Use of cannabinoids in the treatment of inflammatory skin diseases

Abstract

The present invention relates to the use of one or more cannabinoids in the treatment of an inflammatory skin disease. Preferably the one or more cannabinoids are taken from the group consisting of: cannabidiol (CBD), cannabidiolic acid (CBDA), cannabidivarin (CBDV), cannabigerol (CBG), cannabigervarin (CBGV) and tetrahydrocannabivarin (THCV). The inflammatory skin disease may be caused by one or more of the following: microbial infection-induced dermatitis; solar dermatitis; atopic dermatitis; and allergic contact dermatitis.

Claims

1. A method of treating an inflammatory skin disease comprising administering cannabidiolic acid (CBDA) to a subject in need thereof, wherein the CBDA is administered at a dose of between 10 and 1000 mg, and wherein the inflammatory skin disease is microbial infection-induced dermatitis.

2. The method according to claim 1, wherein the CBDA is in the form of a highly purified extract of cannabis such that it is present at greater than 95% of the total extract (w/w).

3. The method according to claim 1, wherein the CBDA is synthetically produced.

4. The method according to claim 1, wherein the CBDA is used concomitantly with one or more other medicaments.

5. The method according to claim 4, wherein the one or more other medicaments is a corticosteroid.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which

(2) FIG. 1 shows the effects of phytocannabinoids on PolyIC-induced cytokine expression in HaCaT keratinocytes;

(3) FIG. 2 shows the effects of phytocannabinoids on PolyIC-induced cytokine release in HaCaT keratinocytes;

(4) FIG. 3 shows the effects of phytocannabinoids on PolyIC-induced cytokine expression and release in HPV-Ker keratinocytes;

(5) FIG. 4 shows the effects of phytocannabinoids on PolyIC-induced cytokine release in NHEK keratinocytes;

(6) FIG. 5 shows the effects of phytocannabinoids on UVB-induced cytokine expression and release in HaCaT keratinocytes;

(7) FIG. 6 shows the effects of phytocannabinoids on UVB-induced cytokine release in HPV-Ker keratinocytes;

(8) FIG. 7 shows the effects of phytocannabinoids on UVB-induced cytokine release in NHEK keratinocytes;

(9) FIG. 8 shows the effects of phytocannabinoids on SEB/TSLP-induced cytokine release in HPV-Ker keratinocytes;

(10) FIG. 9 shows an ELISA assay for MCP-2 release in the supernatants of poly-(I:C)-stimulated HaCaT cells in presence of vehicle or CBD;

(11) FIG. 10 shows an ELISA assay for MCP-2 release in the supernatants of poly-(I:C)-stimulated HaCaT cells in presence of vehicle or CBC (A), CBG (B), THCV (C) and CBGV (D);

(12) FIG. 11 shows the concentrations of AEA (A), 2-AG (B), PEA (C) and OEA (D) in poly-(I:C)-stimulated HaCaT cells (100 mg.Math.ml-1, 6 h, 37° C.) treated with CBD (20 mM); and

(13) FIG. 12 shows the effect of administration of CBD 24 h (A) 48 h (B) and 72 h (C) after DNFB-induced ACD in mice.

DETAILED DESCRIPTION

Example 1: Assessment of Anti-Inflammatory Effects of Phytocannabinoids (Functional In Vitro Studies)

(14) The following example uses six different phytocannabinoids to determine the effectiveness of them in three different models of skin inflammatory diseases namely; microbial infection-induced dermatitis; solar dermatitis; and atopic dermatitis.

(15) Different skin cell lines (HaCaT; HPV-Ker; and NHEK) were tested in the three different models to determine whether there was a significant difference between them.

(16) There are many different forms of inflammatory skin diseases including acne; alopecia areata; basal cell carcinoma; Bowen's disease; congenital erythropoietic porphyria; contact dermatitis; Darier's disease; dystrophic epidermolysis bullosa; eczema (atopic eczema); epidermolysis bullosa simplex; erythropoietic protoporphyria; fungal infections of nails; Hailey-Hailey disease; herpes simplex; hidradenitis suppurativa; hirsutism; hyperhidrosis; ichthyosis; impetigo; keloids; keratosis pilaris; lichen planus; lichen sclerosus; melisma; pemphigus vulgaris; plantar warts (verrucas); pityriasis lichenoides; polymorphic light eruption; psoriasis; pyoderma gangrenosum; rosacea; scabies; shingles; squamous cell carcinoma; Sweet's syndrome; and vitiligo.

(17) These diseases are all caused by inflammation of the skin and as such cannabinoids demonstrating positive results in one or more of the models of inflammatory skin diseases might suggest that such cannabinoids might be useful agents in the treatment of one or more of the inflammatory skin diseases described above.

(18) Materials and Methods

(19) Materials

(20) The phytocannabinoids cannabidiol (CBD), cannabidiolic acid (CBDA), cannabidivarin (CBDV), cannabigerol (CBG), cannabigervarin (CBGV) and tetrahydrocannabivarin (THCV) were tested for their ability to decrease skin inflammation.

(21) Following viability assays, two concentrations (0.3 and 3 μM) of each phytocannabinoid were selected which did not induce dramatic changes in cellular viability of human keratinocytes.

(22) Lipoteichoic acid from Staphylococcus aureus [LTA; Toll-like receptor 2 (TLR2) activator], polyinosinic-polycytidylic acid [poly-(I:C); plC; TLR3 activator], lipopolysaccharide (LPS; TLR4 activator) and Staphylococcus enterotoxin B (SEB; inflammation inductor in atopic dermatitis) were obtained from Sigma-Aldrich (St. Louis, Mo., USA).

(23) Thymic stromal lymphopoietin (TSLP; inflammation inductor in atopic dermatitis) were purchased from eBioscience, Ltd. (Hatfield, Ireland, United Kingdom) and were diluted in water.

(24) Cell Cultures

(25) Human immortalized keratinocytes (HPV-Kers and HaCaTs) were cultured in serum-free EpiLife medium (Life Technologies Hungary Ltd., Budapest, Hungary) supplemented with Human Keratinocyte Growth Supplement (HKGS; in 1:100; Life Technologies Hungary Ltd.) and antibiotics (preformed mixture of penicillin and streptomycin in 1:100; PAA Laboratories GmbH, Pasching, Austria and Fungizone® Antimicotic (in 1:200; Life Technologies Hungary Ltd.) or in Dulbecco's Modified Eagle Medium (DMEM; Life Technologies Hungary Ltd.) supplemented with 10 (V/V) % fetal bovine serum (FBS; Life Technologies Hungary Ltd.) and the above mentioned antibiotics mixture with Fungizone® antimicotic, respectively.

(26) For establishing primary normal human epidermal keratinocyte cultures (NHEKs), human skin samples were obtained after obtaining written informed consent from healthy individuals undergoing dermatosurgery, adhering to Helsinki guidelines, and after obtaining Institutional Research Ethics Committee's permission.

(27) NHEKs were isolated after overnight dermo-epidermal separation in 2.4 IU/ml dispase (Roche Diagnostics, Berlin, Germany) by short trypsin (0.05%, Sigma-Aldrich) digestion. Cells were cultured in the same medium than the immortalized keratinocytes: EpiLife serum-free medium supplemented with Human Keratinocyte Growth Supplement, mixture of antibiotics and Fungizone® antimicotic. All cells were cultured at 37° C. in humidified, 5% CO2 containing atmosphere, the medium was changed every other day, and cells were sub-cultured at 70-80% confluence.

(28) Determination of Cytokine Release (ELISA)

(29) Cells were treated as indicated for 6, 24 or 48 hours. Supernatants were collected, and the released amount of interleukin IL-6 and IL-8 were determined using OptEIA kits (BD Pharmingen, Franklin Lakes, N.J., USA) and IL-la (from the R&D Systems, Inc., Minneapolis, United States) according to the manufacturer's protocol.

(30) UVB Irradiation

(31) Culture medium of the cells cultivated in Petri-dishes (d=35 mm) was replaced by 800 μl colourless Sebomed Basal Medium. Lids were removed and cells were then irradiated by using a narrow-band UV-irradiation instrument (Bio-Sun microprocessor-controlled UV irradiation system; Wilber Lourmat, Marne-la-Vallee, France), with a total dose of 40 mJ/cm2 of UVB (312 nm).

(32) Immediately after the irradiation, the medium was replaced with the conventional culture medium of the cells (see above) with or without test compounds or vehicle. Following a 6-hr culture period, cells were harvested for RT-qPCR (supernatants were also collected in all cases).

(33) Quantitative Real-Time Polymerase Chain Reaction (RT-qPCR)

(34) RT-qPCR experiments were performed as described previously (Oláh et al., 2014) on a Roche Light Cycler 480 QPCR System (Roche Applied Sciences) using the 5′ nuclease assay. Total RNA was isolated using TRIzol (LifeTechnologies), DNase treatment was performed according to the manufacturer's protocol, and then 1 μg of total RNA were reverse-transcribed into cDNA by using High Capacity cDNA Kit from Life Technologies Corporation.

(35) PCR amplification was performed by using the TaqMan primers and probes (assay IDs: Hs00174092_m1 for IL-1α, Hs00174097_m1 for IL-1β, Hs00985639_m1 for IL-6, Hs00174103_m1 for IL-8 and Hs00174128_m1 for tumor necrosis factor-α [TNFα]).

(36) As internal control, expression of peptidyl-prolyl isomerase A (PPIA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and actin beta (ACTB) was determined (assay IDs: Hs99999904_m1 for PPIA, Hs99999905_m1 for GAPDH and Hs99999903_m1 for ACTB).

(37) The amount of the transcripts was normalized to those of the housekeeping gene using the ΔCT method. When indicated, the results were then normalized to the expression of the vehicle control or the LTA-treated culture (ΔΔCT method), and were plotted as mean±SD of 3 technical replicates.

(38) Statistical Analysis

(39) Data were analysed and graphs were plotted by using Origin Pro Plus 6.0 software (Microcal, Northampton, Mass., USA), using Student's two-tailed two samples t-test and P<0.05 values were regarded as significant differences.

(40) Results

(41) Determination of Effects of Phytocannabinoids on Inflammatory Response of Human Epidermal Keratinocytes

(42) Selection of Appropriate Models of Skin Inflammatory Diseases

(43) Three different cellular inflammatory models were tested in order to determine the effects of the phytocannabinoids in models of skin inflammatory diseases. These models were:

(44) Inflammation induced by TLR3 activation. This model mimics microbial infection-induced dermatitis. In this model all 3 keratinocyte cell types were tested to determine the phytocannabinoids effects on the cytokines: IL6 and IL8 on all 3 cell types; and IL1α on HaCaT keratinocytes.

(45) Inflammation induced by UVB irradiation. This model mimics solar dermatitis. In this model all 3 keratinocyte cell types were tested to determine the phytocannabinoids effects on the cytokines: IL6 and IL8 on all 3 cell types.

(46) Inflammation induced by the combination of Staphylococcus aureus enterotoxin B (SEB) and thymic stromal lymphopoietin (TSLP). This model mimics atopic dermatitis. In this model HPV-Ker cells were tested to determine the phytocannabinoids effects on the cytokine: IL8.

(47) A. Model of Microbial Infection-Induced Dermatitis

(48) HaCaT Keratinocytes

(49) In HaCaT keratinocytes, all of the phytocannabinoids (at both concentrations) resulted in a marked suppression of the TLR3-activation (by the administration of PolyIC, known ligand of TLR3) induced up-regulation of mRNA expressions of the pro-inflammatory cytokines IL1α, IL6 and IL8 (FIG. 1).

(50) These anti-inflammatory effects were also detected when the release of the above cytokines were tested (FIG. 2); namely, most phytocannabinoids were able to prevent the PolyIC-induced augmented secretion of the pro-inflammatory IL1α, IL6 and IL8.

(51) HPV-Ker Keratinocytes

(52) In HPV-KER cells, the tested phytocannabinoids did not exert any significant effect on the TLR3-activation induced up-regulation of mRNA expressions of the pro-inflammatory cytokines IL6 and IL8 (FIG. 3, upper panels).

(53) However, as a marked contrast, all measured phytocannabinoids prevented the PolyIC-induced augmented secretion of the pro-inflammatory IL8 whereas most phytocannabinoids also exerted anti-inflammatory action on the IL6 release (FIG. 3, lower panels). In the latter case, the only exception was CBGV which, at 0.3 μM, did not affect IL6 levels whilst, at higher concentration (3 μM), significantly further augmented the TLR3-activation induced augmented release of IL6.

(54) NHEKs

(55) Similar to as found on HPV-Ker cells, the tested phytocannabinoids did not exert any significant effect on the TLR3-activation induced up-regulation of mRNA expressions of the pro-inflammatory cytokines IL6 and IL8 (data not shown).

(56) However, the phytocannabinoids induced a very heterogeneous response on the PolyIC-induced augmented secretion of the pro-inflammatory cytokines IL6 and IL8 (FIG. 4). Namely: 1. Administration of CBD, CBDA, and CBG resulted in a dose-dependent suppression of the elevated release of both IL6 and IL8. 2. CBDV did not affect the release of IL6; however, at the higher concentration, it significantly further augmented the TLR3-activation induced augmented release of IL8. 3. CBGV did not affect the release of IL8; however, at the higher concentration, it significantly further augmented the TLR3-activation induced augmented release of IL6. 4. THCV, at low concentration, did not affect the IL6 level but it significantly suppressed the elevated release of IL8. However, at high concentration, it did not affect the release of IL8 but significantly further augmented the TLR3-activation induced augmented release of IL6.
B. Model of Solar Induced Dermatitis

(57) HaCaT Keratinocytes

(58) In HaCaT keratinocytes, the tested phytocannabinoids behaved in different ways on the mRNA and peptide levels of UVB-upregulated expressions of IL6 and IL8, depending on the concentration as described below and in FIG. 5.

(59) CBD: 1. anti-inflammatory effects: 0.3 μM vs. IL6 and IL8 (RT-qPCR) and vs. IL8 (ELISA); 3 μM vs. IL6 (ELISA) 2. pro-inflammatory effects: 3 μM vs. IL8 (RT-qPCR)

(60) CBDA: 1. pro-inflammatory effects: 0.3 μM vs. IL6 and IL8 (both RT-qPCR and ELISA); 3 μM vs. IL8 (both RT-qPCR and ELISA)

(61) CBDV 1. anti-inflammatory effects: 0.3 μM vs. IL6 (RT-qPCR); 3 μM vs. IL6 and IL8 (both RT-qPCR and ELISA)

(62) CBG 1. anti-inflammatory effects: 0.3 μM vs. IL6 (RT-qPCR); 3 μM vs. IL6 (both RT-qPCR and ELISA) 2. pro-inflammatory effects: 3 μM vs. IL8 (ELISA)

(63) CBGV 1. anti-inflammatory effects: 0.3 μM vs. IL6 (RT-qPCR); 3 μM vs. IL6 (both RT-qPCR and ELISA) 2. pro-inflammatory effects: 0.3 μM vs. IL6 (ELISA) and IL8 (both RT-qPCR and ELISA); 3 μM vs. IL8 (both RT-qPCR and ELISA)

(64) THCV 1. anti-inflammatory effects: 0.3 μM vs. IL6 (ELISA) and IL8 (both RT-qPCR and ELISA); 3 μM vs. IL6 (RT-qPCR) 2. pro-inflammatory effects: 3 μM vs. IL6 and IL8 (ELISA)

(65) HPV-Ker Keratinocytes

(66) All of the tested phytocannabinoids significantly and markedly suppressed the UVB-upregulated secretion of IL6 and IL8 (FIG. 6).

(67) Of further importance, none of the phytocannabinoids exerted pro-inflammatory actions.

(68) NHEKs

(69) In NHEKs, only certain phytocannabinoids (CBGV and CBDV vs. IL6; CBDA and CBG vs. IL8), and only at given (mostly lower) concentrations, could significantly suppress the UVB-upregulated expressions of the cytokines (FIG. 7).

(70) At higher concentrations, the phytocannabinoids (CBG and THCV vs. IL6; all vs. IL8) exhibited pro-inflammatory effects.

(71) C. Model of Atopic Dermatitis

(72) In this model, employed on HPV-Ker keratinocytes, higher concentrations of all tested phytocannabinoids exerted significant anti-inflammatory effects as they suppressed the SEB/TSLP-induced up-regulation of IL8 release (FIG. 8).

(73) Of importance, lower concentrations of the phytocannabinoids either did not induce any significant effect (CBDA, CBDV, CBG) or exert pro-inflammatory actions.

(74) Conclusions

(75) Tables 1 to 3 below summarize the results obtained with the tested phytocannabinoids in the various keratinocyte inflammatory models.

(76) The scores for the cannabinoids at the two concentrations are based on a score of 1 point attributed to an anti-inflammatory effect; a score of 0 points being attributed to no effect; and a score of −1 being attributed to a pro-inflammatory effect.

(77) Table 1 demonstrates that in the TLR3-induced inflammation model, which mimics microbial infection-induced dermatitis, all phytocannabinoids exerted remarkable anti-inflammatory actions.

(78) In particular the overall scores suggest that the cannabinoids CBDA and CBG might be most effective at the reduction of inflammation in microbial infection-induced dermatitis.

(79) Table 2 demonstrates in the UVB-induced inflammation model, which mimics solar dermatitis, all phytocannabinoids exerted remarkable anti-inflammatory actions only on HPV-Ker keratinocytes.

(80) Importantly, several phytocannabinoids induced pro-inflammatory actions (or did not cause measurable effects) in the other two models.

(81) The overall scores for the cannabinoids suggest that the compounds CBDA and CBG might be most effective in the reduction of inflammation caused by UV light.

(82) Table 3 demonstrates that in the SEB/TSLP-induced inflammation model, which mimics atopic dermatitis, all phytocannabinoids exerted remarkable, dose-dependent anti-inflammatory actions.

(83) At lower concentrations, certain phytocannabinoids induced augmentation of inflammation suggesting that not all cannabinoids at all concentrations are suitable candidates for use in the treatment of skin inflammatory diseases. The cannabinoids that were most effective were CBDA, CBDV and CBG in the reduction of inflammation caused by SEB/TSLP.

(84) TABLE-US-00002 TABLE 1 Summary of effects of phytocannabinoids on microbial infection-induced dermatitis Cell Cyto- CBD CBDA CBDV CBG CBGV THCV type kine M&M 0.3 3 0.3 3 0.3 3 0.3 3 0.3 3 0.3 3 HaCaT Il1α qPCR ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ELISA — ✓ — ✓ ✓ — — ✓ ✓ ✓ — ✓ IL6 qPCR ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ELISA — — — ✓ — — — ✓ — ✓ — ✓ IL8 qPCR ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ELISA — ✓ ✓ ✓ ✓ ✓ — — — ✓ ✓ ✓ HPV- IL6 ELISA — — ✓ ✓ ✓ ✓ ✓ ✓ — x ✓ ✓ Ker IL8 ELISA ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ NHEK IL6 ELISA — ✓ — ✓ — — — ✓ — x — x IL8 ELISA ✓ ✓ ✓ ✓ — x — ✓ — — ✓ — SCORE 5 8 7 10 7 5 5 9 5 5 7 7 Key: ✓ anti-inflammatory effect — no effect x pro-inflammatory effect

(85) TABLE-US-00003 TABLE 2 Summary of effects of phytocannabinoids on solar induced dermatitis Cell Cyto- CBD CBDA CBDV CBG CBGV THCV type kine M&M 0.3 3 0.3 3 0.3 3 0.3 3 0.3 3 0.3 3 HaCaT IL6 qPCR ✓ — x — ✓ ✓ ✓ ✓ ✓ ✓ — ✓ ELISA — ✓ x — — ✓ — ✓ x ✓ ✓ x IL8 qPCR ✓ x x x — ✓ — — x x ✓ — ELISA ✓ — x x — ✓ — x x x ✓ x HPV- IL6 ELISA ✓ ✓ — ✓ — ✓ ✓ ✓ ✓ ✓ ✓ ✓ Ker IL8 ELISA ✓ ✓ — ✓ — ✓ ✓ ✓ — ✓ ✓ ✓ NHEK IL6 ELISA — — — — ✓ — — x — ✓ — x IL8 ELISA — x ✓ x — x x ✓ — x x x SCORE 5 1 1 −1 2 5 2 3 −1 2 4 0 Key: ✓ anti-inflammatory effect — no effect x pro-inflammatory effect

(86) TABLE-US-00004 TABLE 3 Summary of effects of phytocannabinoids on atopic dermatitis Cell Cyto- CBD CBDA CBDV CBG CBGV THCV type kine M&M 0.3 3 0.3 3 0.3 3 0.3 3 0.3 3 0.3 3 HPV- IL8 ELISA x ✓ — ✓ — ✓ — ✓ x ✓ x ✓ Ker SCORE −1 1 0 1 0 1 0 1 −1 1 −1 1 Key: ✓ anti-inflammatory effect — no effect x pro-inflammatory effect

Example 2: Assessment of Cannabidiol in a Model of Allergic Contact Dermatitis (ACD)

(87) Methods

(88) Cell Culture

(89) The immortalized HaCaT cell line was cultured in DMEM supplemented with glutamine (2 mM), penicillin (400 U.Math.ml-1), streptomycin (50 mg.Math.ml-1) and 10% FBS at 37° C. in humidified 5% CO2.

(90) Poly-(I:C)-Induced Allergic Contact Dermatitis (ACD) in HaCaT Cells

(91) HaCaT cells were plated into twenty-four-well culture plates at a cell density of 2×105 cells per well, and after 1 day were stimulated with poly-(I:C) (100 μg.Math.ml-1) or vehicle (water) and incubated for 6 h at 37° C. in 5% CO2.

(92) To study the effect of CBD, poly-(I:C)-stimulated HaCaT cells were treated with CBD (1, -5, -10- and 20 μM) or vehicle (methanol) for the indicated times.

(93) The effects of the other phytocannabinoids such as cannabidiol acid (CBDA), cannabidivarin (CBDV), cannabidivarinic acid (CBDVA), cannabichromene (CBC), cannabigerol (CBG), cannabigerolic acid (CBGA), cannabigevarin (CBGV), tetrahydrocannabivarin (THCV) and tetrahydrocannabivarinic acid (THCVA) (all tested at 5, -10- and 20 μM) on MCP-2 production in poly-(I:C)-stimulated HaCaT cells were also investigated.

(94) After 6 h the supernatants were used for MCP-2 ELISA assay according to the manufacturer's instructions. Results are expressed as relative fold of pg m1-1 normalized for poly(I:C)-stimulated HaCaT cell values considered as 100% of released MCP-2.

(95) Cell Viability

(96) Cell viability was measured after 6 h in HaCaT cells treated with CBD (20 μM) or vehicle by using the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay.

(97) Briefly, after 6 h HaCaT cells were incubated with MTT (5 mg.Math.ml-1) for 3 h at 37° C. in 5% CO2. After 3 h HaCaT cells were lysed with DMSO and incubated for 6 h at 37° C. in 5% CO2.

(98) Absorbance was measured at 630 nm. Results are expressed as % of cell viability, where optical density values from vehicle-treated cells were defined as 100% of cell viability.

(99) Analysis of Endocannabinoids and Related N-Acylethanolamines

(100) HaCaT cells were plated into six-well culture plates at a cell density of 9×10.sup.5 cells per well, and after 1 day were stimulated with poly-(I:C) (100 μg.Math.ml-1) and treated with CBD (20 μM) or vehicle, and incubated for 6 h at 37° C. in 5% CO2.

(101) After 6 h the resulting cells and supernatants were subjected to measurement of endocannabinoids such as N-arachidonoyl-ethanolamine (anandamide, AEA) and 2-arachidonoyl-glycerol (2-AG), and related N-acylethanolamines such as PEA and N oleoylethanolamine (OEA).

(102) Cells and supernatants were homogenized in a solution of chloroform/methanol/Tris-HCl 50 mM pH 7.4 (2:1:1 by vol.) containing 10 pmol of [2H]8-AEA, and 5 pmol of [2H]5-2-AG, [2H]4-PEA and [2H]2-OEA as internal deuterated standards. The lipid-containing organic phase was pre-purified by open-bed chromatography on silica gel, and fractions obtained by eluting the column with a solution of chloroform/methanol (90:10 by vol.) were analyzed by liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (LC-APCI-MS) by using a Shimadzu HPLC apparatus (LC-10ADVP) coupled to a Shimadzu (LCMS-2020) quadrupole MS via a Shimadzu APCI interface.

(103) LC-APCI-MS analyses of AEA, 2-AG, PEA and OEA were carried out in the selected ion monitoring (SIM) mode using m/z values of molecular ions+1 for deuterated and undeuterated compounds, respectively as follows: 356 and 348 (AEA), 384.35 and 379.35 (2-AG), 304 and 300 (PEA), 328 and 326 (OEA). AEA, 2-AG, PEA and OEA levels were calculated on the basis of their area ratio with the internal deuterated standard signal areas, and their amounts (pmol) were normalized per ml of volume.

(104) DNFB-Induced Allergic Contact Dermatitis (ACD) in Mice

(105) A total of 10 animals per group were used in the experiments described here.

(106) Briefly, DNFB was diluted in acetone/olive oil (4:1 by vol.) immediately before use and eight-ten week-old female C57BL/6J mice were sensitized by painting 50 μl of 0.2% DNFB on the shaved abdomen on two consecutive days. Then, mice were challenged by painting 10 μl of 0.3% DNFB on both sides of one ear on day 5.

(107) Ear swelling was measured 24, 48 and 72 h after this first challenge by measuring the difference in ear thickness between the unchallenged and the challenged ear using an engineer's micrometer. CBD (2.5, -5- and 10 mg.Math.kg-1) was administered intraperitoneally (i.p.) on day 5 (the day of the first challenge with DNFB), 6 and, 7 after the initial sensitization with DNFB. CBD was dissolved in DMSO and Tween-20 1%.

(108) Data Analysis

(109) For the determination of MCP-2, endocannabinoids, PEA and OEA, group means were compared using the one-way ANOVA followed by Newman-Keuls multiple comparison test or the Student's t-test. For the determination of cell viability, group means were compared using the Student's t-test. All determinations were performed at least in triplicate. For the determination of ear thickness, group means were compared using the one-way ANOVA followed by Tukey-Kramer multiple comparison test (n=10).

(110) Materials

(111) HaCaT cell line was purchased from CLS Cell Lines Service. Cell culture media, antibiotics, MTT and DNFB were purchased from Sigma-Aldrich. Poly-(I:C) was purchased from InvivoGen.

(112) CBD, CBDA, CBDV, CBDVA, CBC, CBG, CBGA, CBGV, THCV and THCVA (>99.9% purity) were used.

(113) AM251, AM630 and I-RTX were purchased from Tocris Bioscience. MCP-2 ELISA kit was purchased from RayBiotech, Inc. Deuterated standards-[2H]8-AEA, [2H]5-2-AG, [2H]4-PEA and [2H]2-OEA-were purchased from Cayman Chemical.

(114) Eight to 10-week-old female C57BL/6J mice were purchased from Harlan Sprague Dawley Inc.

(115) Results

(116) MCP-2 Protein Levels in Poly-(I:C)-Stimulated HaCaT Cells

(117) The effects of CBD, CBDA, CBDV, CBDVA, CBC, CBG, CBGA, CBGV, THCV and THCVA on MCP-2 protein levels in poly-(I:C)-stimulated HaCaT cells were investigated.

(118) HaCaT cells stimulated for 6 h with poly-(I:C) (100 μg.Math.ml-1) and treated with the vehicle of the phytocannabinoids produced significantly higher levels of the MCP-2 chemokine (FIGS. 9 and 10) as compared to vehicle-stimulated HaCaT cells (data not shown).

(119) When HaCaT cells were co-stimulated with poly-(I:C) and CBD (1, -5-, 10- and 20 μM), a strong concentration-dependent reduction of MCP-2 protein levels as compared to poly-(I:C)-stimulated HaCaT cells treated with vehicle of CBD was observed (FIG. 9).

(120) The maximum effect was observed at highest concentration tested of CBD (20 μM), as compared to poly-(I:C)-stimulated HaCaT cells treated with vehicle of CBD (FIG. 9).

(121) On the contrary, when HaCaT cells were administered with poly-(I:C) and CBC or CBG no effect was observed at low concentrations (5 and −10 μM), although at the highest concentration tested (20 μM) CBC or CBG were able to reduce MCP-2 production (FIG. 10A, B).

(122) Likewise, when HaCaT cells were co-administered with poly-(I:C), THCV had no effect at the lowest concentration tested (5 μM), but at 10 μM and 20 μM THCV was able to reduce MCP-2 production (FIG. 100).

(123) CBGV was able to reduce MCP-2 production only at 10 μM (FIG. 10D).

(124) No effect was observed on MCP-2 protein levels after treatment of poly-(I:C)-stimulated HaCaT cells with CBDA, CBDV, CBDVA, CBGA and THCVA as compared to poly-(I:C)-stimulated HaCaT cells treated with the respective vehicles (data not shown).

(125) Likewise, no significant variation was observed on MCP-2 protein levels after that HaCaT cells were treated with CBD or the other phytocannabinoids alone (at highest concentration tested, 20 μM), i.e. in the absence of poly-(I:C), as compared to vehicle-treated HaCaT cells (data not shown), indicating that this concentration of CBD was not cytotoxic.

(126) AEA Levels in Poly-(I:C)-Stimulated HaCaT Cells

(127) The effect of CBD (20 μM) on AEA, 2-AG, PEA and OEA levels in poly-(I:C)-stimulated HaCaT cells was measured.

(128) It was observed that when HaCaT cells were stimulated with poly-(I:C), AEA levels were significantly increased by −3-fold compared to vehicle-treated HaCaT cells, and a nearly statistically significant trend towards elevation of PEA levels (P=0.0633) was observed (FIG. 11A,C).

(129) When poly-(I:C)-stimulated HaCaT cells were treated with CBD (20 μM), AEA levels were increased by −8-fold compared to vehicle-treated HaCaT cells, and by −2.7-fold compared to poly-(I:C)-stimulated HaCaT cells (FIG. 11A).

(130) No consistent effect of CBD was observed on 2-AG and OEA levels in poly-(I:C)-stimulated HaCaT cells (FIG. 11B, D).

(131) Effect of CBD on Ear Skin Oedema in Mice with DNFB-Induced Allergic Contact Dermatitis (ACD)

(132) The anti-inflammatory effect of CBD in an animal model of ACD was tested. It was observed that only CBD at the highest dose tested (10.Math.mg kg-1, i.p). administered on day 5, 6 and 7 reduced the ear thickness measured 48 and 72 h after the first challenge when compared to control mice (FIG. 12).

(133) The 2.5 and 5 mg.Math.kg-1 doses did not show any statistically significant effect (FIG. 12).

CONCLUSIONS

(134) CBD was more potent than other phytocannabinoids tested here (CBDA, CBDV, CBDVA, CBC, CBG, CBGA, CBGV, THCV and THCVA), in the inhibition of MCP-2 chemokine production in poly-(I:C)-stimulated keratinocytes cells.

(135) Furthermore, CBD exerts has been shown to produce anti-inflammatory effects in an in vivo model of allergic contact dermatitis (ACD) and as such is a potentially valuable therapeutic treatment option for this disease.

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