WO2023058016A1 - Non-steroidal anti-inflammatory drugs and cannabinoids and uses thereof - Google Patents

Abstract

Non-steroidal anti-inflammatory drugs and cannabinoids are provided. Accordingly, there is provided an article of manufacture comprising as active ingredients a non-steroidal anti-inflammatory drug and a composition comprising phytocannabinoids, wherein the phytocannabinoids comprise at least 80 % CBD and at least one of CBG and THCV. Also provided are methods of using same in the treatment of disease e.g. inflammation, pain and/or fever. Also provided is an article of manufacture comprising as active ingredients a steroid and a composition comprising phytocannabinoids; and its use in therapy.

Description

NON-STEROIDAL ANTI-INFLAMMATORY DRUGS AND CANNABINOIDS AND USES

THEREOF

RELATED APPLICATIONS

This application claims the benefit of priority of U.S. Provisional Patent Application No. 63/251,759 filed on 4 October 2021, the contents of which are incorporated herein by reference in their entirety.

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates to non-steroidal antiinflammatory drugs and cannabinoids and uses thereof.

The search of novel and more effective treatment for multiple diseases, including pain and inflammation, is of intense research.

Marijuana (Cannabis sativa) contains more than 500 constituents, among them phytocannabinoids, terpenes and flavonoids [ElSohly et al., Phytochemistry of Cannabis sativa L. Phytocannabinoids, Springer (2017) 1-36]. Cannabinoids were previously suggested as immune modulators and have been used for e.g. systemic treatment of pain and inflammation.

Non-steroidal anti-inflammatory drugs (NSAIDs) are a group of drugs that work by inhibiting the activity of cyclooxygenase enzymes (COX-1 or COX-2). NSAIDs have been largely used for the treatment of e.g. pain, fever, and inflammation.

Additional background art includes:

Anil et al. Sci Rep. 2021 Jan 14; 11(1): 1462; and

International Patent Application Publication No. W02020/121312; WO2018/163164; and WO2018/163163.

SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present invention there is provided an article of manufacture comprising as active ingredients a non-steroidal anti-inflammatory drug (NSAID) and a composition comprising phytocannabinoids, wherein the phytocannabinoids comprise at least 80 % CBD and at least one of CBG and THCV.

According to some embodiments of the invention, the NSAID and the composition comprising phytocannabinoids are provided in a co-formulation. According to some embodiments of the invention, the NSAID and the composition comprising phytocannabinoids are provided in separate formulations.

According to an aspect of some embodiments of the present invention there is provided a method of treating a disease selected from the group consisting of inflammation, pain and fever in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a non-steroidal anti-inflammatory drug (NSAID) and a therapeutically effective amount of a composition comprising phytocannabinoids, wherein the phytocannabinoids comprise at least 80 % CBD and at least one of CBG and THCV, thereby treating the disease in the subject.

According to an aspect of some embodiments of the present invention there is provided a non-steroidal anti-inflammatory drug (NSAID) and a composition comprising phytocannabinoids, wherein the phytocannabinoids comprise at least 80 % CBD and at least one of CBG and THCV, for use in treating a disease selected from the group consisting of inflammation, pain and fever in a subject in need thereof.

According to an aspect of some embodiments of the present invention there is provided a method of treating a disease selected from the group consisting of inflammation, pain, edema and fever in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a non-steroidal anti-inflammatory drug (NSAID) and a therapeutically effective amount of a composition comprising phytocannabinoids, wherein the phytocannabinoids comprise at least 80 % CBD and at least one of CBG and THCV, thereby treating the disease in the subject.

According to an aspect of some embodiments of the present invention there is provided a non-steroidal anti-inflammatory drug (NSAID) and a composition comprising phytocannabinoids, wherein the phytocannabinoids comprise at least 80 % CBD and at least one of CBG and THCV, for use in treating a disease selected from the group consisting of inflammation, pain, edema and fever in a subject in need thereof.

According to some embodiments of the invention, the pain is selected from the group consisting of inflammatory pain, acute pain, chronic pain, arthritis, pain following nerve injury, cancer associated pain, chemotherapy associated pain, migraine, low back pain and toothache.

According to some embodiments of the invention, the inflammation is selected from the group consisting of inflammation of the respiratory system, colon inflammation, rheumatoid arthritis, osteoarthritis, extra-articular rheumatism and ankylosing spondylitis. According to some embodiments of the invention, the inflammation is inflammation of the respiratory system or colon inflammation.

According to some embodiments of the invention, the inflammation is at a stage of cytokine storm.

According to some embodiments of the invention, the edema comprises leg and/or hand edema.

According to an aspect of some embodiments of the present invention there is provided an article of manufacture comprising as active ingredients a steroid and a composition comprising phytocannabinoids, wherein the phytocannabinoids comprise at least 80 % CBD and at least one of CBG and THCV.

According to some embodiments of the invention, the steroid and the composition comprising phytocannabinoids are provided in a co-formulation.

According to some embodiments of the invention, the steroid and the composition comprising phytocannabinoids are provided in separate formulations.

According to an aspect of some embodiments of the present invention there is provided a method of treating a disease selected from the group consisting of inflammation of the respiratory system, colon inflammation and cytokine storm in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a steroid and a therapeutically effective amount of a composition comprising phytocannabinoids, wherein the phytocannabinoids comprise at least 80 % CBD and at least one of CBG and THCV, thereby treating the disease in the subject.

According to an aspect of some embodiments of the present invention there is provided asteroid and a composition comprising phytocannabinoids, wherein the phytocannabinoids comprise at least 80 % CBD and at least one of CBG and THCV, for use in treating a disease selected from the group consisting of inflammation of the respiratory system, colon inflammation and cytokine storm in a subject in need thereof.

According to some embodiments of the invention, the disease is not cancer.

According to some embodiments of the invention, the NSAID is selected from the group consisting of ibuprofen and diclofenac.

According to some embodiments of the invention, the NSAID is diclofenac.

According to some embodiments of the invention, the NSAID and the composition comprising phytocannabinoids have a combined additive or synergistic anti-inflammatory effect as compared to each of the NSAID and the composition comprising phytocannabinoids when administered as a single agent.

According to some embodiments of the invention, the NSAID and the composition comprising phytocannabinoids have a combined additive or synergistic effect on pain reduction as compared to each of the NSAID and the composition comprising phytocannabinoids when administered as a single agent.

According to some embodiments of the invention, the steroid is selected from the group consisting of budesonide and dexamethasone.

According to some embodiments of the invention, the steroid and the composition comprising phytocannabinoids have a combined additive or synergistic anti-inflammatory effect as compared to each of the steroid and the composition comprising phytocannabinoids when administered as a single agent.

According to some embodiments of the invention, the composition comprising phytocannabinoids is a synthetic composition.

According to some embodiments of the invention, the at least 80 % CBD is at least 90 %.

According to some embodiments of the invention, the phytocannabinoids comprise 90 - 95 % CBD.

According to some embodiments of the invention, when the phytocannabinoids comprise the CBG the phytocannabinoids comprise at least 2% CBG.

According to some embodiments of the invention, when the phytocannabinoids comprise the CBG the phytocannabinoids comprise at least 5 % CBG.

According to some embodiments of the invention, when the phytocannabinoids comprise the CBG the phytocannabinoids comprise 5 - 7 % CBG.

According to some embodiments of the invention, when the phytocannabinoids comprise the CBG the phytocannabinoids comprise 5.5 - 6.5 % CBG.

According to some embodiments of the invention, when the phytocannabinoids comprise the CBG, a concentration ratio of the CBD and the CBG is 20 : 1 - 10 : 1.

According to some embodiments of the invention, when the phytocannabinoids comprise the CBG, a concentration ratio of the CBD and the CBG is 17 : 1 - 13 : 1.

According to some embodiments of the invention, when the phytocannabinoids comprise the THCV the phytocannabinoids comprise at least 0.2 % THCV. According to some embodiments of the invention, when the phytocannabinoids comprise the THCV the phytocannabinoids comprise less than 1 % THCV.

According to some embodiments of the invention, when the phytocannabinoids comprise the THCV the phytocannabinoids comprise 0.3 - 0.5 % THCV.

According to some embodiments of the invention, when the phytocannabinoids comprise the THCV, a concentration ratio of the CBD and the THCV is 300 : 1 - 100 : 1.

According to some embodiments of the invention, when the phytocannabinoids comprise the THCV, a concentration ratio of the CBD and the THCV is 250 : 1 - 200 : 1.

According to some embodiments of the invention, the composition comprising phytocannabinoids is devoid of Tetrahydrocannabinol (THC).

According to some embodiments of the invention, the composition comprising phytocannabonids is devoid of phytocannabinoids other than the CBD, CBG and/or THCV.

According to some embodiments of the invention, the composition comprising phytocannabinoids is devoid of cannabis active ingredients other than the phytocannabinoids.

According to some embodiments of the invention, the composition comprising phytocannabinoids is provided as a concentration of at least 10 pg / ml of the phytocannabinoids.

Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced. In the drawings:

FIGs. 1A-D demonstrate the combined anti-inflammatory effect of FCBD:std and ibuprofen (Figure 1A), budesonide (Figure IB), dexamethasone (Figure 1C) or diclofenac (Figure ID) on macrophages treated with TNFa, manifested by a decreased level of IL- 8 secreted from of KG1 cells. The anti-inflammatory activity was determined by ELISA as a function of level of IL- 8 (pg / cell) in the supernatant 4 hours following treatment (n = 3). Synergy was calculated using the Bliss independence drug interaction model. Synergy is apparent when the experimental (observed) value of IL-8 reduction is higher than the calculated (expected) value. Delta between the observed and expected values, calculated using the Bliss model are shown in the Y axis.

FIGs. 2A-D demonstrate the combined anti-inflammatory effect of FCBD:std and ibuprofen (Figure 2A), budesonide (Figure 2B), dexamethasone (Figure 2C) or diclofenac (Figure 2D) on lung epithelial cells treated with TNFa, manifested by a decreased level of IL-8 secreted from A549 cells. The anti-inflammatory activity was determined by ELISA as a function of level of IL- 8 (pg / cell) in the supernatant 4 hours following treatment (n = 3). Synergy was calculated using the Bliss independence drug interaction model. Synergy is apparent when the experimental (observed) value of IL-8 reduction is higher than the calculated (expected) value. Delta between the observed and expected values, calculated using the Bliss model are shown in the Y axis.

FIGs. 3A-D demonstrate the combined anti-inflammatory effect of FCBD:std and ibuprofen (Ibu, Figure 3A), budesonide (Bud, Figure 3B), dexamethasone (Dexa, Figure 3C) or diclofenac (Diclo, Figure 3D) on co-cultures of macrophages (KG1 cells) and lung epithelial cells (A549 cells) treated with TNFa, manifested by a decreased level of IL-8 secreted from the cells. The anti-inflammatory activity was determined by ELISA as a function of level of IL-8 (pg / cell) in the supernatant 4 hours following treatment. Control - vehicle control (1 % methanol, 0.5 % DMSO, 0.2 % methanol + 0.5% DMSO and 1% DMSO for Ibu, Bud, Dexa and Diclo, respectively). TNFa - solvent control+ TNFa. Means ± SE (n=3) are shown. Levels of means with different letters are significantly different from all combinations of pairs according to Tukey-Kramer honest significant difference (HSD; P < 0.05).

FIG. 4 demonstrates the combined anti-inflammatory effect of FCBD:std and diclofenac (Diclo) on co-cultures of macrophages (KG1 cells) and lung epithelial cells (A549 cells) treated with TNFa, manifested by a decreased level of IL-6 secreted from the cells. The antiinflammatory activity was determined by ELISA as a function of level of IL-6 (pg / cell) in the supernatant 4 hours following treatment. Control - vehicle control 1% methanol (1% DMSO vehicle control is not significantly different from 1% methanol, not shown). TNFa - solvent control+ TNFa. Means ± SE (n=3) are shown. Levels of means with different letters are significantly different from all combinations of pairs according to Tukey-Kramer honest significant difference (HSD; P < 0.05).

FIGs. 5-6 demonstrate the relative inhibition level (%) of COX-1 (Figure 5) and COX-2 (Figure 6) activity in the presence of FCBD:std, ibuprofen (Ibu) or diclofenac (Diclo). Vehicle control - 1% methanol (1 % DMSO vehicle control is not significantly different from 1 % methanol, not shown).

FIGs. 7A-F show representative confocal images of the 3D epithelial cells and macrophages co-cultures. Figures 7A-B show angle (Figure 7A) and side (Figure 7B) views of a 8-pm PET membrane seeded with A549 cells (Bar = 50 pm). Figure 7C shows side view of ECM seeded with macrophages (Bar = 50 pm). Figure 7D shows top view of a monolayer of A549 (Bar = 50 pm). Figure 7E shows top view of macrophages in ECM (Bar = 50 pm). Figure 7F shows ciliated A549 cells on the membrane; arrows point to ciliate structures (Bar = 20 pm). Nuclei are stained by Hoechst and membrane by Dil perchlorate. Merged- both nuclei and membrane staining.

FIGs. 8A-C demonstrate the combined anti-inflammatory effect of FCBD:std and ibuprofen (Ibu, Figure 8 A) or diclofenac (Diclo, Figures 8B-C) on 3D co-cultures of macrophages (KG1 cells) and lung epithelial cells (A549 cells) treated with TNFa, manifested by decreased level of IL-8 or IL-6 secreted from the cells. The anti-inflammatory activity was determined by ELISA as a function of level of IL-8 or IL-6 (pg / cell) in the supernatant 4 hours following treatment (n = 3). Control - vehicle control (1% methanol and 1% DMSO for Ibu and Diclo, respectively). TNFa - solvent control+ TNFa. Means ± SE (n=3) are shown. Levels of means with different letters are significantly different from all combinations of pairs according to Tukey-Kramer honest significant difference (HSD; P < 0.05).

FIGs. 9A-D demonstrate the dose-response anti-inflammatory effect of ibuprofen (Figure 9A), budesonide (Figure 9B), dexamethasone (Figure 9C) or diclofenac (Figures 9D) on macrophages treated with TNFa, manifested by decreased level of IL- 8 secreted from of KG1 cells. The anti-inflammatory activity was determined by ELISA as a function of level of IL-8 (pg / cell) in the supernatant 4 hours following treatment. The IC50 values were calculated from 5P logistic curve fit by GraphPad Prism version 6.1. Error bars indicate ± SE (n = 3).

FIGs. 10A-D demonstrate the dose-response anti-inflammatory effect of ibuprofen (Figure 10A), budesonide (Figure 10B), dexamethasone (Figure 10C) or diclofenac (Figures 10D) on A549 lung epithelial cells treated with TNFa, manifested by decreased level of IL-8 secreted from the cells. The anti-inflammatory activity was determined by ELISA as a function of level of IL-8 (pg / cell) in the supernatant 4 hours following treatment. The IC50 values were calculated from 5P logistic curve fit by GraphPad Prism version 6.1. Error bars indicate ± SE (n = 3).

FIG. 11 is a schematic representation of the structure of the 3D printed model. Black middle arrow - a 8 pm PEM membrane attached to the printed ECM.

FIG. 12 demonstrates the combined anti-inflammatory effect of FCBD:std and diclofenac on mRNA expression levels of IL-6 in paws of carrageenan-induced edema model, as determined by quantitative PCR. Shown are gene transcript values as a ratio between the target gene versus mouse GAPDH as a reference gene. Values were calculated relative to the average expression of target genes in treated versus control using the 2AACt method. Levels with different letters are significantly different from all combinations of pairs by Tukey-Kramer honest significant difference (HSD; P < 0.05).

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates to non-steroidal antiinflammatory drugs and cannabinoids and uses thereof.

Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.

Marijuana (Cannabis sativa) contains more than 500 constituents, among them phytocannabinoids, terpenes and flavonoids. Cannabinoids were previously suggested as immune modulators and have been used for e.g. systemic treatment of pain and inflammation.

The present inventors have now uncovered synergistic anti-inflammatory effects of a combination of Non-steroidal anti-inflammatory drugs (NSAIDs) and specific compositions comprising phytocannabinoids on lung epithelial cells and macrophages.

Specifically, as is illustrated in the Examples section which follows, the present inventors prepared a synthetic composition referred to herein as “FCBD:std” containing combination of phytocannabinoid mimicking a liquid chromatography FCBD fraction of a cannabis extract described in Anil et al. Sci Rep. 2021 Jan 14; 11(1): 1462, which contain CBD (93.5 %), CBG (6.1 %) and THCV (0.4 %). Following, the inventors demonstrated that combined treatment with this composition and NSAIDs (e.g. ibuprofen or diclofenac) has synergistic anti-inflammatory effects, manifested by reduced secretion of IL-8 and IL-6 in single and co-cultures of lung epithelial cells and macrophages (Examples 1-4 of the Examples section which follows). Further, the inventors demonstrated that combined treatment with this composition and NSAIDs (e.g. diclofenac) has synergistic therapeutic effects in a carrageenan-induced paw edema mouse model (Example 5-6 of the Examples section which follows).

Consequently, specific embodiments of the present invention propose novel articles of manufactures comprising NSAIDs and phytocannabinoids and their use in therapy.

Thus, according to an aspect of the present invention there is provided an article of manufacture comprising as active ingredients a non-steroidal anti-inflammatory drug (NSAID) and a composition comprising phytocannabinoids, wherein said phytocannabinoids comprise at least 80 % CBD and at least one of CBG and THCV.

According to specific embodiments, the NSAID and the composition comprising phytocannabinoids are provided in a co-formulation.

According to specific embodiments, the NSAID and the composition comprising phytocannabinoids are provided in separate formulations.

Alternatively or additional, the present inventors have now uncovered synergistic antiinflammatory effects of a combination of steroids and specific compositions comprising phytocannabinoids on lung epithelial cells.

Specifically, as is illustrated in the Examples section which follows, the present inventors demonstrated that combined treatment with FCBD:std and steroids (e.g. budesonide or dexamethasone) has synergistic anti-inflammatory effects on lung epithelial cell, manifested by reduced secretion of IL- 8 in cultures of lung epithelial cells (Example 2 of the Examples section which follows). Further, this combined treatment was not synergistic in reducing macrophage activity and even decreased the anti-inflammatory activity of each of the agents (Example 1 of the Examples section which follows).

Consequently, specific embodiments of the present invention propose novel articles of manufactures comprising steroids and phytocannabinoids and their use in therapy.

Thus, according to an alternative or an additional aspect of the present invention there is provided an article of manufacture comprising as active ingredients a steroid and a composition comprising phytocannabinoids, wherein said phytocannabinoids comprise at least 80 % CBD and at least one of CBG and THCV. According to specific embodiments, the steroid and the composition comprising phytocannabinoids are provided in a co-formulation.

According to specific embodiments, the steroid and said composition comprising phytocannabinoids are provided in separate formulations.

Non-steroidal anti-inflammatory drugs (“NSAIDs”) are a class of drugs well known in the art. See, for example, The Pharmacological Basis of Therapeutics, 8th edition, Goodman et al., Chapter 26 (1990). These drugs work by inhibiting the activity of cyclooxygenase enzymes (COX-1 and/or COX-2).

According to specific embodiments, the NS AID inhibits COX-1.

According to specific embodiments, the NSAID inhibits COX-2.

Non-limiting examples of NSAID include salicylates, for example, aspirin, pyrazolone derivatives, for example, phenylbutazone, onyphenbutazone, antipyrine, aminopyrine, dipyrone and apazone, indomethacin, sulindac, fenamates, for example, mefenamic, meclofenamic, flufenamic, tolfenamic and etofenamice acids, aryl acetic acid and propionic acid compounds, for example, 2-(p-isobutylphenyl)propionic acid (generic name ibuprofen), a-methyl-4-(2- thienylcarbonyl)benzene acetic acid (generic name suprofen), 4,5-diphenyl-2-oxazole propionic acid (generic name oxprozin), rac-6-chloro-alphamethyl-carbazole-2-acetic acid (generic name carprofen), 2-(3-phenyloxyphenyl)-propionic acid, the calcium salt dihydrate thereof (these compounds being referred to generically as fenoprofen and fenoprofen calcium), 2-(6-methoxy-2- naphthyl)propionic acid (generic name naproxen, the generic name of the sodium salt is naproxen sodium), 4-(l,3-dihydro-l-oxo-2H-isoindol-2-yl)-a-methylbenzene acetic acid (generic name indoprofen), 2-(3-benzoylphenyl)propionic acid (generic name ketoprofen), and 2-(2-fluoro-4- biphenylyl)propionic acid (generic name flurbiprofen), and l-5-(4-methylbenzoyl)-lH-pyrrole-2- acetic acid (generic name tolmetin). Non-steroidal anti-inflammatory drugs also include, for example, sodium 5-(4-chlorobenzoyl)-l,4-dimethyl-lH-pyrrole-2-acetate dihydrate (generically referred to as zomepirac sodium), 4-hydroxy-2-methyl-N-(2-pyridyl-2H-l,2-benzothiazine-3- carboxamide- 1,1 -di oxide (generic name piroxicam), 2',4'-difluoro-4-hydroxy-3- biphenylcarboxylic acid (generic name difhmisal), or 1 -isoprop yl-7-methyl-4-phenyl-2( 1H)- quinozolinone (generic name proquazone), phenylacetic acid derivatives, for example, diclofenac, etodolac and nabumetone.

Non-limiting examples of NSAIDs that can be used with specific embodiments of the invention include diclofenac, etodolac, ketorolac, bromfenac, ibuprofen, fenoprofen, fluriboprofen, ketoprofen, naproxen, suprofen, meclofenamate, mefenamic acid, piroxicam, meloxicam, indomethacin, sulindac, phenylbutazone, firocoxib, oxyphenbutazonetolmetin, celecoxib, rofecoxib, valdecoxib or a pharmaceutically acceptable salt, metabolite, or prodrug thereof, or a combination thereof.

According to specific embodiments, the NSAID is selected from the group consisting of ibuprofen and diclofenac.

According to specific embodiments, the NSAID is diclofenac.

According to specific embodiment, the steroid is a corticosteroid.

Non-limiting examples of steroids that can be used with specific embodiments of the invention include beclomethasone, budesonide, dexamethasone, flunisolide, triamcinolone acetonide, meprednisone, 21 -acetoxypregnenolone, alclometasone, algestone, amcinonide, betamethasone, chloroprednisone, clobetasol, clobetasone, clocortolone, cloprednol, corticosterone, cortisone, cortisone 21 P-cyclopentaneproprionate, cortisone phosphate, cortivazol, deflazacort, desonide, desoximetasone, diflorasone, diflucortolone, difluprednate, enoxolone, fluazacort, flucloronide, flumethasone, flunisolide, fluocinolone acetonide, fluocinonide, fluocortin butyl, fluocotolone, fluorometholone, fluperolone acetate, fluprednidene acetate, fluprednisolone, flurandrenolide, formocortal, halcinonide, halometasone, halopredone acetate, hydrocortamate, hydrocortisone, isoflupredone, mazipredone, medrysone, meprednisone, methylprednisolone, mometasone furoate, prednicarbate, prednisolone, prednisone, prednival, prednylidene, tixocortol, tomatidine, triamcinolone, triamcinolone acetonide, and the like),

According to specific embodiments, the steroid is selected from the group consisting of betamethasone, budesonide, cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone, salicylic acid derivative-containing corticosteroids, and tolazoline and dimethyl sulfoxide.

According to specific embodiments, the steroid is selected from the group consisting of budesonide and dexamethasone.

According to a specific embodiment, the term “phytocannabinoid” refers to a meroterpenoid with a resorcinyl core typically decorated with a para-positioned isoprenyl, alkyl, or aralkyl side chain originated from a cannabis plant, acidic or decarboxylated acid forms thereof. The term also reads on synthetic analogs or derivatives of the plant originated substance.

Alternatively or additionally, the term “phytocannabinoid” refers to a cannabinoid selected from the list provided in Table 11 hereinbelow originated from a cannabis plant, acidic or decarboxylated acid forms thereof. The term also reads on synthetic analogs or derivatives of the plant originated substance.

Table 11: List of phytocannabinoids (modified from Berman P, Futoran K, Lewitus GM, Mukha D, Benami M, Shlomi T, Meiri D. A new ESLLC/MS approach for comprehensive metabolic profiling of phytocannabinoids in Cannabis. Scientific reports. 2018 Sep 24; 8(l):l-5, the contents of which are fully incorporated herein by reference)

According to specific embodiments, at least 50 % of the cannabinoids or cannabis-derived compounds in the composition comprising phytocannabinoids are phytocannabinoids.

According to specific embodiments, at least 55 %, at least 60 % or at least 65 % of the cannabinoids or cannabis-derived compounds in the composition comprising phytocannabinoids are phytocannabinoids.

According to specific embodiments, 50 - 100 %, 60 - 90 % or 60 - 80 % of the cannabinoids or cannabis-derived compounds in the composition comprising phytocannabinoids are phytocannabinoids. According to specific embodiments, 60 - 70 % of the cannabinoids or cannabis-derived compounds in the composition comprising phytocannabinoids are phytocannabinoids.

According to specific embodiments, 90 - 100 % of the cannabinoids or cannabis-derived compounds in the composition comprising phytocannabinoids are phytocannabinoids. The compositions comprising phytocannabinoids disclosed herein comprise phytocannabinoids wherein the phytocannabinoids comprise CBD and at least one of CBG and THCV.

According to specific embodiments, the phytocannabinoids in the composition comprise CBD and CBG.

According to specific embodiments, the phytocannabinoids in the composition comprise CBD and THCV.

According to specific embodiments, the phytocannabinoids in the composition comprise CBD, CBG and THCV.

Cannabidiol (CBD) (CAS No. 13956-29-1), as used herein, encompasses native CBD (i.e. originating from the Cannabis plant), or synthetic or naturally occurring analogs or derivatives thereof. According to specific embodiments, any CBD analog may be used in accordance with specific embodiments of the present teachings as long as it comprises the anti-inflammatory and/or macrophage activating activities described herein (alone, or as part of a composition discussed herein).

Exemplary CBD analogs include, but are not limited to, (-)-DMH-CBD-l l-oic acid, HU- 308 (commercially available e.g. from Tocris Bioscience, 3088), 0-1602 (commercially available e.g. from Tocris Bioscience 2797/10), DMH-CBD (commercially available e.g. from Tocris Bioscience, 1481) [as discussed in detail in Burstein S, Bioorg Med Chem. (2015) 23(7): 1377-85], Abn-CBD, HUF-101. CBDV, CBDM, CBND-C5, CBND-C3, 6-Hydroxy-CBD-triacetate or CBD- aldehyde-diacetate [as discussed in detail in An Overview on Medicinal Chemistry of Synthetic and Natural Derivatives of Cannabidiol, Frontiers in Pharmacology, June 2017 | Volume 8 | Article 422],

According to specific embodiments, the CBD is not CBDV.

According to specific embodiments, the CBD comprises native CBD.

Pure or synthetic CBD can be commercially obtained from e.g. Restek catalog no. 34011.

Cannabigerol (CBG) (CAS No. 25654-31-3), as used herein, encompasses native CBG (i.e. originating from the Cannabis plant), or synthetic or naturally occurring analogs or derivatives thereof. According to specific embodiments, any CBG analog may be used in accordance with specific embodiments of the present teachings as long as it comprises the antiinflammatory and/or macrophage activating activities described herein (alone, or as part of a composition discussed herein).

According to specific embodiments, the CBG comprises native CBG. Pure or synthetic CBG can be commercially obtained from e.g. Restek catalog no. 34091.

Tetrahydrocannabivarin (THCV) (CAS No. 31262-37-0), as used herein, encompasses native THCV (i.e. originating from the Cannabis plant), or synthetic or naturally occurring analogs or derivatives thereof. According to specific embodiments, any THCV analog may be used in accordance with specific embodiments of the present teachings as long as it comprises the anti-inflammatory and/or macrophage activating activities described herein (alone, or as part of a composition discussed herein).

An exemplary THCV analog include, but is not limited to, A8-THCV.

According to specific embodiments, the THCV comprises native THCV.

Pure or synthetic THCV can be commercially obtained from e.g. Restek catalog no. 34100.

As used herein, a “percent (%) of a phytocannabinoid” in the compositions disclosed herein refers to the concentration as presented in percentage (w/v) of the recited phytocannabinoid out of the total phytocannabinoids (and only the phytocannabinoids) in the composition, as can be determined by the peak area according to a HPLC profile of the composition.

Methods of determining presence or absence of a compounds in the composition, as well as the concentration of a compound in the composition are well known in the art, such as, but not limited to ultraviolet-visible spectroscopy ("UV-Vis"), infrared spectroscopy ("IR"), and the like; mass-spectrometry ("MS") methods such as, but not limited to, time-of-flight MS; quadrupole MS; electrospray MS, Fourier-transform MS, Matrix- Assisted Laser Desorption/Ionization ("MALDI"), and the like; chromatographic methods such as, but not limited to, gaschromatography ("GC"), liquid chromatograph ("LC"), high-performance liquid chromatography ("HPLC"), and the like; and combinations thereof (e.g., GC/MS, LC/MS, HPLC/UV-Vis, and the like), and other analytical methods known to persons of ordinary skill in the art.

According to specific embodiments, determining presence or absence of a compound in the composition and/or the concentration of a compound in the composition is effected by analytical high pressure liquid chromatography (HPLC).

According to specific embodiments, the phytocannabinoids comprise at least 80 % CBD.

According to specific embodiments, the phytocannabinoids comprise at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 % or at least 93 % CBD.

According to specific embodiments, the phytocannabinoids comprise at least 90 % CBD. According to specific embodiments, the phytocannabinoids comprise less than 99 %, less than 98 %, less than 97 %, less than 96 % less than 95 %, less than 94 % CBD.

According to specific embodiments, the phytocannabinoids comprise less than 95 % CBD.

According to specific embodiments, the phytocannabinoids comprise 93.5 ± 10 % CBD.

According to specific embodiments, the phytocannabinoids comprise 90 - 95 % CBD.

According to specific embodiments, the phytocannabinoids comprise 93.5 % CBD.

According to specific embodiments, the phytocannabinoids comprise at least 2 % CBG.

According to specific embodiments, the phytocannabinoids comprise at least 3 % CBG.

According to specific embodiments, the phytocannabinoids comprise at least 4 %, at least 4.5 %, at least 5 %, at least 5.5 % or at least 6 % CBG.

According to specific embodiments, the phytocannabinoids comprise at least 5 % CBG.

According to specific embodiments, the phytocannabinoids comprise less than 10 %, less than 8 %, less than 7 % CBG.

According to specific embodiments, the phytocannabinoids comprise 2 - 10 %, 3 - 9 % or 4 - 8 % CBG.

According to specific embodiments, the phytocannabinoids comprise 5 - 7 % CBG.

According to specific embodiments, the phytocannabinoids comprise 5.5 - 6.5 % CBG.

According to specific embodiments, the phytocannabinoids comprise about 6.1 % CBG.

According to specific embodiments, the phytocannabinoids comprise at least 0.1 % THCV.

According to specific embodiments, the phytocannabinoids comprise at least 0.2 %, at least 0.25 %, at least 0.3 %, at least 0.35 % THCV.

According to specific embodiments, the phytocannabinoids comprise less than 5 %, less than 4 %, less than 3 %, less than 2 %, less than 1 %, less than 0.5 % THCV.

According to specific embodiments, the phytocannabinoids comprise less than 1 % THCV.

According to specific embodiments, the phytocannabinoids comprise 0.1 - 1 %, 0.2 - 0.9 %, 0.2 - 0.8 %, 0.2 - 0.7 % or 0.3 - 0.6 % THCV.

According to specific embodiments, the phytocannabinoids comprise 0.3 - 0.5 % THCV.

According to specific embodiments, the phytocannabinoids comprise about 0.4 % THCV.

According to specific embodiments, a CBD : CBG concentration ratio in the composition or fraction is 20 : 1 - 10 : 1. According to specific embodiments, a CBD : CBG concentration ratio in the composition or fraction is 17 : 1 - 13 : 1.

According to specific embodiments, a CBD : CBG concentration ratio in the composition or fraction is about 15 : 1.

According to specific embodiments, a CBD : THCV concentration ratio in the composition or fraction is 300 : 1 - 100 : 1.

According to specific embodiments, a CBD : THCV concentration ratio in the composition or fraction is 250 : 1 - 200 : 1.

According to specific embodiments, a CBD : THCV concentration ratio in the composition or fraction is about 233 : 1.

According to specific embodiments, the concentration ratio is determined by g / 1 : g / 1 or pg / ml : pg / ml.

According to specific embodiments, the phytocannabinoids comprise 90 - 95 % CBD, 5.5 - 6.5 % CBG and 0.3 - 0.5 % THCV.

According to a specific embodiment, the phytocannabinoids comprise about 93.5 % CBD, about 6.1 % CBG and about 0.4 % THCV.

According to specific embodiments, the composition comprising phytocannabinoids is devoid of Tetrahydrocannabinol (THC).

According to specific embodiments, the composition comprising phytocannabinoids is devoid of phytocannabinoids other than CBD, CBG and/or THCV.

According to specific embodiments, the composition comprising phytocannabinoids is devoid of cannabinoids or cannabis active ingredients other than phytocannabinoids.

According to specific embodiments, the composition comprising phytocannabinoids comprises cannabinoids or cannabis active ingredients other than phytocannabinoids.

According to specific embodiments, the composition comprising phytocannabinoids comprises at least one terpene or terpenoid.

According to specific embodiments, the composition comprising phytocannabinoids comprises at least one of the terpenes and terpenoids listed in Table 12 hereinbelow.

According to specific embodiments, the composition comprising phytocannabinoids comprises at least one of the terpenes and terpenoids listed in Table 12 hereinbelow in a concentration ratio according to Table 12 ± 10 %. According to specific embodiments, the composition comprising phytocannabinoids comprises at least one of the terpenes and terpenoids listed in Table 12 hereinbelow in a concentration ratio according to Table 12.

According to specific embodiments, the composition comprising phytocannabinoids comprises at least two, at least three, at least four, at least five, at least six or all of the terpenes and terpenoids listed in Table 12 hereinbelow.

According to other specific embodiments, the composition comprising phytocannabinoids is devoid or terpenes and/or terpenoids.

Table 12: Terpenes and terpenoids that can be part of the composition of some embodiments of the invention.

The composition comprising phytocannabinoids of some embodiments of the invention may be a synthetic composition, a compositions comprising purified cannabinoids or a fraction of a cannabis extract.

According to specific embodiments, the composition is not a cannabis extract.

According to specific embodiments, the composition is a synthetic composition.

Herein, the term “synthetic composition” refers to a chemically defined composition which can include active ingredients which are chemically synthesized and/or purified to a level of purity of at least 99 %.

As used herein “a chemically defined composition” refers to a composition in which all the constituents are known by structure and optionally concentration.

According to specific embodiments, the phytocannabinoids are purified from cannabis.

According to specific embodiments, the phytocannabinoids are synthetic phytocannabinoids .

According to specific embodiments, the composition is a cannabis derived fraction.

As used herein “a fraction” refers to a portion of the extract that contains only certain chemical ingredients of the extract but not all. According to specific embodiments, the composition is a liquid chromatography fraction of a cannabis extract.

According to specific embodiments, the liquid chromatography comprises high pressure liquid chromatography (HPLC) or flash chromatography.

According to specific embodiments, the liquid chromatography fraction of cannabis extract comprises a liquid chromatography pooled fractions of cannabis extract comprising active ingredients detectable by a detector operated at 220 nm, wherein the active ingredients comprise the compounds disclosed herein.

According to specific embodiments, the liquid chromatography fraction is obtainable by subjecting the cannabis extract to flash chromatography comprising a Flash chromatography apparatus equipped with a diode array detector, a C18 functionalized silica column, a 85% methanol in water mobile phase, at a flow rate of 60 ml / min.

According to specific embodiments, the fraction is collected between about 26-31 minutes of the flash chromatography.

According to specific embodiments, the detector is a diode array detector.

According to specific embodiments, the detector is a 1260 MWD-VL detector.

Cannabis is a genus of flowering plants in the family Cannabaceae that includes three different species, Cannabis sativa, Cannabis indica and Cannabis ruderalis. The term Cannabis encompasses wild type Cannabis and also variants thereof, including cannabis chemovars which naturally contain different amounts of the individual cannabinoids. For example, some Cannabis strains have been selectively bred to produce high or low levels of THC or CBD and other cannabinoids.

According to specific embodiments, the Cannabis plant is a wild-type plant.

According to specific embodiments, the Cannabis plant is transgenic.

According to specific embodiments, the Cannabis plant is genomically edited.

According to specific embodiments, the Cannabis plant is Cannabis sativa (C. sativa).

According to specific embodiments, the Cannabis plant is C. sativa strain Arbel (obtained from IMC, Israeli Medical Cannabis, Israel).

The extract may be derived from a cultivated Cannabis plant (i.e. not grown in their natural habitat) or may be derived from Cannabis plants which grow in the wild.

The tissue of the Cannabis plant from which the extract is typically obtained is the inflorescence. Accordingly, the extract may be obtained from the complete flower head of a plant including stems, stalks, bracts, and flowers. However, it will be appreciated that a cannabis extract of some embodiments the invention may be obtained from only part of the inflorescence, such as from the bracts and/or flowers.

According to specific embodiments, the extract is obtained from a fresh plant (i.e. a plant not heated prior to the extraction process). Fresh plants include plants taken immediately following harvesting (e.g., up to an hour or several hours) for extraction as well as plants frozen immediately after harvesting (e.g. at about -70 °C to -90 °C, e.g. at -80 °C, for any required length of time) prior to extraction.

According to specific embodiments, the extract is obtained from fresh inflorescence.

According to specific embodiments, the extract is obtained from a frozen inflorescence (e.g. frozen immediately after harvesting at about -70 °C to -90 °C, e.g. at -80 °C, for any required length of time). Thus, for example, the extract may be obtained from a cryopreserved inflorescence, or from an inflorescence frozen in liquid nitrogen or in dry ice.

According to specific embodiments, the extract is obtained from an inflorescence which has not been subjected to heating (such as heating at e.g. at 120 °C to 180 °C, e.g. at 150 °C, for any length of time, such as for 1-5 hours).

According to specific embodiments, the extract is obtained from dry Cannabis inflorescence. Drying the inflorescence may be carried out using any method known in the art, such as by pulverizing with liquid nitrogen or with dry-ice/alcohol mixture.

According to specific embodiments, the dry inflorescence is obtained from the grower.

According to specific embodiments, the polar solvent comprises a polar, protic solvent (e.g., ethanol or methanol). In some embodiments, the polar solvent comprises a polar, aprotic solvent (e.g., acetone). Polar solvents suitable for use with specific embodiments of the present invention include, but are not limited to, ethanol, methanol, n-propanol, iso-propanol, a butanol, a pentanol, acetone, methylethylketone, ethylacetate, acetonitrile, tetrahydrofuran, dimethylformamide, dimethylsulfoxide, water, and combinations thereof.

According to specific embodiments, the polar solvent is ethanol (e.g. absolute ethanol i.e. above 99.8 %, or in the range of 99-70 % in water).

The concentration or amount of a polar solvent used to Cannabis inflorescence can be varied. Generally, the ratio of a Cannabis inflorescence to a polar solvent (weight to volume) is the amount of a polar solvent sufficient to extract about 70 % or more, about 75 % or more, about 85 % or more, about 90 % or more, about 95 % or more, about 97 % or more, or about 99 % or more of a composition having a cytotoxic activity. In some embodiments, the ratio of polar solvent to Cannabis inflorescence is about 1 : 2 to about 1 : 20 (w / v), e.g. about 1 : 4 to about 1 : 10 (w / v).

In particular embodiments, the extract is an ethanol extract.

In particular embodiments, absolute ethanol is added to the inflorescence at a sample-to- absolute ethanol ratio of 1:4 (w/v).

In some embodiments, the Cannabis inflorescence is contacted with a polar solvent (e.g. ethanol) for about 15 minutes or more, about 30 minutes or more, about 1 hour or more, about 2 hours or more, or about 5 hours or more.

According to specific embodiments, the Cannabis inflorescence is contacted with a polar solvent (e.g. ethanol) for about 30 minutes.

Temperature can also be controlled during the contacting. In some embodiments, the Cannabis inflorescence is contacted with a polar solvent at temperature of about 15 °C to about 35 °C, or about 20 °C to about 25 °C.

According to specific embodiments, the Cannabis inflorescence is contacted with a polar solvent (e.g. ethanol) while being constantly mixed e.g. on a shaker.

In some embodiments, the process of obtaining the composition of some embodiments of the present invention comprises isolating a liquid extract (i.e. filtered extract) from the mixture (i.e. crude extract) comprising the liquid extract and solids. Suitable means for isolating the liquid extract (i.e. filtered extract) include those known in the art of organic synthesis and include, but are not limited to, gravity filtration, suction and/or vacuum filtration, centrifuging, setting and decanting, and the like. In some embodiments, the isolating comprises filtering a liquid extract through a porous membrane, syringe, sponge, zeolite, paper, or the like having a pore size of about 1-5 pm, about 0.5-5 pm, about 0.1-5 pm, about 1-2 pm, about 0.5-2 pm, about 0.1-2 pm, about 0.5-1 pm, about 0.1-1 pm, about 0.25-0.45 pm, or about 0.1-0.5 pm (e.g. about 2 pm, about 1 pm, about 0.45 pm, or about 0.25 pm).

According to a specific embodiment, the crude extract is filtered through a 0.45-pm syringe filter such as that commercially available from Merck, Darmstadt, Germany.

According specific embodiments, process of obtaining the composition of some embodiments of the present invention comprises drying (i.e. removal of the polar solvent) and/or freezing the filtered extract following generation thereof.

The method for drying the filtered extract (i.e. removing the polar solvent) is not particularly limited, and can include solvent evaporation at a reduced pressure (e.g., sub- atmospheric pressure) and/or an elevated temperature (e.g., above about 25 °C). In some embodiments, it can be difficult to completely remove a polar solvent from a liquid extract by standard solvent removal procedures such as evaporation. In some embodiments, processes such as co-evaporation, lyophilization, and the like can be used to completely remove the polar solvent from a liquid fraction to form a dry powder, dry pellet, dry granulate, paste, and the like. According to a specific embodiment, the polar solvent is evaporated with a vacuum evaporator.

According to specific embodiments, the extract (e.g. the filtered extract) is subjected to a decarboxylation step. Decarboxylation may be effected by heating the extract in a pressure tube in the oven at 220 °C for 10 minutes.

Following generation of the filtered extract, specific embodiments of the process of obtaining the composition of some embodiments of the present invention comprises additional purification steps so as to further purify active agents from the extract.

Thus, for example, fractionating the filtered extract. Fractionating can be performed by processes such as, but not limited to: column chromatography, preparative high performance liquid chromatography ("HPLC"), flash chromatography, reduced pressure distillation, and combinations thereof.

According to a specific embodiment, fractionating is performed by HPLC or flash chromatography .

In some embodiments, fractionating comprises re-suspending the filtered extract in a polar solvent (such as methanol, as discussed above), applying the polar extract to a separation column, and isolating the Cannabis fraction by column chromatography (e.g. preparative HPLC, flow cytometry).

An eluting solvent is applied to the separation column with the polar extract to elute fractions from the polar extract. Suitable eluting solvents for use include, but are not limited to, methanol, ethanol, propanol, acetone, acetic acid, carbon dioxide, methylethyl ketone, acetonitrile, butyronitrile, carbon dioxide, ethyl acetate, tetrahydrofuran, di-iso-propylether, ammonia, triethylamine, N,N-dimethylformamide, N,N-dimethylacetamide, and the like, and combinations thereof.

According to an alternative or an additional embodiment, liquid chromatography is performed on a reverse stationary phase.

According to an alternative or an additional embodiment, liquid chromatography comprises high performance liquid chromatography (HPLC) or flash chromatography, as further described hereinabove.

The fractions or extract obtained may be immediately used or stored until further use. According to specific embodiments, the fraction or extract is kept frozen, e.g. in a freezer, until further use (e.g. at about -20 °C to -90 °C, at about -70 °C to -90 °C, e.g. at -80 °C), for any required length of time.

According to other specific embodiments, the fraction or extract is immediately used (e.g. within a few minutes e.g., up to 30 minutes).

The extracts and/or fractions may be used separately. Alternatively, different extracts (e.g. from different plants or from separate extraction procedures) may be pooled together. Likewise, different fractions (from the same extract, from different extracts, from different plants and/or from separate extraction procedures) may be pooled together.

The term “pooled” as used herein refers to collected from the liquid chromatography (e.g. HPLC, flash chromatography) either as a single fraction or a plurality of fractions.

According to specific embodiments, the composition comprising phytocannabinoids do not effect Cyclooxygenase 1 (COXI) and/or Cyclooxygenase 2 (COX2) activity, when administered as a single agent.

According to specific embodiments, the composition comprising phytocannabinoids do not significantly decrease or inhibit COXI and/or COX2 activity, when administered as a single agent.

Methods of determining COXI and COX2 activity are well known in the art and are also commercially available from e.g. Abeam, BioVision, Inc., Cayman Chemical, Enzo Life Sciences. Non-limiting commercially available kits include the Cyclooxygenase 1 (COXI) Inhibitor Screening Assay Kit (Catalog No. ab204698, abeam) and the Cyclooxygenase 2 (COX2) Inhibitor Screening Assay Kit (Catalog No. K547-100, BioVision, Inc.).

The compositions comprising phytocannabinoids of some embodiments of the invention have an anti-inflammatory effect on lung epithelial cells and/or colon epithelial cell, when administered as a single agent.

According to specific embodiments, the composition comprising phytocannabinoids has a combined additive or synergistic anti-inflammatory effect on lung epithelial cells and/or colon epithelial cells as compared to each of the phytocannabinoids CBD, CBG and THCV when administered as a single agent.

As used herein the term “anti-inflammatory effect on lung epithelial cells and/or colon epithelial cells” refers to a significant decrease in inflammation of the indicated cells in the presence of the composition in comparison to same in the absence of the composition. Such an effect may be manifested by, for example, but not limited to reduction of expression and/or secretion of pro-inflammatory cytokines which may be determined by e.g. PCR, ELISA, flow cytometry.

The compositions comprising phytocannabinoids of some embodiments of the invention have an anti-inflammatory effect on macrophages, when administered as a single agent.

According to other specific embodiments, the composition comprising phytocannabinoids does not have an anti-inflammatory effect on macrophages, when administered as a single agent.

According to specific embodiments, the composition comprising phytocannabinoids has a combined additive or synergistic anti-inflammatory effect on macrophages as compared to each of the phytocannabinoids CBD, CBG and THCV when administered as a single agent.

As used herein, the term “anti-inflammatory effect on macrophages” refers to a significant decrease in macrophage activation in the presence of the composition in comparison to same in the absence of the composition or fraction.

As used herein the term “macrophage activation” or “activating macrophages” refer to the process of stimulating a macrophage that results in cellular maturation, cytokine production, phagocytosis and/or induction of regulatory or effector functions. Methods of determining macrophage activation are well known in the art and include for example the phorbol 12- myristate 13-acetate (PMA) differentiation protocol [see e.g. Starr, T., Bauler, T. J., et al. PLoS ONE 13, e0193601 (2018)]. Such an effect may be manifested by, for example, but not limited to:

(i) macrophage polarization, which may be determined by e.g. microscopy;

(ii) macrophage phagocytosis, which may be determined by e.g. internalization of silica particles;

(iii) expression and/or secretion of pro-inflammatory cytokines, which may be determined by e.g. PCR, western blot, ELISA, flow cytometry; and/or

(iv) expression of receptors that are associated with phagocytosis, which may be determined by e.g. PCR, western blot, immuno staining.

The compositions comprising phytocannabinoids of some embodiments of the invention have an anti-inflammatory effect in edema, as corroborated by a carrageenan-induced paw edema mouse model.

According to specific embodiments, the composition comprising phytocannabinoids has a combined additive or synergistic anti-inflammatory effect in edema as compared to each of the phytocannabinoids CBD, CBG and THCV when administered as a single agent. Such an effect may be manifested by, for example, but not limited to reduction of edema, edema- associated pain or molecularly by markers such as reduction of expression and/or secretion of pro-inflammatory cytokines which may be determined by e.g. PCR, ELISA, flow cytometry.

Non-limiting Examples of pro-inflammatory cytokines, which may be used with specific embodiments of the invention, include IL-6, IL-8, IL-7, TNFa, CCL2 and CCL7.

According to specific embodiments, the pro-inflammatory cytokine is selected from the group consisting of IL-6 and IL-8.

According to a specific embodiment, the decrease is in at least 2 %, 5 %, 10 %, 30 %, 40 % or even higher say, 50 %, 60 %, 70 %, 80 %, 90 % or 100 %. According to specific embodiments, the decrease is at least 1.5 fold, at least 2 fold, at least 3 fold, at least 5 fold, at least 10 fold, or at least 20 fold as compared to same in the absence of the composition.

The combination of NSAID and composition comprising phytocannabinoids of some embodiments of the invention have a combined additive or synergistic anti-inflammatory effect (e.g. on lung epithelial cells, colon epithelial cells and/or macrophages) as compared to each of the NSAID and the composition comprising phytocannabinoids when administered as a single agent.

Consequently, according to an aspect of the present invention, there is provided a method of inhibiting inflammation, the method comprising contacting a lung epithelial cell, a colon epithelial cell and/or a macrophage with the combination of NSAID and composition comprising phytocannabinoids disclosed herein.

The combination of steroid and composition comprising phytocannabinoids of some embodiments of the invention have a combined additive or synergistic anti-inflammatory effect (e.g. on lung epithelial cells and/or colon epithelial cells) as compared to each of the steroid and the composition comprising phytocannabinoids when administered as a single agent.

Consequently, according to an aspect of the present invention, there is provided a method of inhibiting inflammation, the method comprising contacting a lung epithelial cell and/or colon epithelial cell with the combination of steroid and composition comprising phytocannabinoids disclosed herein.

According to specific embodiments, the contacting is effected in-vitro or ex- vivo.

According to other specific embodiments, the contacting is effected in-vivo.

According to specific embodiments, the method comprises determining inflammation (or anti-inflammatory effect). Methods of determining inflammation and macrophage activation are known in the art and are further described hereinabove and in the Examples section which follows.

Additionally or alternatively, the combination of NSAID and composition comprising phytocannabinoids of some embodiments of the invention have a combined additive or synergistic effect on pain reduction as compared to each of said NSAID and said composition comprising phytocannabinoids when administered as a single agent.

Methods of determining an effect on pain reduction are well known in the art and include for example the Von Frey/hot plate assay, the formalin assay, thermal assay (e.g. tail withdrawal, hot plate, tail flick).

Additionally or alternatively, the combination of NSAID and composition comprising phytocannabinoids of some embodiments of the invention have a combined additive or synergistic effect on edema reduction as compared to each of said NSAID and said composition comprising phytocannabinoids when administered as a single agent.

Methods of determining an effect on edema are well known in the art and may be corroborated for example by the carrageenan-induced paw edema mouse model.

As the combinations of composition comprising phytocannabinoids and NSAID or steroid disclosed herein are endowed with e.g. anti-inflammatory effects, specific embodiments suggest their use in treating a disease that can benefit from such treatment in a subject in need thereof.

Hence, according to an aspect of the present invention, there is provided a method of treating a disease selected from the group consisting of inflammation, pain, edema and fever in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a non-steroidal anti-inflammatory drug (NSAID) and a therapeutically effective amount of a composition comprising phytocannabinoids, wherein said phytocannabinoids comprise at least 80 % CBD and at least one of CBG and THCV, thereby treating the disease in the subject.

According to an additional or an alternative aspect of the present invention, there is provided a non-steroidal anti-inflammatory drug (NSAID) and a composition comprising phytocannabinoids, wherein said phytocannabinoids comprise at least 80 % CBD and at least one of CBG and THCV, for use in treating a disease selected from the group consisting of inflammation, pain, edema and fever in a subject in need thereof.

According to an additional or an alternative aspect of the present invention, there is provided a method of treating a disease selected from the group consisting of inflammation, pain and fever in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a non-steroidal anti-inflammatory drug (NSAID) and a therapeutically effective amount of a composition comprising phytocannabinoids, wherein said phytocannabinoids comprise at least 80 % CBD and at least one of CBG and THCV, thereby treating the disease in the subject.

According to an additional or an alternative aspect of the present invention, there is provided a non-steroidal anti-inflammatory drug (NSAID) and a composition comprising phytocannabinoids, wherein said phytocannabinoids comprise at least 80 % CBD and at least one of CBG and THCV, for use in treating a disease selected from the group consisting of inflammation, pain and fever in a subject in need thereof.

According to an additional or an alternative aspect of the present invention, there is provided a method of treating a disease selected from the group consisting of respiratory inflammation and cytokine storm in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a steroid and a therapeutically effective amount of a composition comprising phytocannabinoids, wherein said phytocannabinoids comprise at least 80 % CBD and at least one of CBG and THCV, thereby treating the disease in the subject.

According to an additional or an alternative aspect of the present invention, there is provided a steroid and a composition comprising phytocannabinoids, wherein said phytocannabinoids comprise at least 80 % CBD and at least one of CBG and THCV, for use in treating a disease selected from the group consisting of respiratory inflammation and cytokine storm in a subject in need thereof.

As used herein, the term “subject” or “subject in need thereof’ includes mammals, preferably human beings at any age or gender which suffer from the pathology e.g. inflammation, pain, edema, fever, cytokine storm. According to specific embodiments, this term encompasses individuals who are at risk to develop the pathology.

According to specific embodiments, the subject is diagnosed with the pathology.

According to specific embodiments, the subject exhibit at least one symptom of the disease.

According to specific embodiments, the subject does not have cancer or is not diagnosed with cancer.

As used herein, the term “treating” refers to curing, reversing, attenuating, alleviating, minimizing, suppressing or halting the deleterious effects of a disease or disorder (e.g. inflammation, pain, edema, fever, cytokine storm). Those of skill in the art will understand that various methodologies and assays can be used to assess the development of a pathology, and similarly, various methodologies and assays may be used to assess the reduction, remission or regression of a pathology.

According to a specific embodiment, treating is preventing.

As used herein, the term “preventing” refers to keeping a disease, disorder or condition from occurring in a subject who may be at risk for the disease, but has not yet been diagnosed as having the disease.

According to specific embodiments, the disease is fever.

As used herein, the term “fever” is defined as a body temperature that is above normal body temperature set point for the organism. Generally, a human subject is considered to have a fever if the body temperature is at or above 38° C rectally, 37.5° C orally, or 37.2° C under armpit.

Thus, according to specific embodiments, the fever is a body temperature of above 37° C.

According to specific embodiments, the fever is a body temperature of above 38° C, above 39° C or above 40° C.

According to specific embodiments, the disease is pain.

As used herein the term "pain" refers to all types of pain.

Non-limiting examples of pain include, postherpetic neuralgia, diabetic neuropathy, pruritus, psoriasis, cluster headache, postmastectomy pain syndrome, rhinopathy, oral mucositis, cutaneous allergy, detrusor hyperreflexia, loin pain/hematuria syndrome, neck pain, amputation stump pain, reflex sympathetic dystrophy, pain due to skin tumor and arthritis including rheumatoid arthritis, osteoarthritis, headache, post-surgical pain, oral pain, pain caused by injury, vulvodynia, interstitial cystitis, rhinitis, burning mouth syndrome, oral mucositis, herpes neuralgia, dermatitis, pruritis, tinnitus, phantom or amputation stump pain, acquired immune deficiency syndrome neuropathy, back pain, opioid-resistant pain, visceral pain, bone injury pain, pain during labor and delivery, pain resulting from bums (including sunburn), post-partum pain, migraine, angina pain, genitourinary tract-related pain including cystitis, dysmenorrhea.

According to specific embodiments, the pain is acute pain.

According to specific embodiments, the pain is associated with edema.

According to other specific embodiments, the pain is chronic pain.

According to specific embodiments, the pain is nociceptive pain. As used herein, the term “nociceptive pain” involves direct activation of the nociceptors, such as mechanical, chemical, and thermal receptors, found in various tissues, such as bone, muscle, vessels, viscera, and cutaneous and connective tissue. Nociceptive pain occurs in the setting of an undamaged nervous system, e.g. the afferent somatosensory pathways are considered intact. Non-limiting examples of nociceptive pain include post-operative pain, cluster headaches, dental pain, surgical pain, pain resulting from bums, sunburns, exposure to extremely cold temperatures, bruises, fractures, post-partum pain, angina pain, genitourinary tract related pain, damage by contact with toxic of hazardous chemicals.

According to specific embodiments, the pain is neuropathic pain.

As used herein, the term “neuropathic pain” refers to pain initiated or caused by injury to or dysfunction of the central or peripheral nervous system. According to specific embodiments, the neuropathic pain has typical symptoms such as hyperesthesia (enhanced sensitivity to a natural stimulus), hyperalgesia (abnormal sensitivity to pain), allodynia (widespread tenderness, characterized by hypersensitivity to non-noxious tactile stimuli), and/or spontaneous burning pain. Non-limiting examples of neuropathic pain include, but are not limited to, medication- induced neuropathy and nerve compression syndromes such as carpal tunnel, radiculopathy due to vertebral disk herniation, post- amputation syndromes such as stump pain and phantom limb pain, metabolic disease such as diabetic neuropathy, viral-related neuropathy including herpes zoster and human immunodeficiency virus (HIV) disease, tumor infiltration leading to irritation or compression of nervous tissue, neuritis, as after cancer radiotherapy, autonomic dysfunction from complex regional pain syndrome (CRPS), trigeminal neuralgia, postherpetic neuralgia, and the reflex sympathetic dystrophies including causalgia, mononeuropathies, peripheral nerve injury, central nerve injury, opioid resistant neuropathic pain, bone injury pain, pain during labor and delivery, non-specific lower back pain, multiple sclerosis-related pain, fibromyalgia, acute and chronic inflammatory demyelinating poly radiculopathy, alcoholic polyneuropathy, segmental neuropathy, ischemic optic neuropathy, geniculate neuralgia, occipital neuralgia, periodic migrainous neuralgia, chemotherapy-induced polyneuropathy, brachial plexus avulsion, post-surgical neuropathy including post-mastectomy pain or post-thoracotomy pain, idiopathic sensory neuropathy, nutrition deficiency-related neuropathy, phantom limb pain, post-radiation plexopathy, radiculopathy, for example, sciatica, toxin exposure-related neuropathy, post- traumatic neuralgia, compressive myelopathy, Parkinson's disease-related neuropathy, post- ischemic myelopathy, post- radiation myelopathy, post-stroke pain, post-traumatic spinal cord injury pain, temporomandibular disorder, myofascial pain, and syringomyelia. According to specific embodiments, the pain is selected from the group consisting of inflammatory pain, acute pain, chronic pain, arthritis, pain following nerve injury, cancer associated pain, chemotherapy associated pain, migraine, low back pain and toothache.

According to specific embodiments, the pain is not inflammatory pain.

According to specific embodiments, the pain is not associated with inflammation.

According to specific embodiments, the pain is not associated with inflammation in the vicinity of the origin of the pain.

According to specific embodiments, the pain is not associated with vascular inflammation.

According to specific embodiments, the disease is edema.

As used herein, the term “edema” refers to the abnormal accumulation of fluid under the skin or in one or more body cavities leading to swelling of a selected tissue. Causes may include, but not limited to, venous insufficiency, heart failure, kidney problems, low protein levels, liver problems, deep vein thrombosis, infections, angioedema, certain medications, bums, injuries, trauma, frostbite, surgery, curvature, fractures, hereditary autoimmune disease and compromised lymphatic system. Causes of edema which are generalized to the whole body can cause edema in multiple organs and peripherally. For example, severe heart failure can cause pulmonary edema, pleural effusions, ascites and peripheral edema. Organ-specific edema types include, but not limited to, brain edema, pulmonary edema, pleural edema, corneal edema, periorbital edema, cutaneous edema, myxedema, hand edema, legs (e.g. feet) edema, and lymphedema.

According to specific embodiments, the edema comprises hand edema and/or leg edema.

According to specific embodiments, the edema is a drug-induced edema.

According to specific embodiments, the disease is inflammation.

Inflammation or inflammatory diseases encompassed by specific embodiments of the invention include, but are not limited to, chronic inflammatory diseases and acute inflammatory diseases.

According to specific embodiments, the inflammatory disease is an acute inflammatory disease.

According to specific embodiments, the inflammatory disease is associated with edema.

Inflammatory diseases associated with hypersensitivity

Examples of hypersensitivity include, but are not limited to, Type I hypersensitivity, Type II hypersensitivity, Type III hypersensitivity, Type IV hypersensitivity, immediate hypersensitivity, antibody mediated hypersensitivity, immune complex mediated hypersensitivity, T lymphocyte mediated hypersensitivity and DTH.

Type I or immediate hypersensitivity, such as asthma.

Type II hypersensitivity include, but are not limited to, rheumatoid diseases, rheumatoid autoimmune diseases, rheumatoid arthritis (Krenn V. et al., Histol Histopathol 2000 Jul; 15 (3):791), spondylitis, ankylosing spondylitis (Jan Voswinkel et al., Arthritis Res 2001; 3 (3): 189), systemic diseases, systemic autoimmune diseases, systemic lupus erythematosus (Erikson J. et al., Immunol Res 1998; 17 (l-2):49), sclerosis, systemic sclerosis (Renaudineau Y. et al., Clin Diagn Lab Immunol. 1999 Mar;6 (2): 156); Chan OT. et al., Immunol Rev 1999 Jun; 169: 107), glandular diseases, glandular autoimmune diseases, pancreatic autoimmune diseases, diabetes, Type I diabetes (Zimmet P. Diabetes Res Clin Pract 1996 Oct;34 Suppl:S125), thyroid diseases, autoimmune thyroid diseases, Graves’ disease (Orgiazzi J. Endocrinol Metab Clin North Am 2000 Jun;29 (2):339), thyroiditis, spontaneous autoimmune thyroiditis (Braley-Mullen H. and Yu S, J Immunol 2000 Dec 15; 165 (12):7262), Hashimoto’s thyroiditis (Toyoda N. et al., Nippon Rinsho 1999 Aug;57 (8):181O), myxedema, idiopathic myxedema (Mitsuma T. Nippon Rinsho. 1999 Aug;57 (8): 1759); autoimmune reproductive diseases, ovarian diseases, ovarian autoimmunity (Garza KM. et al., J Reprod Immunol 1998 Feb;37 (2):87), autoimmune anti-sperm infertility (Diekman AB. et al., Am J Reprod Immunol. 2000 Mar;43 (3): 134), repeated fetal loss (Tincani A. et al., Lupus 1998;7 Suppl 2:S 107-9), neurodegenerative diseases, neurological diseases, neurological autoimmune diseases, multiple sclerosis (Cross AH. et al., J Neuroimmunol 2001 Jan 1;112 (1-2):1), Alzheimer’s disease (Oron L. et al., J Neural Transm Suppl. 1997;49:77), myasthenia gravis (Infante AJ. And Kraig E, Int Rev Immunol 1999;18 (l-2):83), motor neuropathies (Kornberg AJ. J Clin Neurosci. 2000 May;7 (3): 191), Guillain-Barre syndrome, neuropathies and autoimmune neuropathies (Kusunoki S. Am J Med Sci. 2000 Apr;319 (4):234), myasthenic diseases, Lambert-Eaton myasthenic syndrome (Takamori M. Am J Med Sci. 2000 Apr;319 (4):204), paraneoplastic neurological diseases, cerebellar atrophy, paraneoplastic cerebellar atrophy, non-paraneoplastic stiff man syndrome, cerebellar atrophies, progressive cerebellar atrophies, encephalitis, Rasmussen’s encephalitis, amyotrophic lateral sclerosis, Sydeham chorea, Gilles de la Tourette syndrome, polyendocrinopathies, autoimmune polyendocrinopathies (Antoine JC. and Honnorat J. Rev Neurol (Paris) 2000 Jan;156 (1):23); neuropathies, dysimmune neuropathies (Nobile-Orazio E. et al., Electroencephalogr Clin Neurophysiol Suppl 1999;50:419); neuromyotonia, acquired neuromyotonia, arthrogryposis multiplex congenita (Vincent A. et al., Ann N Y Acad Sci. 1998 May 13 ;841 :482), cardiovascular diseases, cardiovascular autoimmune diseases, atherosclerosis (Matsuura E. et al., Lupus. 1998;7 Suppl 2:S135), myocardial infarction (Vaarala O. Lupus. 1998;7 Suppl 2:S132), thrombosis (Tincani A. et al., Lupus 1998;7 Suppl 2:S 107-9), granulomatosis, Wegener’s granulomatosis, arteritis, Takayasu’s arteritis and Kawasaki syndrome (Praprotnik S. et al., Wien Klin Wochenschr 2000 Aug 25; 112 (15-16):660); anti-factor VIII autoimmune disease (Lacroix-Desmazes S. et al., Semin Thromb Hemost.2000;26 (2): 157); vasculitises, necrotizing small vessel vasculitises, microscopic polyangiitis, Churg and Strauss syndrome, glomerulonephritis, pauci-immune focal necrotizing glomerulonephritis, crescentic glomerulonephritis (Noel LH. Ann Med Interne (Paris). 2000 May; 151 (3): 178); antiphospholipid syndrome (Flamholz R. et al., J Clin Apheresis 1999; 14 (4): 171); heart failure, agonist-like 0- adrenoceptor antibodies in heart failure (Wallukat G. et al., Am J Cardiol. 1999 Jun 17;83 (12A):75H), thrombocytopenic purpura (Moccia F. Ann Ital Med Int. 1999 Apr-Jun; 14 (2): 114); hemolytic anemia, autoimmune hemolytic anemia (Efremov DG. et al., Leuk Lymphoma 1998 Jan;28 (3-4):285), gastrointestinal diseases, autoimmune diseases of the gastrointestinal tract, intestinal diseases, chronic inflammatory intestinal disease (Garcia Herola A. et al., Gastroenterol Hepatol. 2000 Jan;23 (1): 16), celiac disease (Landau YE. and Shoenfeld Y. Harefuah 2000 Jan 16;138 (2):122), autoimmune diseases of the musculature, myositis, autoimmune myositis, Sjogren’s syndrome (Feist E. et al., Int Arch Allergy Immunol 2000 Sep;123 (1):92); smooth muscle autoimmune disease (Zauli D. et al., Biomed Pharmacother 1999 Jun;53 (5-6):234), hepatic diseases, hepatic autoimmune diseases, autoimmune hepatitis (Manns MP. J Hepatol 2000 Aug;33 (2):326) and primary biliary cirrhosis (Strassburg CP. et al., Eur J Gastroenterol Hepatol. 1999 Jun;l l (6):595).

Type IV or T cell mediated hypersensitivity, include, but are not limited to, rheumatoid diseases, rheumatoid arthritis (Tisch R, McDevitt HO. Proc Natl Acad Sci U S A 1994 Jan 18;91 (2):437), systemic diseases, systemic autoimmune diseases, systemic lupus erythematosus (Datta SK., Lupus 1998;7 (9):591), glandular diseases, glandular autoimmune diseases, pancreatic diseases, pancreatic autoimmune diseases, Type 1 diabetes (Castano L. and Eisenbarth GS. Ann. Rev. Immunol. 8:647); thyroid diseases, autoimmune thyroid diseases, Graves’ disease (Sakata S. et al., Mol Cell Endocrinol 1993 Mar;92 (1):77); ovarian diseases (Garza KM. et al., J Reprod Immunol 1998 Feb;37 (2):87), prostatitis, autoimmune prostatitis (Alexander RB. et al., Urology 1997 Dec;50 (6):893), polyglandular syndrome, autoimmune polyglandular syndrome, Type I autoimmune polyglandular syndrome (Hara T. et al., Blood. 1991 Mar 1;77 (5):1127), neurological diseases, autoimmune neurological diseases, multiple sclerosis, neuritis, optic neuritis (Soderstrom M. et al., J Neurol Neurosurg Psychiatry 1994 May;57 (5):544), myasthenia gravis (Oshima M. et al., Eur J Immunol 1990 Dec;20 (12):2563), stiff-man syndrome (Hiemstra HS. et al., Proc Natl Acad Sci U S A 2001 Mar 27;98 (7):3988), cardiovascular diseases, cardiac autoimmunity in Chagas’ disease (Cunha-Neto E. et al., J Clin Invest 1996 Oct 15;98 (8): 1709), autoimmune thrombocytopenic purpura (Semple JW. et al., Blood 1996 May 15;87 (10):4245), anti-helper T lymphocyte autoimmunity (Caporossi AP. et al., Viral Immunol 1998;11 (1):9), hemolytic anemia (Sallah S. et al., Ann Hematol 1997 Mar;74 (3): 139), hepatic diseases, hepatic autoimmune diseases, hepatitis, chronic active hepatitis (Franco A. et al., Clin Immunol Immunopathol 1990 Mar;54 (3):382), biliary cirrhosis, primary biliary cirrhosis (Jones DE. Clin Sci (Colch) 1996 Nov;91 (5):551), nephric diseases, nephric autoimmune diseases, nephritis, interstitial nephritis (Kelly CJ. J Am Soc Nephrol 1990 Aug;l (2): 140), connective tissue diseases, ear diseases, autoimmune connective tissue diseases, autoimmune ear disease (Yoo TJ. et al., Cell Immunol 1994 Aug;157 (1):249), disease of the inner ear (Gloddek B. et al., Ann N Y Acad Sci 1997 Dec 29;830:266), skin diseases, cutaneous diseases, dermal diseases, bullous skin diseases, pemphigus vulgaris, bullous pemphigoid and pemphigus foliaceus.

Examples of delayed type hypersensitivity include, but are not limited to, contact dermatitis and drug eruption.

Examples of types of T lymphocyte mediating hypersensitivity include, but are not limited to, helper T lymphocytes and cytotoxic T lymphocytes.

Examples of helper T lymphocyte-mediated hypersensitivity include, but are not limited to, Thl lymphocyte mediated hypersensitivity and Th2 lymphocyte mediated hypersensitivity.

Autoimmune diseases

Include, but are not limited to, cardiovascular diseases, rheumatoid diseases, glandular diseases, gastrointestinal diseases, cutaneous diseases, hepatic diseases, neurological diseases, muscular diseases, nephric diseases, diseases related to reproduction, connective tissue diseases and systemic diseases.

Examples of autoimmune cardiovascular diseases include, but are not limited to atherosclerosis (Matsuura E. et al., Lupus. 1998;7 Suppl 2:S135), myocardial infarction (Vaarala O. Lupus. 1998;7 Suppl 2:S132), thrombosis (Tincani A. et al., Lupus 1998;7 Suppl 2:S 107-9), Wegener’s granulomatosis, Takayasu’s arteritis, Kawasaki syndrome (Praprotnik S. et al., Wien Klin Wochenschr 2000 Aug 25; 112 (15-16):660), anti-factor VIII autoimmune disease (Lacroix- Desmazes S. et al., Semin Thromb Hemost.2000;26 (2): 157), necrotizing small vessel vasculitis, microscopic polyangiitis, Churg and Strauss syndrome, pauci-immune focal necrotizing and crescentic glomerulonephritis (Noel LH. Ann Med Interne (Paris). 2000 May; 151 (3): 178), antiphospholipid syndrome (Flamholz R. et al., J Clin Apheresis 1999; 14 (4): 171), antibody- induced heart failure (Wallukat G. et al., Am J Cardiol. 1999 Jun 17;83 (12A):75H), thrombocytopenic purpura (Moccia F. Ann Ital Med Int. 1999 Apr-Jun; 14 (2): 114; Semple JW. et al., Blood 1996 May 15;87 (10):4245), autoimmune hemolytic anemia (Efremov DG. et al., Leuk Lymphoma 1998 Jan;28 (3-4):285; Sallah S. et al., Ann Hematol 1997 Mar;74 (3): 139), cardiac autoimmunity in Chagas’ disease (Cunha-Neto E. et al., J Clin Invest 1996 Oct 15;98 (8): 1709) and anti-helper T lymphocyte autoimmunity (Caporossi AP. et al., Viral Immunol 1998;11 (1):9).

Examples of autoimmune rheumatoid diseases include, but are not limited to rheumatoid arthritis (Krenn V. et al., Histol Histopathol 2000 Jul;15 (3):791 ; Tisch R, McDevitt HO. Proc Natl Acad Sci units S A 1994 Jan 18;91 (2):437) and ankylosing spondylitis (Jan Voswinkel et al., Arthritis Res 2001; 3 (3): 189).

Examples of autoimmune glandular diseases include, but are not limited to, pancreatic disease, Type I diabetes, thyroid disease, Graves’ disease, thyroiditis, spontaneous autoimmune thyroiditis, Hashimoto’s thyroiditis, idiopathic myxedema, ovarian autoimmunity, autoimmune anti-sperm infertility, autoimmune prostatitis and Type I autoimmune polyglandular syndrome, diseases include, but are not limited to autoimmune diseases of the pancreas, Type 1 diabetes (Castano L. and Eisenbarth GS. Ann. Rev. Immunol. 8:647; Zimmet P. Diabetes Res Clin Pract 1996 Oct;34 Suppl:S125), autoimmune thyroid diseases, Graves’ disease (Orgiazzi J. Endocrinol Metab Clin North Am 2000 Jun;29 (2):339; Sakata S. et al., Mol Cell Endocrinol 1993 Mar;92 (1):77), spontaneous autoimmune thyroiditis (Braley-Mullen H. and Yu S, J Immunol 2000 Dec 15;165 (12):7262), Hashimoto’s thyroiditis (Toyoda N. et al., Nippon Rinsho 1999 Aug;57 (8):181O), idiopathic myxedema (Mitsuma T. Nippon Rinsho. 1999 Aug;57 (8):1759), ovarian autoimmunity (Garza KM. et al., J Reprod Immunol 1998 Feb;37 (2):87), autoimmune anti-sperm infertility (Diekman AB. et al., Am J Reprod Immunol. 2000 Mar;43 (3): 134), autoimmune prostatitis (Alexander RB. et al., Urology 1997 Dec;50 (6):893) and Type I autoimmune polyglandular syndrome (Hara T. et al., Blood. 1991 Mar 1;77 (5): 1127).

Examples of autoimmune gastrointestinal diseases include, but are not limited to, chronic inflammatory intestinal diseases (Garcia Herola A. et al., Gastroenterol Hepatol. 2000 Jan;23 (1): 16), celiac disease (Landau YE. and Shoenfeld Y. Harefuah 2000 Jan 16;138 (2):122), colitis, ileitis and Crohn’s disease.

Examples of autoimmune cutaneous diseases include, but are not limited to, autoimmune bullous skin diseases, such as, but are not limited to, pemphigus vulgaris, bullous pemphigoid and pemphigus foliaceus. Examples of autoimmune hepatic diseases include, but are not limited to, hepatitis, autoimmune chronic active hepatitis (Franco A. et al., Clin Immunol Immunopathol 1990 Mar;54

(3):382), primary biliary cirrhosis (Jones DE. Clin Sci (Colch) 1996 Nov;91 (5):551; Strassburg CP. et al., Eur J Gastroenterol Hepatol. 1999 Jun; 11 (6):595) and autoimmune hepatitis (Manns MP. J Hepatol 2000 Aug;33 (2):326).

Examples of autoimmune neurological diseases include, but are not limited to, multiple sclerosis (Cross AH. et al., J Neuroimmunol 2001 Jan 1;112 (1-2): 1), Alzheimer’s disease (Oron L. et al., J Neural Transm Suppl. 1997;49:77), myasthenia gravis (Infante AJ. And Kraig E, Int Rev Immunol 1999;18 (l-2):83; Oshima M. et al., Eur J Immunol 1990 Dec;20 (12):2563), neuropathies, motor neuropathies (Kornberg AJ. J Clin Neurosci. 2000 May;7 (3): 191); Guillain- Barre syndrome and autoimmune neuropathies (Kusunoki S. Am J Med Sci. 2000 Apr;319

(4):234), myasthenia, Lambert-Eaton myasthenic syndrome (Takamori M. Am J Med Sci. 2000 Apr;319 (4):204); paraneoplastic neurological diseases, cerebellar atrophy, paraneoplastic cerebellar atrophy and stiff-man syndrome (Hiemstra HS. et al., Proc Natl Acad Sci units S A 2001 Mar 27;98 (7):3988); non-paraneoplastic stiff man syndrome, progressive cerebellar atrophies, encephalitis, Rasmussen’s encephalitis, amyotrophic lateral sclerosis, Sydeham chorea, Gilles de la Tourette syndrome and autoimmune polyendocrinopathies (Antoine JC. and Honnorat J. Rev Neurol (Paris) 2000 Jan;156 (1):23); dysimmune neuropathies (Nobile- Orazio E. et al., Electroencephalogr Clin Neurophysiol Suppl 1999;50:419); acquired neuromyotonia, arthrogryposis multiplex congenita (Vincent A. et al., Ann N Y Acad Sci. 1998 May 13 ;841 :482), neuritis, optic neuritis (Soderstrom M. et al., J Neurol Neurosurg Psychiatry 1994 May;57 (5):544) and neurodegenerative diseases.

Examples of autoimmune muscular diseases include, but are not limited to, myositis, autoimmune myositis and primary Sjogren’s syndrome (Feist E. et al., Int Arch Allergy Immunol 2000 Sep;123 (1):92) and smooth muscle autoimmune disease (Zauli D. et al., Biomed Pharmacother 1999 Jun;53 (5-6):234).

Examples of autoimmune nephric diseases include, but are not limited to, nephritis and autoimmune interstitial nephritis (Kelly CJ. J Am Soc Nephrol 1990 Aug;l (2): 140).

Examples of autoimmune diseases related to reproduction include, but are not limited to, repeated fetal loss (Tincani A. et al., Lupus 1998;7 Suppl 2:S 107-9).

Examples of autoimmune connective tissue diseases include, but are not limited to, ear diseases, autoimmune ear diseases (Yoo TJ. et al., Cell Immunol 1994 Aug;157 (1):249) and autoimmune diseases of the inner ear (Gloddek B. et al., Ann N Y Acad Sci 1997 Dec 29;830:266).

Examples of autoimmune systemic diseases include, but are not limited to, systemic lupus erythematosus (Erikson J. et al., Immunol Res 1998; 17 (l-2):49) and systemic sclerosis (Renaudineau Y. et al., Clin Diagn Lab Immunol. 1999 Mar;6 (2): 156); Chan OT. et al., Immunol Rev 1999 Jun; 169: 107).

Infectious diseases

Examples of infectious diseases include, but are not limited to, chronic infectious diseases, subacute infectious diseases, acute infectious diseases, viral diseases, bacterial diseases, protozoan diseases, parasitic diseases, fungal diseases, mycoplasma diseases and prion diseases, as further described in details hereinbelow.

Graft rejection diseases

Examples of diseases associated with transplantation of a graft include, but are not limited to, graft rejection, chronic graft rejection, subacute graft rejection, hyperacute graft rejection, acute graft rejection and graft versus host disease.

Allergic diseases

Examples of allergic diseases include, but are not limited to, asthma, hives, urticaria, pollen allergy, dust mite allergy, venom allergy, cosmetics allergy, latex allergy, chemical allergy, drug allergy, insect bite allergy, animal dander allergy, stinging plant allergy, poison ivy allergy and food allergy.

Cancerous diseases

Examples of cancer include but are not limited to carcinoma, lymphoma, blastoma, sarcoma, and leukemia. Particular examples of cancerous diseases but are not limited to: Myeloid leukemia such as Chronic myelogenous leukemia. Acute myelogenous leukemia with maturation. Acute promyelocytic leukemia, Acute nonlymphocytic leukemia with increased basophils, Acute monocytic leukemia. Acute myelomonocytic leukemia with eosinophilia; Malignant lymphoma, such as Birkitt's Non-Hodgkin's; Lymphoctyic leukemia, such as Acute lumphoblastic leukemia. Chronic lymphocytic leukemia; Myeloproliferative diseases, such as Solid tumors Benign Meningioma, Mixed tumors of salivary gland, Colonic adenomas; Adenocarcinomas, such as Small cell lung cancer, Kidney, Uterus, Prostate, Bladder, Ovary, Colon, Sarcomas, Liposarcoma, myxoid, Synovial sarcoma, Rhabdomyosarcoma (alveolar), Extraskeletel myxoid chonodrosarcoma, Ewing's tumor; other include Testicular and ovarian dysgerminoma, Retinoblastoma, Wilms' tumor, Neuroblastoma, Malignant melanoma, Mesothelioma, breast, skin, prostate, and ovarian.

According to specific embodiments, the disease is not cancer.

According to specific embodiments, the disease is not in co-morbidity with cancer.

According to specific embodiments, the inflammation is selected from the group consisting of inflammation of the respiratory system, colon inflammation, rheumatoid arthritis, osteoarthritis, extra-articular rheumatism and ankylosing spondylitis.

According to specific embodiments, the inflammation is inflammation of the respiratory system.

According to specific embodiments, the inflammation of the respiratory system is lung inflammation.

Non-limiting examples of inflammation of the respiratory system diseases include rhinitis, otitis media, laryngopharyngitis, tonsillitis, pneumonia, asthma, COPD (Chronic Obstructive Pulmonary Disease), lung cancer, respiratory viral infection, bronchiectasis, acute pulmonary inflammation, acute lung injury (ALI), acute respiratory distress syndrome (ARDS), sarcoidosis, cystic fibrosis.

According to specific embodiments, the inflammation is colon inflammation.

Non-limiting examples of colon inflammation diseases include inflammatory bowel disease (IBD) e.g. ulcerative colitis, Crohn’s disease; and colon cancer.

According to specific embodiments, the disease (e.g. inflammation) is at a stage prior to cytokine storm.

According to other specific embodiments, the disease (e.g. inflammation) is at a stage of cytokine storm.

According to specific embodiments, the disease is cytokine storm.

As used herein, the term “cytokine storm”, also known as “cytokine release syndrome” or “inflammatory cascade”, refers to a disease characterized by dysregulated of pro-inflammatory cytokines, which causes an excessive production of cytokines leading to a positive feedback loop between cytokines and mediator-releasing cells. Non-limiting examples of symptoms of cytokine storm may include high fever, swelling and redness, extreme fatigue and nausea. Cytokine storm may progress to shock, disseminated intravascular coagulation (DIC), and multiple organ failure through neutrophil activation, blood coagulation mechanism activation, and vasodilation. According to specific embodiments, the inflammation is associated with infectious disease.

As used herein, the term “associated with infectious disease” means that a pathogen infection leads to the disease.

According to specific embodiments, the infectious disease is not in co-morbidity with other inflammatory diseases.

As used herein, the term “infection” or "infectious disease" refers to a disease induced by a pathogen. Non-limiting specific examples of pathogens include, viral pathogens, bacterial pathogens e.g., intracellular mycobacterial pathogens (such as, for example, Mycobacterium tuberculosis), intracellular bacterial pathogens (such as, for example, Listeria monocytogenes), intracellular protozoan pathogens (such as, for example, Leishmania and Trypanosoma), parasitic diseases, fungal diseases, prion diseases.

Methods of analyzing infection are well known in the art and are either based on serology, protein markers, or nucleic acid assays.

According to specific embodiments, the infectious disease is associated with a viral infection.

As used herein, the term “associated with a viral infection” means that a viral infection leads to the disease.

Specific types of viral pathogens causing infectious diseases treatable according to specific embodiments of the present invention include, but are not limited to, retroviruses, circoviruses, parvoviruses, papovaviruses, adenoviruses, herpesviruses, iridoviruses, poxviruses, hepadnaviruses, picornaviruses, caliciviruses, togaviruses, flaviviruses, reoviruses, orthomyxoviruses, paramyxoviruses, rhabdoviruses, bunyaviruses, coronaviruses, arenaviruses, and filoviruses.

Non-limiting examples of viral infections include human immunodeficiency virus (HIV)- induced acquired immunodeficiency syndrome (AIDS), coronavirus, influenza, rhinoviral infection, viral meningitis, Epstein-Barr virus (EBV) infection, hepatitis A, B or C virus infection, measles, papilloma virus infection/warts, cytomegalovirus (CMV) infection, Herpes simplex virus infection, yellow fever, Ebola virus infection, rabies, etc.

According to specific embodiments, the viral infection is respiratory (or lung) viral infection. Non-limiting examples of respiratory viral infections include a Corona virus infection, a respiratory syncytial virus (RSV) infection, an influenza virus infection, a parainfluenza virus infection, an adenovirus infection and a rhinovirus infection.

According to specific embodiments, the viral infection is a Corona virus infection.

According to specific embodiments, a clinical manifestation of Corona virus infection includes symptoms selected from the group consisting of inflammation in the lung, alveolar damage, ARDS, fever, cough, shortness of breath, diarrhea, organ failure, pneumonia, cytokine storm, septic shock and/or blood clots.

As used herein, “Corona virus” refers to enveloped positive- stranded RNA viruses that belong to the family Coronaviridae and the order Nidovirales.

Examples of Corona viruses which are contemplated herein include, but are not limited to, 229E, NL63, OC43, and HKU1 with the first two classified as antigenic group 1 and the latter two belonging to group 2, typically leading to an upper respiratory tract infection manifested by common cold symptoms.

However, Corona viruses, which are zoonotic in origin, can evolve into a strain that can infect human beings leading to fatal illness. Thus, particular examples of Corona viruses contemplated herein are SARS-CoV, Middle East respiratory syndrome Coronavirus (MERS- CoV), and the recently identified SARS-CoV-2 [causing 2019-nCoV (also referred to as “COVID-19”)].

It would be appreciated that any Corona virus strain is contemplated herein even though SARS-CoV-2 is emphasized in a detailed manner.

According to specific embodiments, the Corona virus is SARS-CoV-2.

As used herein the SARS-CoV-2 includes any variants and mutants thereof including, but not limited to, the B.1.1.7 (Alpha), B.1.351 (Beta), B.1.617.2 (Delta), P.l (Gamma), B.1.526 (Iota), B.1.427 (Epsilon), B.1.429 (Epsilon), B.1.617 (Kappa, Delta), B.1.525 (Eta) and P.2 (Zeta).

According to specific embodiments, the combination of agents disclosed herein can be used alone or in combination with other established or experimental therapeutic/prophylactic regimen to the disease. Hence, according to specific embodiments, the composition comprising phytocannabinoids and NSAID or steroid disclosed herein are provided to the individual with additional active agents to achieve an improved therapeutic or preventive effect as compared to treatment with each agent by itself. Thus, for example, for treating a Corona virus infection the combination of agents disclosed herein may be administered in conjunction with e.g. mechanical ventilation, neuromuscular blockers, extracorporeal membrane oxygenation (ECMO), anti-viral drug, anti-fungal drug, anti-bacterial drug, immune-globulin treatment, vaccine. In such therapy, measures (e.g., dosing and selection of the complementary agent) are taken to prevent adverse side effects which may be associated with combination therapies.

Each of the compositions comprising phytocannabinoids, NSAID, steroid or combination thereof described herein can be administered to an organism per se, or in a pharmaceutical composition where it is mixed with suitable carriers or excipients.

As used herein, a "pharmaceutical composition" refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.

Herein the term "active ingredient" refers to the cannabis derived active ingredients e.g. phytocannabinoids, NSAID and/or steroid accountable for the biological effect.

According to specific embodiments, the cannabis derived active ingredients are the only active ingredients in the composition comprising phytocannabinoids.

According to specific embodiments, the phytocannabinoids are the only active ingredients in the composition comprising phytocannabinoids.

According to specific embodiments, the cannabis derived active ingredients e.g. phytocannabinoids, and the NSAID or steroid are the only active ingredients in the combination of agents or article of manufacture disclosed herein.

Hereinafter, the phrases "physiologically acceptable carrier" and "pharmaceutically acceptable carrier" which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. An adjuvant is included under these phrases.

Herein the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

Techniques for formulation and administration of drugs may be found in “Remington’s Pharmaceutical Sciences,” Mack Publishing Co., Easton, PA, latest edition, which is incorporated herein by reference.

Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intracardiac, e.g., into the right or left ventricular cavity, into the common coronary artery, intravenous, intraperitoneal, intranasal, or intraocular injections.

Conventional approaches for drug delivery to the central nervous system (CNS) include: neurosurgical strategies (e.g., intracerebral injection or intracerebro ventricular infusion); molecular manipulation of the agent (e.g., production of a chimeric fusion protein that comprises a transport peptide that has an affinity for an endothelial cell surface molecule in combination with an agent that is itself incapable of crossing the BBB) in an attempt to exploit one of the endogenous transport pathways of the BBB; pharmacological strategies designed to increase the lipid solubility of an agent (e.g., conjugation of water-soluble agents to lipid or cholesterol carriers); and the transitory disruption of the integrity of the BBB by hyperosmotic disruption (resulting from the infusion of a mannitol solution into the carotid artery or the use of a biologically active agent such as an angiotensin peptide). However, each of these strategies has limitations, such as the inherent risks associated with an invasive surgical procedure, a size limitation imposed by a limitation inherent in the endogenous transport systems, potentially undesirable biological side effects associated with the systemic administration of a chimeric molecule comprised of a carrier motif that could be active outside of the CNS, and the possible risk of brain damage within regions of the brain where the BBB is disrupted, which renders it a suboptimal delivery method.

Alternately, one may administer the pharmaceutical composition in a local rather than systemic manner, for example, via injection of the pharmaceutical composition directly into a tissue region of a patient.

Pharmaceutical compositions of some embodiments of the invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with some embodiments of the invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.

For injection, the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank’s solution, Ringer’s solution, or physiological salt buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

For oral administration, the pharmaceutical composition can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the pharmaceutical composition to be formulated as an oil-based formulation, tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient. Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

The pharmaceutical composition can be formulated for inhalation. For example, the compositions can be formulated as vapors or aerosols that can be inhaled into the lungs. Vapor formulations include liquid formulations that are vaporized when loaded into a suitable vaporization device.

Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

Pharmaceutical compositions which can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration. For transdermal administration, the composition can be formulated in a form of a gel, a cream, an ointment, a paste, a lotion, a milk, a suspension, an aerosol, a spray, a foam, a serum, a swab, a pledget, a pad or a patch. Formulations for transdermal delivery can typically include carriers such as water, liquid alcohols, liquid glycols, liquid polyalkylene glycols, liquid esters, liquid amides, liquid protein hydrolysates, liquid alkylated protein hydrolysates, liquid lanolin, lanolin derivatives, glycerin, mineral oil, silicone, petroleum jelly, lanolin, fatty acids, vegetable oils, parabens, waxes, and like materials commonly employed in topical compositions. Various additives, known to those skilled in the art, may be included in the transdermal formulations of the invention. For example, solvents may be used to solubilize certain active ingredients substances. Other optional additives include skin permeation enhancers, opacifiers, anti-oxidants, gelling agents, thickening agents, stabilizers, and the like.

For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

For administration by nasal inhalation, the active ingredients for use according to some embodiments of the invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

The pharmaceutical composition described herein may be formulated for parenteral administration, e.g., by bolus injection or continues infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative. The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran.

Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.

Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use.

The pharmaceutical composition of some embodiments of the invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.

The administration of the composition comprising phytocannabinoids and the administration of the NSAID or steroid can be effected in the same route or in separate routes.

The administration of the composition comprising phytocannabinoids may be prior to, following or concomitant with the NNSAID or steroid.

Multiple rounds of administration of the composition comprising phytocannabinoids and/or the NSAID or steroid can be administered.

The compositions comprising phytocannabinoids, NSAID, steroid or combination thereof described herein can be administered to a subject in need thereof in a variety of other forms including a nutraceutical composition.

As used herein, a "nutraceutical composition" refers to any substance that may be considered a food or part of a food and provides medical or health benefits, including the prevention and treatment of disease. In some embodiments, a nutraceutical composition is intended to supplement the diet and contains at least one or more of the following ingredients: a vitamin; a mineral; an herb; a botanical; a fruit; a vegetable; an amino acid; or a concentrate, metabolite, constituent, or extract of any of the previously mentioned ingredients; and combinations thereof.

In some embodiments, a nutraceutical composition of the present invention can be administered as a "dietary supplement," as defined by the U.S. Food and Drug Administration, which is a product taken by mouth that contains a "dietary ingredient" such as, but not limited to, a vitamin, a mineral, an herb or other botanical, an amino acid, and substances such as an enzyme, an organ tissue, a glandular, a metabolite, or an extract or concentrate thereof.

Non-limiting forms of nutraceutical compositions of the present invention include: a tablet, a capsule, a pill, a softgel, a gelcap, a liquid, a powder, a solution, a tincture, a suspension, a syrup, or other forms known to persons of skill in the art. A nutraceutical composition can also be in the form of a food, such as, but not limited to, a food bar, a beverage, a food gel, a food additive/supplement, a powder, a syrup, and combinations thereof.

Pharmaceutical or nutraceutical compositions suitable for use in context of some embodiments of the invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients effective to prevent, alleviate or ameliorate symptoms of a disorder or prolong the survival of the subject being treated.

Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

For any preparation used in the methods of the invention, the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays. For example, a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.

Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals.

A non-limiting example of an animal model for SARS-CoV-2 is the transgenic mouse expressing human ACE2 (see e.g, Bao et al. (2020) Nature 583: 830-833.

The doses determined in the mouse animal model can be converted for the treatment other species such as human and other animals diagnosed with the disease, using conversion Tables known to the skilled in the art.

The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 P-l).

Dosage amount and interval may be adjusted individually to provide levels of the active ingredient sufficient to induce or suppress the biological effect (minimal effective concentration, MEC). The MEC will vary for each preparation, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations. Depending on the severity and responsiveness of the condition to be treated, dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.

The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.

According to specific embodiments, the composition comprising phytocannabinoids is provided as a concentration of at least 1 pg I ml, at least 5 pg / ml, at least 10 pg / ml, at least 15 pg / ml, at least 20 pg / ml, at least 25 pg / ml, at least 30 pg / ml, at least 40 pg / ml of said phytocannabinoids .

According to specific embodiments, the composition comprising phytocannabinoids is provided as a concentration of at least 10 pg / ml of said phytocannabinoids.

According to specific embodiments, the composition comprising phytocannabinoids is provided as a concentration of at least 20 pg / ml of said phytocannabinoids.

According to specific embodiments, the NSAID or steroid is provided in an amount below the gold standard for treatment as a single agent (e.g. in order to prevent known side effects).

Compositions of some embodiments of the invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert. Compositions comprising a preparation of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as is further detailed above.

As used herein the term “about” refers to ± 10 %.

The terms "comprises", "comprising", "includes", "including", “having” and their conjugates mean "including but not limited to".

The term “consisting of’ means “including and limited to”. The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging/ranges between” a first indicate number and a second indicate number and “ranging/ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

When reference is made to particular sequence listings, such reference is to be understood to also encompass sequences that substantially correspond to its complementary sequence as including minor sequence variations, resulting from, e.g., sequencing errors, cloning errors, or other alterations resulting in base substitution, base deletion or base addition, provided that the frequency of such variations is less than 1 in 50 nucleotides, alternatively, less than 1 in 100 nucleotides, alternatively, less than 1 in 200 nucleotides, alternatively, less than 1 in 500 nucleotides, alternatively, less than 1 in 1000 nucleotides, alternatively, less than 1 in 5,000 nucleotides, alternatively, less than 1 in 10,000 nucleotides.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984); “Nucleic Acid Hybridization" Hanies, B. D., and Higgins S. J., eds. (1985); "Transcription and Translation" Hames, B. D., and Higgins S. J., eds. (1984); "Animal Cell Culture" Freshney, R. I., ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1- 317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference.

MATERIALS AND METHODS

Standard/material preparation and use - The cannabinoid standards at a concentration of 1 mg / mL in methanol included cannabidiol (CBD, Restek catalog no. 34011) cannabigerol (CBG, Restek catalog no. 34091) and tetrahydrocannabivarin (THCV, Restek catalog no. 34100). The working stock concentration of THCV at 100 pg / mL was prepared in complete DMEM growth media. A composition refered to herein as “FCBD:std” which includes combination of CBD (93.5 %), CBG (6.1 %) and THCV (0.4 %) was prepared at a stock concentration of 60 pg / mL by N2 gas drying of CBD and mixing the dried CBD with CBG and THCV in complete DMEM growth media. Phorbol 12-myristate 13-acetate (PMA) (P1585; Sigma Aldrich, USA) was dissolved in DMSO at a stock concentration of 5 pg / mL. Ibuprofen (14883; Sigma Aldrich, USA) was dissolved in methanol at a stock concentration of 50 mg / mL. Diclofenac sodium (619; British Pharmacopoeia Chemical Reference Substance) was dissolved in DMSO at a stock concentration of 50 mg / mL. Budesonide (B l 157300; European Pharmacopoeia Reference Standard) was dissolved in DMSO at a stock concentration of 200,100,20 pg / mL. Dexamethosone (D4902; Sigma Aldrich, USA) was dissolved in DMSO at a stock concentration of 1000,200,100,20 pg / mL. TNFa (300-01A; PeproTech, Rocky Hill, NJ, USA) was dissolved in water at a stock concentration of 100 pg / mL. Analytical grade methanol was used at final concentration of 0.8 to 0.2 % (v/v) according to the indicated concentration of the treatment. The highest methanol concentration in each experiment was used for vehicle control. Bio grade Dimethyl Sulfoxide was used at a final concentration of 1 to 0.5 % (v/v) according to the indicated concentration of the treatment. Ultra-pure deionized water (MS grade) was used as received without further purification.

Cell cultures - The lung cancer cell line A549 (ATCC® CCL-185™) was cultured in DMEM (01-055-1 A, Biological Industries, Israel) growth media supplemented with 10 % FBS, 1 % glutamic acid, 1 % pen-strep. The plasmocin. Macrophage cell line KG1 (ATCC® CCL- 246™) was cultured in DMEM growth media supplemented with 20 % FBS, 1 % glutamic acid, 1 % pen-strep and plasmocin. 10 ng / mL PMA in DMEM media supplemented with 10 % FBS, 1 % glutamic acid, 1 % pen-strep and plasmocin was used as stimulating environment for the differentiation of KG1 cells. Differentiated cells with typical morphology were attached to the plate surface within 2 days of initiation⁴². The differentiated KG1 cells were trypsinized and used in the experiments described hereinbelow.

Determination of IL levels and cell viability - IL-6 and IL-8 levels were determined as described previously⁴³ with the following modifications:

For cono-cultures, A549 or KG1 cells were plated at 2.4 x 10⁴ cells per well in DMEM complete media (100 pL) in 96-well cell culture plates. The cells were incubated overnight at 37 °C in air and 5 % CO2 in a humidified incubator. Cell excitation was performed with 300 ng / mL TNFa and treatments were performed with FCBD:std, Diclofenac, Ibuprofen, Dexamethasone and/or Budesonide in 100 pL Complete DMEM. For co-cultures, the cell number of A549 to differentiated KG1 was adjusted to 3 : 1 ratio [37]. A549 cells were plated at 1.2 x 10⁴ cells per well in DMEM complete media (100 pL) in a 96-well cell culture plate and were incubated overnight in a humidified incubator. Subsequently, the differentiated KG1 cells were added at 0.4 x 10⁴ cells per well on top of A549 cells and the co-culture was incubated overnight in a humidified incubator. Cell excitation was performed with 300 ng / mL TNFa and treatments were performed with FCBD:std, Diclofenac, Ibuprofen, Dexamethasone and/or Budesonide in 100 pL Complete DMEM.

IL-6 and IL-8 secretion levels were analyzed following 4 hours of incubation of the mono- or coculture. Specifically, supernatant samples were collected and tested using IL-6 and IL-8 ELISA kits (DY206 and DY208, respectively, R&D Systems, Minneapolis, MN, USA). For cell viability, an Alamar Blue (resazurin) assay was performed on each well as described previously⁴³. For dose response assays, data points were connected by non-linear regression lines of the sigmoidal dose-response relation. GraphPad Prism version 6.1 (www(dot)graphpad(dot)com/scientific-software/prism/, GraphPad Software Inc., San Diego, USA) was employed to produce dose-response curves and IC50 doses were calculated using nonlinear regression analysis.

3D model - Hydrogels (AGFCH) included Sigma- Aldrich products with the combination of alginate (40 % v/v), gelatin (10 % v/v), fibrinogen-collagen (45 % v/v) and hyaluronic acid (5 % v/v). Alginate (W201502; Sigma-Aldrich, USA) was dissolved in 10 % glycerol in PBS at stock concentration of 22.5 mg / mL, gelatin (G9764; Bio-Basic, USA) was dissolved in 10 % glycerol in PBS at stock concentration of 45 mg / mL, fibrinogen (F38791; Sigma-Aldrich, USA) was dissolved in PBS at stock concentration of 50 mg / mL, collagen (C9791; Sigma- Aldrich, USA) was dissolved in 0.1 M acetic acid at stock concentration of 2.2 mg / mL and hyaluronic acid (08185 ; Sigma- Aldrich, USA) was dissolved in water at stock concentration of 2 mg / mL. The 3D model was prepared on ThinCert™ - 24 well, PET membrane, 8.0 pm pore size (662638, Greiner bio-one). The gel (AGFCH) was mixed with 0.8xl0⁴ differentiated KG1 cell per membrane and printed on the basal side of Thincert and cross-linked using CaCh (A610050; Bio-Basic, USA) and thrombin (SRP6557; Sigma-Aldrich, USA) for 5 minutes. The printed structure on the Thincert was washed with PBS and transferred to 24 well plate having 300 pl complete DMEM growth media. The 2.4xl0⁴ A549 cells were added to the apical side of the Thincert and allowed to grow as a monolayer for 2 days in complete DMEM growth media. The membrane of the Thincert was removed using sterial surgical blade and transferred to a 96 well plate. Treatments were performed for 4 hours and supernatant samples were collected and tested using IL-6 and IL-8 ELISA kits (DY206 and DY208 respectively, R&D Systems, Minneapolis, MN, USA). Structures were stained using EasyProbe Hoechst and Dil perchlorate membrane staining as described hereinbelow. To assess cell viability of 3D structure, the membrane was washed with PBS and stained with Alamar Blue (resazurin, AR002; R&D Systems) assay.

Cellular staining and confocal microscopy - The 3D model on the tissue culture inserts was washed with PBS and the membrane was removed from the insert using surgical blade. The membrane was washed with PBS and incubated for 40 minutes with Dil perchlorate for membrane staining and Hoechst for nuclear staining (C017 and FP027, respectively; ABP Bioscience Rockville, MD, USA). Cell microscopy and image acquisition was carried out using a Leica SP8 laser scanning microscope (Leica, Wetzlar, Germany), equipped with a 405, 488 and 552 nm solid state lasers, HCX PL APO CS 10x/0.40 or HC PL APO CS 63x/1.2 water immersion objectives (Leica, Wetzlar, Germany) and Leica Application Suite X software (LASX, Leica, Wetzlar, Germany). Hoechst, 5(6)-Carboxyfluorescein and membrane - emission signals were detected with PMT and HyD (hybrid) detectors in ranges of 415-490 nm, 500-535 nm and 565-660 nm, respectively.

Determination of COX activity - Inhibition of COX activity was determined using a Cyclooxygenase 1 (COXI) Inhibitor Screening Assay Kit (ab204698, abeam) and a Cyclooxygenase 2 (COX2) Inhibitor Screening Assay Kit (Catalog No. K547-100, BioVision, Inc.). Fluorometric detection method was applied in 10 pL COX assay buffer, according to manufacturer instructions.

Statistical analysis - Data was processed using the JMP statistical package (www(dot)jmp(dot)com/en_us/home(dot)html, SAS Inc, NC, USA). Comparisons between two groups were made using the Student’s T-Test. Comparisons between more than 2 groups were made with analysis of variance (ANOVA) followed by Tukey- Kramer's honest significant difference (HSD) test as post hoc. Values are shown as mean ± standard error (s.e.m.). P values < 0.05 were considered significant.

EXAMPLE 1

A SYNERGISTIC EFFECT OF A COMBINATION OF FCBD:std AND NSAIDS ON MACROPHAGE ACTIVITY

A synthetic composition referred to herein as “FCBD:std” mimicking a liquid chromatography FCBD fraction of a cannabis extract described in Anil et al. Sci Rep. 2021 Jan 14; 11(1):1462, which contain CBD (93.5 %), CBG (6.1 %) and THCV (0.4 %) was prepared. Following, the anti-inflammatory effect of a combined treatment with FCBD:std and the NSAIDs ibuprofen or diclofenac; or the steroidal drugs budesonide or dexamethasone, on differentiated monocytes (macrophages; KG1 cells) treated with TNFa, manifested by reduced secretion of IL-8, was evaluated.

As a reference, ibuprofen or budesonide only treatment had a very low anti-inflammatory effect, whereas dexamethasone or diclofenac only treatment had an IC50 value of 263.3 ng / mL and 151.1 pg/mL, respectively (Figures 9A-D).

FCBD:std at a concentration of 20 pg / mL in combination with ibuprofen at a concentration of 300, 400 or 500 pg / mL of, demonstrated an anti-inflammatory effect which was 3.6, 3.6 and 5.7 fold, respectively, higher compared to treatment with ibuprofen alone at these concentrations (Table 1 hereinbelow). Based on Bliss independence drug interaction model, a significant synergy (manifested once the experimental [observed] value of IL-8 reduction was higher than the calculated [expected] value) was apparent with FCBD:std (20 or 30 pg / mL) and ibuprofen (100-500 pg/mL) (Figure 1A and Table 9 hereinbelow).

FCBD:std at a concentration of 30 pg / mL with diclofenac at a concentration of 50, 75 or 100 pg / mL of showed 1.2, 2.0 and 1.9 fold, respectively, higher activity compared to treatment with diclofenac alone at the same concentration (Table 4 hereinbelow). Synergy between FCBD:std and diclofenac was apparent mainly at 30 pg / mL FCBD:std and 50-100 pg / mL diclofenac (Figure ID and Table 9 hereinbelow).

The combination of FCBD:std at a concentration of 30 pg / mL with budesonide at a concentration of 50 ng / mL showed 2.0 fold higher activity compared to treatment with budesonide alone at the same concentration (Table 2 hereinbelow). However, the delta value of experimental [observed] value of IL-8 reduction minus the calculated [expected] value of the combination between FCBD:std and budesonide was not significantly different in comparison to treatment with each of the components alone (Figure IB and Table 9 hereinbelow). Combinations of FCBD:std and budesonide in other concentrations led to a significant inhibition of activity (e.g., 25 pg / mL FCBD:std and 500 ng / mL budesonide; Figure IB; Table 9 hereinbelow).

The combination of FCBD:std at a concentration of 30 pg / mL with dexamethasone at a concentration of 2000 ng / mL resulted in activity that was 3.7 fold higher compared to dexamethasone alone at the same concentration (Table 3 hereinbelow). However, the delta value of experimental [observed] value of IL-8 reduction minus the calculated [expected] value of the combination between FCBD:std and dexamethasone was not significantly different in comparison to treatment with each of the components alone (Figure 1C and Table 9 hereinbelow). Moreover, inhibition rather than enhancement of activity was apparent in combinations of FCBD:std and dexamethasone in other concentrations (e.g., 20 pg / mL FCBD:std and 250 ng / mL of budesonide; Figure 1C and Table 9 hereinbelow).

Taken together, a combination of FCBD:std with NSAIDs demonstrated a synergistic anti-inflammatory activity on macrophages. In comparison, a combination of FCBD:std with steroids was not synergistic and even decreased the anti-inflammatory activity of each of the agents at some of the examined concentrations. EXAMPLE 2

A SYNERGISTIC EFFECT OF A COMBINATION OF FCBD:std AND NSAIDS OR STEROIDS ON LUNG EPITHELIAL CELLS INFLAMMATION

In the next step, the anti-inflammatory effect of a combined treatment with FCBD:std and the NSAIDs ibuprofen or diclofenac; or the steroidal drugs budesonide or dexamethasone, on TNFa treated A549 lung epithelial cells, manifested by reduced secretion of IL-8, was evaluated.

As a reference, ibuprofen or diclofenac only treatment had a low anti-inflammatory activity, whereas budesonide or dexamethasone only treatment reduced IL-8 levels secreted from A549 cells with an IC50 value of 221.3 ng / mL or 214.3 ng/mL, respectively (Figures 10A-D).

The combined treatment with FCBD:std at a concentration of 20 pg / mL and ibuprofen at a concentration of 400 or 500 pg/mL demonstrated an anti-inflammatory effect which was 2.0 and 2.2 fold, respectively, higher than that of each of the compounds alone (Table 5 hereinbelow). Based on Bliss independence drug interaction model, a significant synergy was apparent for the combined treatment with FCBD:std and ibuprofen, especially at the above concentrations (Figure 2 A and Table 10 hereinbelow).

Combined treatment with FCBD:std at a concentration of 5 pg / mL and diclofenac at a concentration of 50, 100, 200 or 300 pg / mL showed 1.4, 1.5, 1.8 and 1.5 fold, respectively, higher activity than diclofenac alone, respectively (Table 8 hereinbelow). Synergy was significant at these concentrations as well as with 10 pg / mL FCBD:std and 50 or 200 pg / mL of diclofenac (Figure 2D; and Table 10 hereinbelow).

FCBD:std at a concentrations of 5 or 20 pg / mL with budesonide at a concentration of 100 ng / mL resulted in activity that was 1.8 and 2.0 fold, respectively, higher than that of each of the compounds alone. FCBD:std at a concentration of 10 pg / mL with budesonide at a concentration of 50 or 100 ng / mL resulted in activity that was 1.5 and 1.6 fold, respectively, higher compared to that of budesonide alone (Table 6 hereinbelow). Based on Bliss independence drug interaction model, a significant synergy was apparent for the combined treatment with FCBD:std and budesonide at these concentrations (Figure 2B and Table 10 hereinbelow).

Combined treatment of FCBD:std with dexamethasone led to only minor increase in activity (Table 7 hereinbelow). A significant synergy was detected mainly at 10 pg / mL FCBD:std combined with 1000 ng / mL dexamethasone (Figure 2C and Table 10 hereinbelow). Taken together, a combination of FCBD:std with NSAIDs or steroids demonstrated a synergistic anti-inflammatory activity on lung epithelial cells.

EXAMPLE 3

A SYNERGISTIC ANTI-INFLAMMATORY EFFECT OF A COMBINATION OF FCBD:std AND NSAIDS ON CO-CULTURES OF MACROPHAGES AND LUNG EPITHELIAL CELLS

To evaluate effective anti-inflammatory treatment of both lung epithelial cells and macrophages, a co-culture of A549 and macrophages (differentiated KG1) was established and treated with TNFa. Following, the effect of FCBD:std and NSAIDs treatment on secretion of IL-8 and IL-6;and COX-1 and COX-2 activity was evaluated.

IL-8 secreted levels

In these settings, treatment with FCBD:std alone had only a minor effect on IL-8 secretion, ibuprofen at 400 pg / mL even led to increased IL-8 secretion per cell, and dexamethasone or budesonide significantly decreased IL-8 secretion (Figure 3A).

A combination of 20 or 25 pg/mL FCBD:std with 400 pg / mL ibuprofen reduced IL-8 substantially. This activity was 2.4 and 2.2 fold, respectively, higher than that of the individual compounds (Figure 3A).

All combinations of FCBD:std at a dose of 20 or 25 pg / mL with diclofenac at a concentration of 50 or 100 pg / mL substantially reduced IL-8 secreted levels. For 20 pg / mL FCBD:std and 50 or 100 pg / mL diclofenac, this activity was 2.9 fold greater than that of the individual compounds at the same concentrations; and for 25 pg / mL FCBD:std and 50 or 100 pg / mL diclofenac, this activity was 3.2 and 2.9 fold, respectively, greater than that of the individual compounds at the same concentrations (Figure 3D).

In contrast, combinations of FCBD:std with dexamethasone or budesonide did not show any additive effect on IL-8 secreted levels (Figures 3B-C).

IL-6 secreted levels

In these settings, FCBD:std and diclofenac as single agents led to a minor non- significant increase in IL-6 secreted levels (Figure 4).

All examined combinations of FCBD:std (5, 20 or 25 pg / mL) and diclofenac (50 or 100 pg / mL) led to a substantial reduction of IL-6 levels secreted in the co-culture (Figure 4). COX-2 and COX-2 activity

While ibuprofen and diclofenac substantially inhibited COX-1 and COX-2 enzyme activity, FCBD:std did not inhibit activity of both enzymes at the examined concentration (Figures 5-6).

Taken together, a combination of FCBD:std with NSAIDs demonstrated a synergistic anti-inflammatory activity on co-cultures of lung epithelial cells and macrophages. In comparison, a combination of FCBD:std with steroids seems not to be synergistic at the examined concentrations.

EXAMPLE 4

A SYNERGISTIC ANTI-INFLAMMATORY EFFECT OF A COMBINATION OF FCBD:std AND NSAIDS IN A 3D MODEL OF MACROPHAGES AND LUNG EPITHELIAL CELLS CO-CULTURES

To establish 3D co-cultures of A549 and macrophages that may resemble alveoli as possible, differentiated KG1 cells (i.e., macrophages) immersed in extra-cellular matrix (ECM) were printed on a 8-pm polyethylene terephthalate (PET) membrane (Figure 11). Following, an A549 culture was established on the 8-pm PET membrane to form cell monolayer (Figures 7A-B and 7D). The A549 cells were ciliated (Figure 7F). Macrophages were visualized in the ECM (Figure 7C and 7E) but also in medium (not shown), suggesting that macrophages were roaming the 3D structure.

Following, the 3D structures of monolayer A459 and ECM macrophages were treated with TNFa followed by FCBD:std, ibuprofen or diclofenac or combination thereof, and the antiinflammatory effect of the treatment, manifested by reduced secretion of IL-8 and/or IL-6, was evaluated (Figures 8A-C).

IL-8 secreted levels

Only a minor decrease in IL-8 secreted level was recorded in these settings following treatment with FCBD:std or diclofenac as a single agent; and no apparent decrease was recorded following treatment with ibuprofen as a single agent (Figures 8A-B).

A combined treatment with FCBD:std and ibuprofen or diclofenac significantly and substantially reduced secretion of IL-8 (Figures 8A-B).

IL-6 secreted levels

A minor decrease in IL-6 secreted level was recorded following treatment with diclofenac as a single agent. However, a combined treatment with FCBD:std and diclofenac significantly reduced secretion of IL-6 (Figure 8C).

Taken together, a combination of FCBD:std with NSAIDs demonstrated a synergistic anti-inflammatory activity in a 3D model of epithelial lung cells and macrophages co-culture.

EXAMPLE 5

A SYNERGISTIC EFFECT OF A COMBINATION OF FCBD:std AND NSAIDS ON PAIN REUDCTION

The in-vivo effect of a combined treatment with FCBD:std and NSAID (e.g. diclofenac) on alleviation of pain and edema is evaluated in a carrageenan-induced ICR female mice model. Treatment is provided orally or IP. Following, pain is assessed by a Von Frey/hot plate; and edema is assessed by gross paw assessment and histological analysis.

EXAMPLE 6

A SYNERGISTIC EFFECT OF A COMBINATION OF FCBD:std AND NSAIDS IN A CARRAGEENAN-INDUCED PAW EDEMA MOUSE MODEL

Material and methods: .-carrageenan-induced paw edema mice model - 6-8 weeks old female ICR mice (Envigo, Israel) were divided into 3 treatment groups (n = 3 / group; equal mean body weight was maintained between groups). All 9 animals were subcutaneously injected with 1 % carrageenan (C1013 Sigma-Aldrich, USA), at a volume of 30 pl, to the left plantar of the left foot. Animals were treated topically with Voltaren Emulgel 1 % diclofenac (5148694/02-2023, Diclofenac diethilamine 1.16 % corresponding to diclofenac sodium 1 % GSK); FCBD:std solution (CBG: 20164-1, CBD: ISO60156-1, THCV: 18091-1, Cayman Chemical Company, USA) in a concentration of 100 mg / mL dissolved in DMSO; or FCBD:std mixed with Voltaren Emulgel (FCBD:std solution in a concentration of 100 mg / mL was dissolved in 100 pL DMSO and mixed with 1 ml Voltaren Emulgel as an emulsion, final concentration of FCBD:std 10 mg / mL). Treatment was effected 2 hours prior to induction with carrageenan and at 2, 4 and 6 hours after induction. All animals were treated topically, by spreading the gel (100 pL) on the paw. The right leg served as a control without induction. Vehicle control was 10 % DMSO in saline, 100 pL was spread per paw. The study was terminated 8 hours post beginning of treatments. Mice were sacrificed with CO2. Both feet from each animal were excised and freezed in liquid nitrogen in Eppendorf tubes and store in an airtight container at -70°C or colder. RNA extraction and purification - 100 mg of mouse leg tissue was taken for RNA extraction. Tissues were finely grounded in liquid nitrogen using a sterile mortar and pestle. Fine ground tissues were dissolved in 1 ml TRI reagent and collected in 1.5 ml microcentrifuge tubes. Tissues were vortexed and left to stand at room temperature for 5 minutes. Following, samples were centrifuged for 5 minutes at 10, 000 RPM, 4 °C. For the phase separation, 150 -200 pl of chloroform per ml of Tri reagent used were added to the centrifuged samples. Thereafter, samples were vortexed and left to stand at room temperature for 2-3 minutes. Chloroform mixed samples were centrifuged for 15 minutes, 13,200 rpm, 4 °C to separate into a lower red phenolchloroform properly and a colorless upper aqueous phase which contains the RNA. The upper phase (±400 pl) was collected in a new autoclaved 1.5 ml centrifuge tube for RNA precipitation. 500 pl of chilled 2-propanol was added to the collected aqueous phase. The mixture was gently vortex and incubated at room temperature for 10 minutes at 13,200 RPM, 40C. The supernatant was properly removed without touching the pellet (precipitated RNA). Precipitated RNA was properly re-suspended in chilled 75 % ethanol per 1 ml of TRI reagent used. Re-suspended samples were centrifuged for 10 min at 10000 RPM, 4°C. Ethanol (75%) was removed without touching the pellet (precipitated RNA). Following, RNA was purified according to standard protocol of GeneJET Purification Kits (Thermo Fisher Scientific).

PCR analysis - Following purification, RNA was reverse-transcribed in a total volume of 20 pL (PB30.11-10, qPCRBIO). PCR was performed in triplicates using a qPCR SyGreen Blue Mix (PB20.16-20, qPCRBIO) and StepOnePlus system (Applied Biosystems Thermo Fisher Scientific, USA). The expression of IL-6 used as target gene was normalized to GAPDH used as reference gene in the 2^AACt method presenting the differences (A) in threshold cycle (Ct) between the target gene and GAPDH gene. AACt = ACt treatment- ACt control.

Results:

Treatment with Voltaren Emulgel 1% diclofenac as a monotherapy lead to reduction in mRNA levels of the pro -inflammatory cytokine, IL-6, in comparison to DMSO treated control (Figure 12). Nevertheless, a combined treatment with both Voltaren Emulgel 1 % diclofenac and FCBD:std lead to a reduction in IL-6 expression levels to a much greater extent (Figure 12).

Taken together, a combination of FCBD:std with NSAIDs had a combined improved effect on inflammation in a carrageenan-induced paw edema mouse model. Table 1: Fold change in IL-8 levels per cell relative to vehicle (methanol 0.8 %)+TNFa control secreted from KG1 cells following 4 hours treatment with of FCBD:std and/or Ibuprofen (Ibu)

Table 2: Fold change in IL-8 levels per cell relative to vehicle (methanol 0.2% and DMSO 0.5%)+TNFa control secreted from KG1 cells following 4 hours treatment with FCBD:std and/or budesonide (Bud)

Table 3: Fold change in IL-8 levels per cell relative to vehicle (methanol 0.2% and DMSO 0.5%)+TNFa control secreted from KG1 cells following 4 hours treatment with FCBD:std and/or dexamethasone (Dexa)

Table 4: Fold change in IL-8 levels per cell relative to vehicle (methanol 0.2% and DMSO 0.5%)+TNFa control secreted from KG1 cells following 4 hours treatment with FCBD:std and/or diclofenac (Diclo)

Table 5: Fold change in IL-8 levels per cell relative to vehicle (methanol 0.2% and DMSO 0.5%)+TNFa control secreted from A549 cells following 4 hours treatment with FCBD:std and/or Ibuprofen (Ibu)

Table 6: Fold change in IL-8 levels per cell relative to vehicle (methanol 0.2% and DMSO 0.5%)+TNFa control secreted from A549 cells following 4 hours treatment with FCBD:std and/or budesonide (Bud)

Table 7: Fold change in IL-8 levels per cell relative to vehicle (methanol 0.2% and DMSO 0.5%)+TNFa control secreted from A549 cells following 4 hours treatment with FCBD:std and/or dexamethasone (Dexa)

Table 8: Fold change in IL-8 levels per cell relative to vehicle (methanol 0.2% and DMSO 0.5%)+TNFa control secreted from A549 cells following 4 hours treatment with FCBD:std and/or Diclofenac (Diclo)

Table 9: Synergistic effects on IL-8 secreted from KG1 cells of combined treatment with FCBD:std and Ibuprofen (a), Dexamethasone (b), budesonide (c) or diclofenac (d), determined by the experimental (observed) and the calculated (expected) values. The delta between observed and experimental values was calculated according to the Bliss independence drug interaction model, and delta values are graphically presented in Figures 1A-D. Levels of the experimental (observed) values with different letters are significantly different from all combinations of pairs according to Tukey-Kramer honest significant difference (HSD; P < 0.05).

Table 10: Synergistic effects on IL-8 secreted from A549 cells of combined treatment with FCBD:std and Ibuprofen (a), Dexamethasone (b), budesonide (c) or diclofenac (d), determined by the experimental (observed) and the calculated (expected) values. The delta between observed and experimental values was calculated according to the Bliss independence drug interaction model, and delta values are graphically presented in Figures 1A-D. Levels of the experimental (observed) values with different letters are significantly different from all combinations of pairs according to Tukey-Kramer honest significant difference (HSD; P < 0.05).

Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

It is the intent of the applicant(s) that all publications, patents and patent applications referred to in this specification are to be incorporated in their entirety by reference into the specification, as if each individual publication, patent or patent application was specifically and individually noted when referenced that it is to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting. In addition, any priority document(s) of this application is/are hereby incorporated herein by reference in its/their entirety. REFERENCES

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Claims

69 WHAT IS CLAIMED IS:

1. An article of manufacture comprising as active ingredients a non-steroidal antiinflammatory drug (NSAID) and a composition comprising phytocannabinoids, wherein said phytocannabinoids comprise at least 80 % CBD and at least one of CBG and THCV.

2. The article of manufacture of claim 1, wherein said NSAID and said composition comprising phytocannabinoids are provided in a co-formulation.

3. The article of manufacture of claim 1, wherein said NSAID and said composition comprising phytocannabinoids are provided in separate formulations.

4. A method of treating a disease selected from the group consisting of inflammation, pain and fever in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a non-steroidal anti-inflammatory drug (NSAID) and a therapeutically effective amount of a composition comprising phytocannabinoids, wherein said phytocannabinoids comprise at least 80 % CBD and at least one of CBG and THCV, thereby treating the disease in the subject.

5. A non-steroidal anti-inflammatory drug (NSAID) and a composition comprising phytocannabinoids, wherein said phytocannabinoids comprise at least 80 % CBD and at least one of CBG and THCV, for use in treating a disease selected from the group consisting of inflammation, pain and fever in a subject in need thereof.

6. A method of treating a disease selected from the group consisting of inflammation, pain, edema and fever in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a non-steroidal antiinflammatory drug (NSAID) and a therapeutically effective amount of a composition comprising phytocannabinoids, wherein said phytocannabinoids comprise at least 80 % CBD and at least one of CBG and THCV, thereby treating the disease in the subject.

7. A non-steroidal anti-inflammatory drug (NSAID) and a composition comprising phytocannabinoids, wherein said phytocannabinoids comprise at least 80 % CBD and at least 70 one of CBG and THCV, for use in treating a disease selected from the group consisting of inflammation, pain, edema and fever in a subject in need thereof.

8. The method of any one of claims 4 and 6 or the NSAID and composition comprising phytocannabinoids for use of any one of claims 5 and 7, wherein said pain is selected from the group consisting of inflammatory pain, acute pain, chronic pain, arthritis, pain following nerve injury, cancer associated pain, chemotherapy associated pain, migraine, low back pain and toothache.

9. The method of any one of claims 4 and 6 or the NSAID and composition comprising phytocannabinoids for use of any one of claims 5 and 7, wherein said inflammation is selected from the group consisting of inflammation of the respiratory system, colon inflammation, rheumatoid arthritis, osteoarthritis, extra- articular rheumatism and ankylosing spondylitis.

10. The method of any one of claims 4 and 6 or the NSAID and composition comprising phytocannabinoids for use of any one of claims 5 and 7, wherein said inflammation is inflammation of the respiratory system or colon inflammation.

11. The method or the NSAID and composition comprising phytocannabinoids for use of any one of claims 4-10, wherein said inflammation is at a stage of cytokine storm.

12. The method or the NSAID and composition comprising phytocannabinoids for use of any one of claims 6-11, wherein said edema comprises leg and/or hand edema.

13. An article of manufacture comprising as active ingredients a steroid and a composition comprising phytocannabinoids, wherein said phytocannabinoids comprise at least 80 % CBD and at least one of CBG and THCV.

14. The article of manufacture of claim 13, wherein said steroid and said composition comprising phytocannabinoids are provided in a co-formulation. 71

15. The article of manufacture of claim 13, wherein said steroid and said composition comprising phytocannabinoids are provided in separate formulations.

16. A method of treating a disease selected from the group consisting of inflammation of the respiratory system, colon inflammation and cytokine storm in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a steroid and a therapeutically effective amount of a composition comprising phytocannabinoids, wherein said phytocannabinoids comprise at least 80 % CBD and at least one of CBG and THCV, thereby treating the disease in the subject.

17. A steroid and a composition comprising phytocannabinoids, wherein said phytocannabinoids comprise at least 80 % CBD and at least one of CBG and THCV, for use in treating a disease selected from the group consisting of inflammation of the respiratory system, colon inflammation and cytokine storm in a subject in need thereof.

18. The method, the NSAID and composition comprising phytocannabinoids for use or the steroid and composition comprising phytocannabinoids for use of any one of claims 4-12 and 16-17, wherein said disease is not cancer.

19. The article of manufacture, the method or the NSAID and composition comprising phytocannabinoids for use of any one of claims 1-12 and 18, wherein said NSAID is selected from the group consisting of ibuprofen and diclofenac.

20. The article of manufacture, the method or the NSAID and composition comprising phytocannabinoids for use of any one of claims 1-12 and 18, wherein said NSAID is diclofenac.

21. The article of manufacture, the method or the NSAID and composition comprising phytocannabinoids for use of any one of claims 1-12 and 18-20, wherein said NSAID and said composition comprising phytocannabinoids have a combined additive or synergistic anti-inflammatory effect as compared to each of said NSAID and said composition comprising phytocannabinoids when administered as a single agent. 72

22. The article of manufacture, the method or the NSAID and composition comprising phytocannabinoids for use of any one of claims 1-12 and 18-21, wherein said NSAID and said composition comprising phytocannabinoids have a combined additive or synergistic effect on pain reduction as compared to each of said NSAID and said composition comprising phytocannabinoids when administered as a single agent.

23. The article of manufacture, the method or the steroid and composition comprising phytocannabinoids for use of any one of claims 13-18, wherein said steroid is selected from the group consisting of budesonide and dexamethasone.

24. The article of manufacture, the method or the steroid and composition comprising phytocannabinoids for use of any one of claims 13-18 and 23, wherein said steroid and said composition comprising phytocannabinoids have a combined additive or synergistic antiinflammatory effect as compared to each of said steroid and said composition comprising phytocannabinoids when administered as a single agent.

25. The article of manufacture, the method, the NSAID and composition comprising phytocannabinoids for use or the steroid and composition comprising phytocannabinoids for use of any one of claims 1-24, wherein said composition comprising phytocannabinoids is a synthetic composition.

26. The article of manufacture, the method, the NSAID and composition comprising phytocannabinoids for use or the steroid and composition comprising phytocannabinoids for use of any one of claims 1-25, wherein said at least 80 % CBD is at least 90 %.

27. The article of manufacture, the method, the NSAID and composition comprising phytocannabinoids for use or the steroid and composition comprising phytocannabinoids for use of any one of claims 1-25, wherein said phytocannabinoids comprise 90 - 95 % CBD.

28. The article of manufacture, the method, the NSAID and composition comprising phytocannabinoids for use or the steroid and composition comprising phytocannabinoids for use of any one of claims 1-27, wherein when said phytocannabinoids comprise said CBG said phytocannabinoids comprise at least 2% CBG. 73

29. The article of manufacture, the method, the NSAID and composition comprising phytocannabinoids for use or the steroid and composition comprising phytocannabinoids for use of any one of claims 1-28, wherein when said phytocannabinoids comprise said CBG said phytocannabinoids comprise at least 5 % CBG.

30. The article of manufacture, the method, the NSAID and composition comprising phytocannabinoids for use or the steroid and composition comprising phytocannabinoids for use of any one of claims 1-29, wherein when said phytocannabinoids comprise said CBG said phytocannabinoids comprise 5 - 7 % CBG.

31. The article of manufacture, the method, the NSAID and composition comprising phytocannabinoids for use or the steroid and composition comprising phytocannabinoids for use of any one of claims 1-30, wherein when said phytocannabinoids comprise said CBG said phytocannabinoids comprise 5.5 - 6.5 % CBG.

32. The article of manufacture, the method, the NSAID and composition comprising phytocannabinoids for use or the steroid and composition comprising phytocannabinoids for use of any one of claims 1-31, wherein when said phytocannabinoids comprise said CBG, a concentration ratio of said CBD and said CBG is 20 : 1 - 10 : 1.

33. The article of manufacture, the method, the NSAID and composition comprising phytocannabinoids for use or the steroid and composition comprising phytocannabinoids for use of any one of claims 1-32, wherein when said phytocannabinoids comprise said CBG, a concentration ratio of said CBD and said CBG is 17 : 1 - 13 : 1.

34. The article of manufacture, the method, the NSAID and composition comprising phytocannabinoids for use or the steroid and composition comprising phytocannabinoids for use of any one of claims 1-33, wherein when said phytocannabinoids comprise said THCV said phytocannabinoids comprise at least 0.2 % THCV.

35. The article of manufacture, the method, the NSAID and composition comprising phytocannabinoids for use or the steroid and composition comprising phytocannabinoids for use 74 of any one of claims 1-34, wherein when said phytocannabinoids comprise said THCV said phytocannabinoids comprise less than 1 % THCV.

36. The article of manufacture, the method, the NSAID and composition comprising phytocannabinoids for use or the steroid and composition comprising phytocannabinoids for use of any one of claims 1-35, wherein when said phytocannabinoids comprise said THCV said phytocannabinoids comprise 0.3 - 0.5 % THCV.

37. The article of manufacture, the method, the NSAID and composition comprising phytocannabinoids for use or the steroid and composition comprising phytocannabinoids for use of any one of claims 1-36, wherein when said phytocannabinoids comprise said THCV, a concentration ratio of said CBD and said THCV is 300 : 1 - 100 : 1.

38. The article of manufacture, the method, the NSAID and composition comprising phytocannabinoids for use or the steroid and composition comprising phytocannabinoids for use of any one of claims 1-37, wherein when said phytocannabinoids comprise said THCV, a concentration ratio of said CBD and said THCV is 250 : 1 - 200 : 1.

39. The article of manufacture, the method, the NSAID and composition comprising phytocannabinoids for use or the steroid and composition comprising phytocannabinoids for use of any one of claims 1-38, wherein said composition comprising phytocannabinoids is devoid of Tetrahydrocannabinol (THC).

40. The article of manufacture, the method, the NSAID and composition comprising phytocannabinoids for use or the steroid and composition comprising phytocannabinoids for use of any one of claims 1-39, wherein said composition comprising phytocannabonids is devoid of phytocannabinoids other than said CBD, CBG and/or THCV.

41. The article of manufacture, the method, the NSAID and composition comprising phytocannabinoids for use or the steroid and composition comprising phytocannabinoids for use of any one of claims 1-40, wherein said composition comprising phytocannabinoids is devoid of cannabis active ingredients other than said phytocannabinoids.

42. The article of manufacture, the method, the NSAID and composition comprising phytocannabinoids for use or the steroid and composition comprising phytocannabinoids for use of any one of claims 1-41, wherein said composition comprising phytocannabinoids is provided as a concentration of at least 10 pg / ml of said phytocannabinoids.