Topical composition with active compounds from <i>Cannabis sativa </i>and <i>Calendula officinalis </i>for reduction of skin lesions

Abstract

Disclosed is a topical composition including essential combination of synergistically acting phyto-active materials, non-psychotropic phytocannabinoids from the plant of Cannabis sativa: Cannabidiol, Cannabidiolic acid, Cannabivarin Cannabigerol in combination with extract of Calendula flower and the formulation of the base to ensure the features of anti-inflammation, anti-oxidation, emollient, and bactericidal components. The topical composition is an emollient dedicated for reduction of skin lesions caused by atopic dermatitis, urticaria, radiotherapy and UV induced skin damage and acne. In addition the topical composition could reduce secretion of fats, facilitate deep skin hydration, reduce pores and exert soothing effect.

Claims

1. A topical composition comprising: about 8.76% Caprylic/capric triglyceride, 5.64% Aloe barbadensis leaf juice, 0.94% Stearic acid, 0.52% Glycerol monostearate, 0.25% Trietanolamine, 0.4% Vitamin E, 3.33% Olive europaea Fruit Oil, 3.33% Glycerol, 0.6% Acrylates/C10-30 alkyl acrylate crosspolymer, 0.07% Calendula Officinalis Flower Extract, 0.8% Phenoxyethanol, and water, wherein the composition further comprises 0.75-1% of a Cannabis sativa extract in decarboxylated form and containing 0.25% of CBD, and 0.75-1% of the Cannabis sativa extract in non-carboxylated form and containing 0.25% CBDA, wherein the Cannabis sativa extract comprises about 80% of CBDA from the total cannabinoids, and THC is less than 0.1%.

2. A topical composition according to claim 1, wherein the Cannabis sativa extract is made from any non-psychotropic Cannabis sativa variety.

3. The topical composition according to claim 2, wherein the Cannabis sativa extract is made from the Benico variety.

4. A topical composition according to claim 1 with a dynamic viscosity from 2100 to 2300 mPa and a pH of about 6.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows human keratinocyte's cell viability to the exposure of diluted Cannabis sativa active materials after 24, 48 and 72 hours of incubation under cell optimal culturing conditions. Sample C- cell viability without exposure to any additives.

(2) FIG. 2 shows estimated GAG and sGAG produced in response to the keratinocyte exposure to diluted active materials after 24 hours of incubation under cell optimal culturing conditions. Control corresponds to non-treated human keratinocyte culture.

(3) FIG. 3 shows the difference between treated and untreated paw edema after 4, 6 and 24 hours. “Preparation 1” reduces paw edema rate (in comparison to the control group) by 43.2%-57.5%. Data presented as group averages ± SN. Differences between control/edema and phytocannabinoid preparation groups are considered significant at p<0.05*.

(4) FIG. 4 shows the difference between treated and untreated paw edema after 4, 6 and 24 hours. “Preparation 2” statistically significantly reduced paw edema (up to 44.4%). Data presented as group averages ± SN. Differences between control/edema and phytocannabinoid preparation groups are considered significant at p<0.05*.

DETAILED DESCRIPTION OF THE INVENTION

(5) Materials and Methods

(6) Preparation of Cannabis sativa Extract

(7) Cannabis extract is produced using pulverized, dried (passive drying process for 52 hours at 35-40° C., to reach residual moisture level of approximate 10%) biomass of Cannabis sativa “Benico” variety which is derived from the mixture comprising upper plant parts—blossoms and leaves. However, other non-psychotropic Cannabis sativa varieties can be used, where THC concentration rages from 0.0001 to 1%. The CBD and CBDA enriched lipophilic fraction (extract) is produced using an approach of supercritical extraction with CO.sub.2 with the following parameters: pressure—465 bar, extraction temperature—70° C., process length—120 minutes. The CBD resin is produced by decarboxylation of CBDA resin at 160° C. for 5-6 hours.

(8) The amount of major cannabinoids: CBD, CBDA, CBG, CBV, in the cannabis extract is determined according to the ST/NAR/40 “Recommended Methods for the Identification and Analysis of Cannabis Products” (United Nations, New York, 2009). 10 mg of the resin is homogenised in the 1 mL mixture of methanol and chloroform (v/v, 9:1), for 15 min in the ultrasound bath. After centrifugation for 10 min at max g to separate insolubilities, the prepared samples were analysed using Shimadzu HPLC chromatography system with 30AC automatic injector, CTO-20AC column thermostat, DGU 20A5 vacuum degas unit, LC-30AD pump and SPD-M20A diode matrix detector. The analysed cannabinoids were fractionated in Supelco Discovery HS C18 (25×4.6 mm, 5 μm; RP) with C18 pre-column under the isocratic elution conditions. The temperature of the column—30° C., mobile phase: acetonitrile 0.1%, flow 0.8 ml/min., separation time 30 min.; injection volume—20 μl; detection at the two channels 225 nm and 306 nm. The data was analysed with Lab Solutions software. The following internal standards were used from Cerilliant: Cannabinol, 1.0 mg/mL (C-046), Cannabidiolic acid (CBDA), 1.0 mg/mL (C-144), Cannabidivarin (CBDV), 1.0 mg/mL (C-140), Cannabigerol (CBG), 1.0 mg/mL (C-141).

(9) Characteristics of Cannabis sativa Extract—

(10) Protein fraction (Kjeldahl Method)—0.63%, Lipids (Soxlet Method) 96.60%, water (Gravimetric Method (103 dgr centigrade))—1.73%. Sum of CBD/CBDA/CBDV/CBG ˜18%, where the extract comprises 80% of CBDA from the total cannabinoids, THC<0.1%.

(11) Preparation of Topical Composition

(12) TABLE-US-00001 Vendor and Compound Content % specifications Caprylic/capric triglyceride 8.76 Aloe barbadensis leaf juice 5.64 Stearic acid 0.94 Glycerol monostearate 0.52 Triethanolamine 0.25 Vitamin E 0.4 Water (Distilled) 74.89 Olive europaea (Olive) Fruit Oil 3.33 Glycerol 3.33 Acrylates/C10-30 Alkyl Acrylate 0.6 Crosspolymer (Carbopol ® Ultrez 21) Calendula officinalis Flower Extract 0.07 Decarboxylated Cannabis sativa 0.25 CBD 15-18% CBD, extract CBD (2.5 mg/mL) 0.75 resin [Satimed] Non- carboxylated Cannabis sativa .sup. 0.25 CBDA 15-18% CBDA, extract CBDA (2.5 mg/mL) 0.75 resin [Satimed] Phenoxyethanol 0.8

(13) Initially, separately the groups of the following components are mixed: (i) required amounts of Caprylic/capric triglycerides, vitamin E and Olive europaea (Olive) fruit oil, (ii) glycerol, Aloe barbadensis leaf juice and water, (iii) Stearic acid, Glycerol monostearate, (iv) Phenoxyethanol is dissolved in water, (v) Triethanolamine is dissolved in water (vi) and separately Calendula officinalis flower extract is dissolved in water followed by 2 time filtration through the filter (0.45-0.75 μm). Finally the required amount of Carbopol® Ultrez 21 is dissolved in the largest amounts of water and incubated with mixing for rehydration and soaking. The groups of components (i), (ii) and (iii) are added to reactor and incubated under the slow mixing at 65±2° C. After the temperature is equilibrated Phenoxyethanol solution is added. The homogenisation at 3000 rpm is performed for 3 minutes. After homogenisation the triethanolamine solution is added and homogenisation at 4000 rpm is repeated for 4-5 minutes. The analogical homogenisation cycles are repeated after introduction of Calendula officinalis Flower Extract solution and decarboxylated and non-decarboxylated extracts of Cannabis sativa. The homogenisation is finalized at 5000 rpm for 10 minutes at 65° C. Finally the Carbopol® Ultrez 21 solution and required amount of residual water is added. The temperature is adjusted to 70° C. and homogenisation continued at 2000 rpm for 10 minutes. The dynamic viscosity of the product was monitored using Rotational Viscometer (Fungilab, SMART Series H) and pH using Consort (C832) pH-meter. The composition is packaged into vials by maintaining the reactor temperature of 65° C. and slow stirring—60 rpm.

EXAMPLES

Example 1

(14) Experimental Procedures

(15) Cell Culturing.

(16) Primary cell line of Human keratinocytes Heka (Gibco, ThermofisherScientic) was cultivated in GIBCO EpiLife serum-free cell culture medium with S7 supplement (ThermofisherScientic) in a microwell plate of 24 vials, 20,000 cells/per well, at 37° C., 5% CO.sub.2. The viable cells after adhesion to the well surface were subjected to 2 μl of Cannabis sativa extract which is used in described topical composition. The extract was prepared after several dissolving steps in ethanol to reach dilution levels from 10.sup.2 to 10.sup.7. The blank control—raw Cannabis sativa seed oil was prepared analogically. The Cannabis sativa extract comprises 18% of cannabinoids CBD/CBDA/CBDV/CBG, and 80% of CBDA from the total cannabinoid level.

(17) Cell Viability.

(18) Living cells were determined using the CCK-8 kit (Cell Counting Kit, Dojingo) while following the manufacturer's instructions. The amount of formazan resulted by activity of dehydrogenases dye was measured in the diluted media spectrophotometrically at 450 nm.

(19) Quantification of sGAG and GAG.

(20) The evaluation of production of extracellular, membrane and intracellular glycosaminoglycans was performed using Blyscan ELISA kits for quantification of sulfonated and non-sulfonated glycosaminoglycans and hyaluronic acid using The Purple-Jelley Hyaluronan Assay (Biocolor, Life Science Assays) after 24 and 48 hours, respectively. For membrane and intracellular analysis of sGAG and GAG, normalized amounts cells were harvested and fractionated. Assays were carried out by following instructions of manufacturer using 50 and 100 μl of harvested supernatant.

(21) Results:

(22) Cannabis sativa extract from the topical composition stimulates skin hydration via stimulation of synthesis of glycosaminoglycans and proliferation of human epidermal keratinocytes in vitro.

(23) Transepidermal water loss occurs due to water mobility and ability to move to the skin surface where it evaporates when exposed to air. sGAG production by keratinocytes maintain its content in the skin, which ensures accumulation of water and prevent dryness.

(24) In FIG. 1, plot of absorption units reflect the response of human keratinocyte's cell viability to the exposure of diluted Cannabis sativa active materials after 24, 48 and 72 hours of incubation under cell optimal culturing conditions. Sample C—cell viability without exposure to any additives.

(25) Experimental data shows that active materials of Cannabis sativa stimulate viability of human keratinocytes. The highest effect was observed after 24 h of cell treatment with cannabinoid entourage.

(26) In FIG. 2, the plot shows estimated GAG and sGAG produced in response to the keratinocyte exposure to diluted active materials after 24 hours of incubation under cell optimal culturing conditions. Control corresponds to non-treated human keratinocyte culture.

(27) Experimental data shows that active materials of Cannabis sativa stimulate production of sGAG and GAG by human keratinocytes. The highest effect was observed after 24 h of cell treatment with the highest dilution cannabinoid entourage compared to non-treated culture. This indicates that keratinocytes exposed to the cannabinoids CBD/CBDA/CBDV/CBG increase production of GAG and sGAG molecules which are very important in protection of skin moisture balance.

Example 2

(28) Experimental Procedures

(29) The effects of two phytocannabinoid preparations (JSC SATIMED): “Preparation 1” (in which the concentration of phytocannabinoids is 40 mg/ml, extract diluted 4.5 fold with raw Cannabis sativa seed oil) and “Preparation 2” (10 mg/ml of phytocannabinoids, extract diluted 10 fold with raw Cannabis sativa seed oil), were analysed on the model of acute inflammation (paw edema) in mice. Three single doses of preparation “Preparation 1” (0.33 mg/kg, 1 mg/kg and 10 mg/kg phytocannabinoids) and one dose of “Preparation 2” (0.23 mg phytocannabinoids) were tested. The experiment was performed on 48 mice (BALB/c strain ♀, 6-7 weeks old, average weight 23 g), which were divided into 6 groups (6 mice per group), and in all of them paw edema was induced:

(30) 1 group. Edema/control—no treatment

(31) 2 group. 50 mg/kg prednisolone/control i.p.

(32) 3 group. 0.33 mg/kg phytocannabinoids “Preparation 1” p.o.

(33) 4 group. 1 mg/kg phytocannabinoids “Preparation 1” p.o.

(34) 5 group. 10 mg/kg phytocannabinoids “Preparation 1” p.o.

(35) 6 group. 0.23 mg phytocannabinoids “Preparation 2” (topical application) (i.p.—intraperitoneally, p.o.—orally)

(36) Prior to the study, the thickness of the right hind foot of each mouse was measured by a digital micrometre (Mitutoyo, Japan). Acute inflammation (paw edema) was induced to all mice by an injection of 20 μl of 1% λ—carrageenan into their right paw. The effects of phytocannabinoids on acute inflammation (paw edema) were measured by analysing the differences between the thickness of the affected and unaffected paw. The test and control materials were administered to animals one hour after carrageenan injection. Digital micrometre measurements were carried out after 4, 6 and 24 hours of carrageenan injection.

(37) Results:

(38) Cannabis sativa extract of topical composition reduces inflammation of Paw edema in mice model after oral administration and topical treatment.

(39) Paw edema was reduced by all analysed doses of “Preparation 1”. The most effective inhibition was observed at 1 mg/kg phytocannabinoid dose, which reduced the rates of paw edema (in comparison to the control group) by 43.2%-57.5% depicted in FIG. 3.

(40) Local application of preparation “Preparation 2” statistically significantly reduced paw edema (up to 44.4%) as shown in FIG. 4.

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