅ alkyl, straight or branched Ci-C _$ acyl, straight or branched C _\ -Cs amide;

R ₈ , and R ₉ are independently selected from H, OH, straight or branched C _\ -Cs alkyl, straight or branched Q-Csalkoxy, -NH ₃ , straight or branched Q-Cs amine; R ₁₀ is selected from H, a straight or branched Cj-Cs alkyl; and Rn and R ₁₂ are independently selected from H, OH, straight or branched C C ₅ alkyl, straight or branched C _\ -Cs alkoxy, -NH ₃ , straight or branched C _\ -Cs amine.

In yet a further aspect the invention provides a compound having the general formula (III):

wherein

is a single or double bond; provided that at least one of is a double bond;

Ri is selected from -R<;OR ₇ , -C(=0)R ₈ , -OC(=0)R ₉ ;

R ₂ is a straight or branched C _\ -Cs alkyl;

R ₃ and R4 are each independently selected from H, straight or branched Q-C5 alkyl, -OR ₁₀ , -C(=0)Rn, -OC(=0)R ₁₂ ; provided that at least one of R ₃ and R4 is different than H;

R ₅ is selected from a straight or branched C ₆ -C ₁₂ alkyl, a straight or branched C ₅ - C ₉ alkoxy, a straight or branched C!-C ₇ ether, each being optionally substituted by at least one substituent selected from -OH, -NH ₃ , straight or branched C -Cs amine, halogen, phenyl, aryl, heteroaryl, cycloalkyl and heterocycloalkyl;

Re is a straight or branched C _\ -Cs alkylene;

R ₇ is selected from H, -C(=0)OH, -C(=0)NH ₃ , -C(=0)H, straight or branched C C ₅ alkyl, straight or branched C _! -C acyl, straight or branched Cj-Cs amide;

R , and R ₉ are independently selected from H, OH, straight or branched C _\ -Cs alkyl, straight or branched Q-Csalkoxy, -NH3, straight or branched Cj-C ₅ amine;

R ₁₀ is selected from H, a straight or branched Q-C5 alkyl; and Rn and R ₁₂ are independently selected from H, OH, straight or branched C C ₅ alkyl, straight or branched Q-Cs alkoxy, -NH ₃ , straight or branched C C ₅ amine. In some embodiments, one of of a compound of any one of formulae (I),

(II) or (III) is a double bond. In some specific embodiments, said one double bond a compound of any one of formulae (I) or (II) is between carbon 1 and 2. In other specific embodiments, said one double bond a compound of any one of formulae (I) or (II) is between carbon 3 and 4. In further specific embodiments, said one double bond a compound of any one of formulae (I) or (II) is between carbon 5 and 6. In some specific embodiments, said one double bond a compound of any one of formulae (I) or (III) is between carbon 2 and 3. In other specific embodiments, said one double bond a compound of any one of formulae (I) or (III) is between carbon 4 and 5. In further specific embodiments, said one double bond a compound of any one of formulae (I) or (III) is between carbon 6 and 1.

In further embodiments, ^{1 2} of a compound of any one of formulae (I) or

(II) is a double bond. In other embodiments, ^{1 6} of a compound of any one of formulae (I) or (III) is a double bond. In some further embodiments, all other bonds, other than the ones indicated in embodiments herein above, are single bonds.

In further embodiments, when either ^{1 2} (for compounds having the formulae (I) or (II)) or ¹ 6 (for compounds having the formulae (I) or (III)), is a double bond, substituents on positions 3 and 4 are in the trans configuration. In other embodiments, when either ^{1 2} (for compounds having the formulae (I) or (II)) or

^{1 6} (for compounds having the formulae (I) or (III)), is a double bond, substituents on positions 3 and 4 are in the cis configuration.

In some embodiments, R _\ is -ReOR ₇ ; R6 is a straight or branched C C ₅ alkylene; and R ₇ is H. In other embodiments, R _\ is -C(=0)R ₈ and Rs is selected from H, OH, straight or branched Ci-C ₅ alkyl, straight or branched Q-Csalkoxy, -NH ₃ , straight or branched Cj-Cs amine. In further embodiments, R ₃ and R4 are each independently selected from H, -OR ₁₀ , and -OC(=0)R ₁₂ ; Ri ₀ is selected from H, a straight or branched C1-C5 alkyl; and R ₁₂ is selected from H, OH, straight or branched -Cs alkyl, -NH ₃ , straight or branched Ci-C ₅ amine. In further embodiments, R ₅ is a straight or branched C ₆ -C ₁₂ alkyl.

In some embodiments the invention encompasses a compound having the general formula (IV):

wherein , R ₃ , R ₄ and R ₅ are as defined herein above.

In other embodiments the invention encompasses a compound having the general formula (V):

wherein , R3, R ₄ and R ₅ are as defined herein above.

In some embodiments, one of of a compound of any one of formulae (IV) or (V) is a double bond. In some specific embodiments, said one double bond a compound of any one of formula (IV) is between carbon 1 and 2. In other specific embodiments, said one double bond a compound of any one of formula (IV) is between carbon 3 and 4. In further specific embodiments, said one double bond a compound of any one of formula (IV) is between carbon 5 and 6. In some specific embodiments, said one double bond a compound of any one of formula (V) is between carbon 2 and 3. In other specific embodiments, said one double bond a compound of any one of formula (V) is between carbon 4 and 5. In further specific embodiments, said one double bond a compound of any one of formula (V) is between carbon 6 and 1.

In further embodiments, ^{1 2} of a compound of formula (IV) is a double bond. In other embodiments, ¹ 6 ₀ f a compound of formula (V) is a double bond. In some further embodiments, all other bonds, other than the ones indicated in embodiments herein above, are single bonds.

In further embodiments, when either ^l 2 (for compounds having the formula (IV) or ^l 6 (for compounds having the formula (V)), is a double bond, substituents on positions 3 and 4 are in the trans configuration. In other embodiments, when either ^{1 2} (for compounds having the formula (IV)) or ^l 6 (for compounds having the formulae (V)), is a double bond, substituents on positions 3 and 4 are in the cis configuration. In some embodiments of a compound of formulae (IV) or (V), R ₃ and R4 are each independently selected from H, -OR ₁₀ , and -OC(=0)R ₁₂ ; R ₁₀ is selected from H, a straight or branched C C ₅ alkyl; and R ₁₂ is selected from H, OH, straight or branched C _\ -C$ alkyl, -NH ₃ , straight or branched C _\ -C _* , amine. In other embodiments of a compound of formulae (IV) or (V), R ₅ is selected from a straight or branched C ₆ -C ₁₂ alkyl.

In some embodiments a compound of the invention is (-)-8,9-dihydro-CBD-7- oic acid diacetate (HU-444), having the formula:

In other embodiments a compound of the invention is (-)-8,9 dihydro-CBD-7-oic acid (HU-445), having the formula:

As used herein, the term "alkyl" refers to a straight or branched chain hydrocarbon having from one to five carbon atoms, or from one to seven carbon atoms, or from five to nine carbon atoms, or from six to twelve carbon atoms. Examples of "alkyl" as used herein include, but are not limited to, methyl, ethyl, propyl, n-butyl, n- pentyl, isobutyl, and isopropyl, tert-butyl, and the like.

As used herein the term "alkylene" refers to a saturated, divalent, branched or straight hydrocarbon group having from one to five carbon atoms. Non-limiting examples of C _1-5 -alkylene groups include, methylene, ethylene, 1,2-propylene, 1,3- propylene, butylene, isobutylidene, pentylene, hexylene and the like.

As used herein the term "ester" is meant to encompass an -COOR group wherein R is an alkyl as defined herein above.

A used herein the term "ether" refers to an -R'OR group, wherein R' is a C1-C7 straight or branched alkylene group and R is a C1-C7 straight or branched alkyl group.

As used herein, the term "alkoxy" refers to an RO- group, where R is alkyl as defined above. As used herein the term "C1-C7 amide" refers to a monoalkyl amide (-CONHR) or dialkyl amide (-CONRR), wherein R and R' are independently a C C ₇ straight or branched alkyl.

As used herein the term "C1-C5 amine" refers to an -NHR or -NRR' group wherein R and R' are independently a C _\ -Cs straight or branched alkyl.

As used herein the term "C7-C5 alkoxy" refers to a -OR group wherein R is a Ci- C ₅ alkyl. As used herein the term "C7-C5 acyl" refers to a -C(=0)R group wherein R is a straight or branched Q-C5 alkyl.

The term "optionally substituted" as used herein means that the groups in question are either unsubstituted or substituted with one or more of the substituents such as for example those specified above, -OH, -NH ₃ , straight or branched Q-C ₅ amine, phenyl, substituted phenyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, halogen (-F, - CI, -Br, -I). When the groups are substituted with more than one substituent the substituents may be the same or different and said substitution may occur at any position on the substituted group (i.e. at a terminal or any mid-chain position or both).

In further embodiments a compound of the invention is intended or indicated for use in the treatment of a condition, disease, disorder or symptom associated with inflammation. The term "inflammation" as used herein is meant to encompass any response of vascular tissues to stimuli, such as pathogens, damaged cells, or irritants. Inflammation can be either acute or chronic. Acute inflammation is the initial response of the body to harmful stimuli and is achieved by the increased movement of plasma and leukocytes (especially granulocytes) from the blood into the injured tissues. A cascade of biochemical events propagates and matures the inflammatory response, involving the local vascular system, the immune system, and various cells within the injured tissue. Prolonged inflammation, known as chronic inflammation, leads to a progressive shift in the type of cells present at the site of inflammation and is characterized by simultaneous destruction and healing of the tissue from the inflammatory process.

Causes of inflammation or any associated disease, disorder or symptom, include but are not limited to: burns, chemical irritants, frostbite, toxins, infection by pathogens, physical, blunt or penetrating, immune reactions due to hypersensitivity, ionizing radiation, foreign bodies, including splinters, dirt and debris etc.

In some embodiments said condition, disease, disorder or symptom associated with inflammation is selected from multiple sclerosis, rheumatoid arthritis, liver inflammation, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, irritable bowel syndrome (IBS), systemic lupus erythematosus (SLE), cutaneous lupus erythematosus, psoriasis, diabetes (Type I and Type II diabetes (IDDM) including the protection of beta cells from destruction), Sjogren's disease, autoimmune thyroid disease, acquired immunodeficiency syndrome (AIDS), sarcoidosis, autoimmune uveitis, autoimmune hepatitis, hypersensitivity lung diseases, hypersensitivity pneumonitis, delayed-type hypersensitivity, interstitial lung disease (ILD) (e.g., idiopathic pulmonary fibrosis, or ILD associated with rheumatoid arthritis or other inflammatory diseases), scleroderma; dermatitis (including atopic dermatitis and eczematous dermatitis), iritis, conjunctivitis, keratoconjunctivitis, idiopathic bilateral progressive sensorineural hearing loss, aplastic anemia, pure red cell anemia, idiopathic thrombocytopenia, polychondritis, Graves ophthalmopathy, amyotrophic lateral sclerosis (ALS), primary biliary cirrhosis, ileitis, chronic inflammatory intestinal disease, celiac disease, Alzheimers's disease or any combinations thereof. In some other embodiments, said condition, disease, disorder or symptom associated with inflammation is selected from rheumatoid arthritis, multiple sclerosis, liver inflammation, inflammatory bowel disease, diabetes and any combinations thereof.

In further embodiments, a compound of the invention is intended or indicated for use in the treatment of a disease or disorder selected from cardiac disfunction in diabetic cardiopathy nephrotoxicity (in some embodiments due to the use of anti-cancer agents), hepatic ischemia/reperfusion injury, cardiac ischemia/reperfusion injury, brain ischemia/reperfusion injury, cancer (in some embodiments breast cancer) and any combinations thereof.

In further embodiments, a compound of the invention is intended or indicated for use in suppressing the generation of at least one of reactive oxygen species, nitric oxygen species, TNFa and any combinations thereof.

In yet other embodiments, a compound of the invention is intended or indicated for use in at least one of enhancing adenosine uptake, 5-HT(lA) activation, PPAR activation and any combinations thereof.

When referring to the suppression of the generation of at least one of reactive oxygen species, nitric oxygen species, TNFa, it should be understood to include any qualitative or quantitative change in the generation of at least one of the species generally found in the inflammation processes and cascades in a patient suffering there from or any combination thereof. Such effects are demonstrated in vitro and in vivo in the below described experimental section of the invention.

Furthermore, when referring to the enhancement of adenosine uptake, 5-HT(l A) activation, PPAR activation it should be understood to encompass it should be understood to include any qualitative or quantitative change in at least one of the anti- inflammation related processes and cascades in a patient suffering therefrom, administered with a compound or composition of the invention. Such effects are demonstrated in vitro and in vivo in the below described experimental section of the invention.

In another one of its aspects the invention provides a composition comprising at least one compound according to the invention. In some embodiments, said composition is a pharmaceutical composition.

In some embodiments, a composition of the invention is intended or indicated for use in the treatment of a condition, disease, disorder or symptom associated with inflammation.

In further embodiments a composition of the invention is intended or indicated for use in suppressing the generation of at least one of reactive oxygen species, nitric oxygen species, TNFa and any combinations thereof.

In yet other embodiments, a composition of the invention is intended or indicated for use in at least one of enhancing adenosine uptake, 5-HT(lA) activation, PPAR activation and any combinations thereof. When referring to pharmaceutical compositions comprising a compound of the subject invention it should be understood to encompass admixtures of a compound of the invention, with any pharmaceutically acceptable auxiliaries known to a person skilled in the art, and optionally other therapeutic agents. The auxiliaries must be "acceptable" in the sense of being compatible with the other ingredients of the composition and not deleterious to the recipients thereof.

Pharmaceutical compositions of the invention include those suitable for oral, rectal, nasal, topical (including transdermal, buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration or administration via an implant. The compositions may be prepared by any method well known in the art of pharmacy. Such methods include the step of bringing in association compounds used in the invention or combinations thereof with any auxiliary agent. Auxiliary agent(s), also named accessory ingredient(s), include those conventional in the art, such as carriers, fillers, binders, diluents, disintegrants, lubricants, colorants, flavouring agents, anti-oxidants, and wetting agents.

Pharmaceutical compositions of the invention suitable for oral administration may be presented as discrete dosage units such as pills, tablets, dragees or capsules, or as a powder or granules, or as a solution or suspension. The active ingredient may also be presented as a bolus or paste. The compositions can further be processed into a suppository or enema for rectal administration.

The invention further includes a pharmaceutical composition, as hereinbefore described, in combination with packaging material, including instructions for the use of the composition for a use as hereinbefore described.

For parenteral administration, suitable compositions include aqueous and nonaqueous sterile injection. The compositions may be presented in unit-dose or multi-dose containers, for example sealed vials and ampoules, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of sterile liquid carrier, for example water, prior to use.

For transdermal administration, e.g. gels, patches or sprays can be contemplated. Compositions or formulations suitable for pulmonary administration e.g. by nasal inhalation include fine dusts or mists which may be generated by means of metered dose pressurized aerosols, nebulisers or insufflators.

The exact dose and regimen of administration of a composition of the invention will necessarily be dependent upon the therapeutic or nutritional effect to be achieved and may vary with the particular formula, the route of administration, and the age and condition of the individual subject to whom the composition is to be administered.

The term "treatment" as used herein refers to the administering of a therapeutic amount of a composition or compound of the invention which is effective to ameliorate undesired symptoms associated with a disease, to prevent the manifestation of such symptoms before they occur, to slow down the progression of the disease, slow down the deterioration of symptoms, to enhance the onset of remission period, slow down the irreversible damage caused in the progressive chronic stage of the disease, to delay the onset of said progressive stage, to lessen the severity or cure the disease, to improve survival rate or more rapid recovery, or to prevent the disease from occurring or a combination of two or more of the above.

As used herein, the term "effective amount" means that amount of a composition or compound of the invention that will elicit the biological or medical response of a tissue, system, animal or human that is being sought, for instance, by a researcher or clinician. The effective amount for purposes disclosed herein is determined by such considerations as may be known in the art. The amount must be effective to achieve the desired therapeutic effect as described above, depending, inter alia, on the type and severity of the disease to be treated and the treatment regime. The effective amount is typically determined in appropriately designed clinical trials (dose range studies) and the person versed in the art will know how to properly conduct such trials in order to determine the effective amount. As generally known, an effective amount depends on a variety of factors including the affinity of a ligand to a receptor, its distribution profile within the body, a variety of pharmacological parameters such as half life in the body, on undesired side effects, if any, on factors such as age and gender, etc.

Furthermore, the term "therapeutically effective amount" means any amount of a composition or compound of the invention which, as compared to a corresponding subject who has not received such amount of a composition or compound of the invention, results in improved treatment, healing, prevention, or amelioration of a disease, disorder, or side effect, or a decrease in the rate of advancement of a disease or disorder. The term also includes within its scope amounts effective to enhance normal physiological function.

In another one of its aspects the invention provides a use of a compound of the invention, for the manufacture of a medicament. In a further aspect, the invention provides a use of a compound of the invention, for the manufacture of a medicament for the treatment of a condition, disease, disorder or symptom associated with inflammation. In yet another aspect, the invention provides a use of a compound of the invention, for the manufacture of a medicament for suppressing the generation of at least one of reactive oxygen species, nitric oxygen species, TNFa and any combinations thereof. In another aspect, the invention provides a use of a compound of the invention, for the manufacture of a medicament for at least one of enhancement of adenosine uptake, 5-HT(l A) activation, PPAR activation and any combinations thereof.

The invention further encompasses a method of treating a condition, disease, disorder or symptom associated with inflammation in a subject in need thereof, said method comprising administering to said subject an effective amount of at least one compound of the invention.

The invention further provides a method of suppressing the generation of at least one of reactive oxygen species, nitric oxygen species, TNFa and any combinations thereof, said method comprising administering to a subject in need thereof, an effective amount of at least one compound of the invention.

It must be noted that, as used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the content clearly dictates otherwise.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", should be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any integer or step or group of integers and steps. BRIEF DESCRIPTION OF THE DRAWINGS

In order to understand the invention and to see how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

Fig. 1 is a scheme of the synthesis of (-) 8,9 dihydro-CBD- 7-oic acid, diacetate

(HU-444) and (-)8,9 dihydro-CBD-7-oic acid (HU-445), in accordance with Example 1.

Figs. 2A-2B show the respective ROI inhibition of HU-444 (2A) and HU-445 (2B) in various concentrations (10, 20, and 40 g/ml).

Figs. 3A-3C show the respective ROI inhibition of HU-444 (3A and 3B) and HU-445 (3C) in various concentrations (1, 2.5, 5, 10, 20, 40 and 60 μg/ml).

Figs. 4A-4B show respectively the inhibition of THP-1 cell migration toward MCP-1 with HU-444 at concentration of 20 and 10 μg/ml (4 A) and the effect of HU- 445 (at 40, 20, 10, 5, 2.5, 1 μg/ml respectively) on TNF levels (4B).

Figs. 5A-5B show the respective inhibiting effect of HU-444 (5 A) and HU-445 (5B) on TNF production in the mice.

Fig. 6 shows the incidences of arthritis in DBA/1 mice immunized with chicken type II collagen.

Fig. 7 shows the effect of treatments with HU-444 (in 2.5, 5, 10 mg/kg dosages) on weight of DBA/1 mice with established chicken type II collagen-induced arthritis. The effect was compared with vehicle and CBD.

Fig. 8 shows the effect of treatments with HU-444 (in 2.5, 5, 10 mg/kg dosages) on clinical arthritis score of DBA/1 mice with established chicken type II collagen- induced arthritis, over ten days. The effect was compared with vehicle and CBD.

Fig. 9 shows the effect of treatments with HU-444 (in 2.5, 5, 10 mg/kg dosages) on clinical arthritis score of DBA/1 mice with established chicken type II collagen- induced arthritis, over ten days. The effect was compared with vehicle and CBD. The effect is shows as the change in clinical score from day 1 (normalised data) over ten days.

Fig. 10 shows the effect of treatments with HU-444 (in 2.5, 5, 10 mg/kg dosages) on clinical arthritis score of DBA/1 mice with established chicken type II collagen-induced arthritis, over ten days. The effect was compared with vehicle and CBD. Fig. 11 shows the effect of treatments with HU-444 (in 2.5, 5, 10 mg/kg dosages) on clinical arthritis score of DBA/1 mice with established chicken type II collagen-induced arthritis, over ten days. The effect was compared with vehicle and CBD. The effect is shows as the change in clinical score from day 1 (normalised data) over ten days.

Fig. 12: shows the effect of treatments with HU-444 (in 2.5, 5, 10 mg/kg dosages) on paw thickness (mm) of DBA/1 mice with established chicken type II collagen-induced arthritis, over ten days. The effect was compared with vehicle and CBD.

Fig. 13 shows the effect of treatments with HU-444 (in 2.5, 5, 10 mg/kg dosages) on paw thickness (mm) of DBA/1 mice with established chicken type II collagen-induced arthritis, over ten days. The effect was compared with vehicle and CBD. The effect is shows as the change in clinical score from day 1 (normalised data) over ten days.

Fig. 14 shows the effect of treatments with HU-444 (in 2.5, 5, 10 mg/kg dosages) on paw thickness (mm) of DBA/1 mice with established chicken type II collagen-induced arthritis, over ten days. The effect was compared with vehicle and CBD. Paw thickness (raw data) over ten days.

Fig. 15 shows the effect of treatments with HU-444 (in 2.5, 5, 10 mg/kg dosages) on paw thickness (mm) of DBA/1 mice with established chicken type II collagen-induced arthritis, over ten days. The effect was compared with vehicle and CBD. The effect is shows as the change in clinical score from day 1 (normalised data) over ten days.

Fig. 16 shows the incidences of arthritis in DBA/1 mice immunized with chicken type II collagen (performed for experiment with 15 mg/kg HU-444).

Fig. 17 shows the effect of treatments with HU-444 (15 mg/kg) on clinical arthritis score of DBA/1 mice with established chicken type II collagen-induced arthritis, over ten days. The effect was compared with vehicle.

Fig. 18 shows the effect of treatments with HU-444 (15 mg/kg) on clinical arthritis score of DBA/1 mice with established chicken type II collagen-induced arthritis, over ten days. The effect was compared with vehicle. The effect is shows as the change in clinical score from day 1 (normalised data) over ten days. Fig. 19 shows the effect of treatments with HU-444 (15 mg/kg) on paw thickness (mm) of DBA/1 mice with established chicken type II collagen-induced arthritis, over ten days. The effect was compared with vehicle.

Fig. 20 shows the effect of treatments with HU-444 (15 mg/kg) on paw thickness (mm) of DBA/1 mice with established chicken type II collagen-induced arthritis, over ten days. The effect was compared with vehicle and CBD. The effect is shows as the change in clinical score from day 1 (normalised data) over ten days.

Fig. 21 shows the joints composite profile (categorized as normal, mild, moderate or severe) of DBA/1 mice with established chicken type II collagen-induced arthritis treated with 15 mg/kg of HU-444 and compared with vehicle.

Fig. 22 shows the joints composite protected vs. damaged profile of DBA/1 mice with established chicken type II collagen-induced arthritis treated with 15 mg/kg of HU-444 and compared with vehicle.

Fig. 23 shows the effect of HU-444 on levels of alanine transferase enzyme in serum of CON-A treated mice. The effect is shown relative to control group.

Fig. 24 shows the effect of HU-444 in various doses (2.5mg, 5mg, lOmg) on the levels of tumour necrosis factor (TNF) in serum of CON-A treated mice. The effect is shown relative to control group.

DETAILED DESCRIPTION OF EMBODIMENTS

The following Examples are representative of techniques employed by the inventors in carrying out aspects of the present invention. It should be appreciated that while these techniques are exemplary of preferred embodiments for the practice of the invention, those of skill in the art, in light of the present disclosure, will recognize that numerous modifications can be made without departing from the spirit and intended scope of the invention.

Example 1: Synthesis of 8,9 dihydro-CBD- 7-oic acid, diacetate (HU-444) and 8.9 dihvdro-CBD-7-oic acid (HU-445) (see also Fig. 1):

(-)-CBD. (-)CBD was isolated from Cannabis sativa resin.

(-)8,9 dihydro-CBD (1). CBD (0.544 g, 1.73 mmol) in ethylacetate (10 ml), Pt (0.021 g), was hydrogenated at 10 psi, for 30 min. The mixture was purified by silica gel chromatography, using petroleum ether and ether as an eluent (0.534 g), Yield (97.5%).

(-)8,9 dihydro-CBD-diacetate (2). Compound !_(0.534 g, 1.689 mmol) was dissolved in pyridine (3 ml) and acetic anhydride (3 ml) and the reaction was stirred over night at room temperature. Then the solution was poured onto iced water (50 ml) and extracted with ether. The combined organic extracts were washed successively with 1M HC1, aqueous sodium bicarbonate and brine, dried over MgS0 ₄ , filtered and evaporated to dryness (0.677 g). Yield (100%).

Mixture 3. Compound 2 (0.677 g, 0.169 mmol) was dissolved in ethanol (4 ml) and selenium (IV) oxide (0.564 g, 5.08 mmol) was added. The mixture was refluxed for 4 hours and monitored by TLC. The ethanol was removed under pressure and the residue was diluted with water and extracted several times with ether, dried over MgS0 ₄ , filtered and evaporated to dryness. The residue was chromatographed on silica gel with petroleum ether - ether to give a mixture (3) (0.378 g), Yield (54 %).

Mixture 4. Dry chromium VI oxide (0.353 g, 3.53 mmol) was added to a stirred solution of dry pyridine (0.951 ml) in dry CH ₂ C1 ₂ (4:1) (4 ml) and stirred at room temperature for 15 min under nitrogen. A solution of the mixture 3 (0.378 g, 0.91 mmol) in dry (CH ₂ C1 ₂ : DMF) (4:1) (1.5 ml) was added and the reaction was stirred at room temperature for 1 hour. Ethanol (2 ml) was added and the reaction was stirred at room temperature for 10 min. The mixture was filtrated on silica gel covered with thin layer of Na ₂ S0 ₄ , washed several times with ethylacetate and evaporated to dryness. The residue was chromatographed on silica gel with petroleum ether - ether to give mixture 4. (0.235 g) Yield (63%).

(-)8,9-dihydro-CBD-7-oic acid diacetate (HU-444). NaC10 ₂ (0.221 g, 2.44 mmol) was added in small quantities to a stirred mixture of compound 4_(0.235 g,), 2- methyl-2-butene (1.5 ml, 14.22 mmol) and a saturated aqueous solution of KH ₂ P04 (0.67 ml) in t-butanol (13.37 ml). The reaction was stirred at room temperature for 5 hours, and monitored by TLC. Water was added (60 ml) and the mixture was extracted several times with ethyl acetate. The organic phase was washed with brine, dried over MgS0 ₄ and filtered. Removal of the solvent under reduced pressure afforded a residue that was chromatographed on silica gel. Elution with petroleum ether:ether gave HU- 444 (0.03 g). Yield (13%). 1H-NMR: δ 6.939 (lH,s,olefm), 6.887 (2H,s,Ar), 3.589 (lH,m,benzyl), 2.512 (3H, m, allyl + benzyl), 2.222 (6H, s. OAc), 1.971 (1H, m), 1.858 (1H, m), 1.544 (5H, br s), 1.324 (5H, m), 0.987 (9H, t, terminal CH ₃ ). GC-MS m/z: 502 (silylation), 472, 430, 415, 400. Mp= 152 °C . [a] ²⁰ _D = -57

(-)8,9 dihydro-CBD-7-oic acid (HU-445). Compound HU-444J0.03 g, 0.069 mmol) was dissolved in ethanol (8 ml). NaB¾ (0.003 g, 0.092 mmol) was added and the reaction was refluxed for 1 hour. The ethanol was removed under pressure, the residue was diluted with water (10 ml) and the solution was extracted with ether. The combined organic extracts were washed with brine, dried over MgS04 and filtered. Removal of the solvents under reduced pressure afforded a residue that was chromatographed on silica gel to give (0.013 g) HU-445. Yield (55%). 'HNMR: δ 6.939 (lH,s,olefin), 6.887 (2H,s,Ar), 4.122 (2H, br s), 3.589 (lH,m,benzyl), 2.512 (3H, m, allyl + benzyl), 2.222 (3H, s. OAc), 1.971 (1H, m), 1.858 (1H, m), 1.544 (5H, br s), 1.324 (5H, m), 0.987 (9H, t, terminal CH ₃ ). GC-MS m/z (after silylation): 560, 488,416,344. Mp= 88-90 C; [a] ²⁰ _D = -56.2

Example 2: In vitro and In vivo effect of HU-444 and HU-445

Raw 264.7 macrophage cell line

Raw 264.7 cells, a monocytic-macrophage cell line derived from BALB/c mice, were obtained from the Addiction Technology Transfer Center (ATTC, Rockville, MD, USA). The cells were cultured in Dulbecco's modified Eagle medium (DMEM) suplemented with 5% fetal calf serum (FCS) and sodium pyrovate, glutamine and antibutics. For activation, the cells were incubated with LPS (E. coli ^g/ml for 24 hs, Sigma, Israel). ROI production by RAW 264.7 cells

Raw 264.7 cells were removed by cell scraper, washed and re-suspended in Hanks' balanced salt solution (without phenol red). For measurement of chemiluminescence, 0.5 ml of cell suspension (5 x 10 ⁵ cells) was added to each luminometer tube, together with various doses of cannabinoids derivative of the invention tested (dissolved in ethanol and diluted with Hanks). The cells were incubated for 20-24 hs and then, 10 μΐ of luminol (Sigma) and 30 μΐ of zymosan (Sigma) were added to the tubes, and the chemiluminescence was measured immediately in a luminometer (Biolumate LB 95, Berhold, Wilbad, Germany[8].

Macrophages

Peritoneal cells were harvested from C57BL/6 female mice four days after intraperitoneal injection of 1.5 ml of thioglycollate medium (Difco). The cells were washed with phosphate-buffered saline, re-suspended in DMEM and supplement, containing 5% FCS, and plated flat-bottom 96-microwell plates at a concentration of 1.2 x 10 ⁵ cells per well. Following 2-3 h of incubation at 37°C, the non-adherant cells were removed by intensive rinsing. About 95% of the adherent cells were macrophages. Treatment of macrophages with cannabinoids

The cannabinoid derivatives of the invention were first dissolved in absolute ethanol (1 mg/50-100 μΐ ethanol), and the solutions were further diluted with DMEM medium. For each cannaboid compound, various nontoxic concentrations were added to the macrophages, followed by addition of 1 μg/ml of lipopolysaccharide (LPS, e. coli, Sigma) for activation. The macrophages were then cultivated in a humid atmosphere with 5% C0 ₂ for 24 hs. The supernatant fluids were harvested and kept at -20°C until assayed for NO and TNFa.

Nitric oxide determination

NO generation was determined by measuring the nitrite accumulated in the supernatants (100 μΐ) of the cannabinoid-treated macrophages as follows. An equal volume (100 μΐ) of Griess reagent (1% sulphanilamide, 0.1% naphthalene diamine HCl, 2% H ₃ P0 ₄ ) was added to each supernatant. Following 10 min of incubation at room temperature, the color production was measured at 550 nm with an ELISA reader. The concentration of the nitrit was calculated according to a standard curve [8].

TNFain-vitro TNFa in the supernatants of the cannabinoid-treated (40, 20, 10, 5,and 1 μΐ/ml) LPS-activated macrophages was determined by ELISA (R&D) according to manufactore instructions.

TNFin vivo

Mice were injected IP with 100 μg LPS, Cannabinoid derivative of the invention was administrated IP simultaneously. Mice were bled 90 min later and TNF in the plasma was measured by Elisa. IN VITRO RESULTS

ROI production by RAW 264.7 cells

Fig. 2 A shows that the reduction in ROI (oxygen radicals) was 61% and 38% at HU-444 concentration of 40 μg/ml and 20μg/ml, respectively.

Fig. 2B shows that the reduction in ROI (oxygen radicals) was 60% , 30% and 39% at HU-445 concentrations of 40 μg/ml, 20 μg/ml and 10μg/ml, respectively.

Nitric oxide determination

Figs. 3A and 3B show that the presence of HU-444 reduced NO generation of RAW cells in the two performed experiments (3A and 3B), by 62-73% and 50-70% at 60 μg/ml and 40 μg/ml HU-444, respectively.

Fig. 3C shows that the presence of HU-445 reduced NO generation of RAW cells, by 78%, 58% and 22% at 40 μg/ml, 20 μg/ml and 10 μg/ml HU-445, respectively.

Chemotaxis

Migration of THP-1 cells towards MCP-1 in Boy den modified chambers was performed with HU-444 as the cannabinoid derivative of the invention. Fig. 4A shows that HU-444 at concentration of 20 and 10 μ πύ inhibited THP-1 cell migration toward MCP-1 by 43% and 35% respectively.

Fig. 4B shows that HU-445 affected TNF levels by 80%, 40% and 27% at 40 μg/ml , 20 μg/ml and 10 μg/ml respectively.

IN VIVO RESULTS Level of TNF in the mice sera after injection of HU-444 was determined in 3 experiments, total of 40 mice. A representative experiment (Fig. 5A) shows that concentration of 2.5 mg/kg and 5 mg/kg HU-444, inhibited TNF production in the mice, by 62%-63%.

Fig. 5B shows that HU-445 affected TNF levels by 55% at 10 μg/ml.

Example 3: Effect of HU-444 on the treatment of established chicken type II collagen-induced arthritis in DBA/1 mice

Male DBA/1 mice were immunized at 16 weeks of age with an emulsion of purified chicken type II collagen and Freund's complete adjuvant (CFA) at the base of the tail, in a single intra dermal injection. No booster was used in this experiment.

In the group receiving 15mg/kg of HU-444 there were twenty male DBA/1 mice immunized with a single i.d. injection of purified chicken type II collagen emulsified in Freund's complete adjuvant and kept in IVC over a 20 h light/dark cycle. They were fed with tap water and laboratory chow ad libitum.

Immunized mice were regularly monitored from day 14 post immunization and mice developing clinical signs of arthritis, as characterised by inflammation in any of their paws were randomly recruited into the different treatment groups.

All treatments were administered as a single intraperitoneal injection, of freshly prepared compounds (prepared every 3 to 4 days as a fresh batch) daily for up to 10 days.

Mice were scored for degree of redness & inflammation in every paw daily and given a clinical score from 0-3. Hind paw thickness was measured in (mm) using a caliper on day of monitoring & Rx. Results presented as raw data of mean ± SEM values of n=9 per group. Alternatively, normalised data in relation to dl of CIA are presented as the means ± SEM. Further results were plotted as an average ± sem of number of mice per group vs day of arthritis.

Mice in all treatment groups were weighed before recruitment into groups and at the end of the experiment, following treatments. Data was plotted as a change in weight.

Treatment Groups

• Vehicle mixture (Ethanol: Cremaphor-EL: Saline, 1 :1:18 v/v/v) (N = 10)

• HU-444 prepared in vehicle mixture • 2.5 mg/kg (N = 10),

• 5.0 mg/kg (N = 9)

• 10.0 mg/kg (N = 9)

• 15.0 mg/kg (N=20)

· CBD prepared in vehicle mixture at 5.0 mg/kg (N = 9)

Results

Fig. 6 shows the incidences of arthritis in DBA/1 mice immunized with chicken type II collagen.

Fig. 7 shows the effect of treatments with HU-444 (in 2.5, 5, 10 mg/kg dosages) on weight of DBA/1 mice with established chicken type II collagen-induced arthritis. The effect was compared with vehicle and CBD.

Fig. 8 shows the effect of treatments with HU-444 (in 2.5, 5, 10 mg/kg dosages) on clinical arthritis score of DBA/1 mice with established chicken type II collagen- induced arthritis, over ten days. The effect was compared with vehicle and CBD.

Fig. 9 shows the effect of treatments with HU-444 (in 2.5, 5, 10 mg/kg dosages) on clinical arthritis score of DBA/1 mice with established chicken type II collagen- induced arthritis, over ten days. The effect was compared with vehicle and CBD. The effect is shows as the change in clinical score from day 1 (normalised data) over ten days.

Fig. 10 shows the effect of treatments with HU-444 (in 2.5, 5, 10 mg/kg dosages) on clinical arthritis score of DBA 1 mice with established chicken type II collagen-induced arthritis, over ten days. The effect was compared with vehicle and CBD.

Fig. 11 shows the effect of treatments with HU-444 (in 2.5, 5, 10 mg/kg dosages) on clinical arthritis score of DBA/1 mice with established chicken type II collagen-induced arthritis, over ten days. The effect was compared with vehicle and CBD. The effect is shows as the change in clinical score from day 1 (normalised data) over ten days.

Fig. 12: shows the effect of treatments with HU-444 (in 2.5, 5, 10 mg/kg dosages) on paw thickness (mm) of DBA/1 mice with established chicken type II collagen-induced arthritis, over ten days. The effect was compared with vehicle and CBD.

Fig. 13 shows the effect of treatments with HU-444 (in 2.5, 5, 10 mg/kg dosages) on paw thickness (mm) of DBA/1 mice with established chicken type II collagen-induced arthritis, over ten days. The effect was compared with vehicle and CBD. The effect is shows as the change in clinical score from day 1 (normalised data) over ten days.

Fig. 14 shows the effect of treatments with HU-444 (in 2.5, 5, 10 mg/kg dosages) on paw thickness (mm) of DBA 1 mice with established chicken type II collagen-induced arthritis, over ten days. The effect was compared with vehicle and CBD. Paw thickness (raw data) over ten days.

Fig. 15 shows the effect of treatments with HU-444 (in 2.5, 5, 10 mg/kg dosages) on paw thickness (mm) of DBA/1 mice with established chicken type II collagen-induced arthritis, over ten days. The effect was compared with vehicle and CBD. The effect is shows as the change in clinical score from day 1 (normalised data) over ten days.

Fig. 16 shows the incidences of arthritis in DBA/1 mice immunized with chicken type II collagen (performed for experiment with 15 mg/kg HU-444).

Fig. 17 shows the effect of treatments with HU-444 (15 mg/kg) on clinical arthritis score of DBA/1 mice with established chicken type II collagen-induced arthritis, over ten days. The effect was compared with vehicle.

Fig. 18 shows the effect of treatments with HU-444 (15 mg/kg) on clinical arthritis score of DBA/1 mice with established chicken type II collagen-induced arthritis, over ten days. The effect was compared with vehicle. The effect is shows as the change in clinical score from day 1 (normalised data) over ten days.

Fig. 19 shows the effect of treatments with HU-444 (15 mg/kg) on paw thickness (mm) of DBA/1 mice with established chicken type II collagen-induced arthritis, over ten days. The effect was compared with vehicle.

Fig. 20 shows the effect of treatments with HU-444 (15 mg/kg) on paw thickness (mm) of DBA/1 mice with established chicken type II collagen-induced arthritis, over ten days. The effect was compared with vehicle and CBD. The effect is shows as the change in clinical score from day 1 (normalised data) over ten days. Fig. 21 shows the joints composite profile (categorized as normal, mild, moderate or severe) of DBA/1 mice with established chicken type II collagen-induced arthritis treated with 15 mg/kg of HU-444 and compared with vehicle.

Fig. 22 shows the joints composite protected vs. damaged profile of DBA/1 mice with established chicken type II collagen-induced arthritis treated with 15 mg/kg of HU-444 and compared with vehicle.

Example 4: Effect of HU-444 on Liver Concanavalin-A (CON-A) injury

Study groups comprised of 8-9 weeks old 16 mice were injected with 25mg/kg CON- A intravenously.

Group A: Four mice injected with 2.5 mg/kg HU-444.

Group B: Four mice injected with 5.0 mg/kg HU-444.

Group C: Four mice injected with 10.0 mg/kg HU-444.

Group D: Four mice injected with the vehicle (mixture of ethanol, cremophor and saline in a 1 : 1 : 18 ratio).

The mice were bled 22hrs later for determination of liver enzyme (alanine transaminase) as well as for tumor necrosis factor (TNF) levels in the serum. The livers were examined histologically. Results

Histology: Controls: large part of the livers was necrotic with massive infiltration of inflammatory cells. In all the HU-444- treated groups - almost no necrosis and much less or negligible amount of infiltration of inflammatory cells. Fig. 23 shows decrease in levels of alanine transferase enzyme of between about

10% (for group receiving 2.5mg/kg of HU-444) to 90% (for group receiving 10.0 mg/kg of HU-444). Fig. 24 shows decrease in tumour necrosis factor (TNF) in serum for all groups administered with HU-444 of about 50%. Conclusion

HU-444 and HU-445 were demonstrated to posses potent anti-inflammatory properties. Both HU-444 and HU-445 suppressed by 60-80% the generation of reactive oxygen species (ROS) and nitric oxygen species (NOS), as well as the production of tumor necrosis factor alfa (TNFa) by mouse macrophages. In the in vivo system, HU- 444 and HU-445 inhibited TNFa production by 60-70%, when applied intraperitoneally to mice.

Moreover, HU-444 markedly suppressed, by 60-70%, the arthritis symptoms when given either intraperitoneally or orally to DBA/1 mice in a murine collagen- induced arthritis model.

HU-444 inhibited markedly the destruction of the livers by inflammation as well as reduced the aminotransaminases levels up to 90%.