Composition Comprising Sea Water and Cannabinoid Loaded Submicroparticles for Pharmaceutical, Nutraceutical and Cosmetic Applications

Abstract

The present invention refers to a composition comprising cannabinoid loaded polymer submicroparticles soluble in aqueous media comprising a polyelectrolyte complex formed by a positive charged polymer, selected from chitosan or a derivative thereof, a negative charged polymer selected from hyaluronic acid or a derivative thereof, and a cannabinoid, or a derivative thereof, or a mixture of cannabinoids, or derivatives thereof, and water, wherein the size of the submicroparticles is 600-750 nm, the weight ratio of positive charged polymer/negative charged polymer is 2.6:1-3.8:1, and the weight ratio polymers/cannabinoid is 100:1 to 1:100, wherein the cannabinoids are arranged in hydrophobic patches formed between the positive charged polymer and the negative charged polymer chains. The method of producing said composition and its nutraceutical, pharmaceutical and cosmetic uses are also contemplated.

Claims

1. A composition comprising cannabinoid loaded polymer submicroparticles soluble in aqueous media characterized in that the submicroparticles comprise: a polyelectrolyte complex formed by: a positive charged polymer selected from chitosan or a derivative thereof, a negative charged polymer selected from hyaluronic acid or a derivative thereof, and a cannabinoid, or a derivative thereof, or a mixture of cannabinoids, or derivatives thereof, and water, wherein the size of the submicroparticles ranges from 600 to 750 nm, the weight ratio of positive charged polymer/negative charged polymer is 2.6:1-3.8:1, and the weight ratio polymers/cannabinoid is 100:1 to 1:100, and wherein the cannabinoids are arranged in hydrophobic patches formed between the positive charged polymer and the negative charged polymer chains.

2. The composition according to claim 1 wherein the pH of the composition ranges from 4 to 7.8, preferably from 4.5 to 5.2.

3. The composition according to claim 1 wherein the weight ratio polymers/cannabinoid is from 10:1 to 1:10, preferably from 5:1 to 1:5.

4. The composition according to claim 1 wherein the cannabinoid is a phytocannabinoid or a derivative thereof, or a mixture of phytocannabinoids, or derivatives thereof, preferably selected from cannabidol, cannabigerol, or a mixture thereof.

5. (canceled)

6. The composition according to claim 1 wherein the negative charged polymer is a hyaluronic acid salt of a molecular weight from 0.5 to 0.75 MDa, preferably sodium hyaluronate.

7. (canceled)

8. The composition according to claim 1 wherein the positive charged polymer is chitosan of a molecular weight from 0.7 to 1 MDa.

9. The composition according to claim 1 wherein the water is sea water.

10. The composition according claim 1 wherein the submicroparticles has a Polydispersity Index (PDI) from 0.25 to 0.9, preferably from 0.35 to 0.5.

11. A method of producing a composition comprising cannabinoid loaded polymer submicroparticles soluble in aqueous media, according to claim 1, characterized in that said method comprises the steps of: a) Obtaining a solution of the positive charged polymer dissolved in an acidic aqueous solution, said solution consisting of acetic acid and sodium acetate, with pH adjusted at 4-4.5, b) Obtaining a solution of the negative charged polymer dissolved in an acidic aqueous solution, said solution selected from a buffer, consisting of an acetic acid and sodium acetate, or water, with pH adjusted at 4-4.5, c) Obtaining a solution of a cannabinoid, or a derivative thereof, or a mixture of cannabinoids, or derivatives thereof, by dissolving said cannabinoids in a solvent selected from ethanol, methanol and 1-isopropanol, d) Addition of the solution obtained in c) to the solution obtained in a), e) Addition of the solution obtained in b) to the solution obtained in d), and f) Shaking and mechanical or magnetic stirring the solution obtained in e) until the solvent is evaporated.

12. The method according to claim 11 wherein, in step a), the solution comprises chitosan in a concentration from 2 to 6%, preferably from 2.5 to 5%. the acidic aqueous solution consists of acetic acid 0.45% and sodium acetate 0.55% and the pH is adjusted with a suitable solution of HCl 5M and/or NaOH 5M.

13. (canceled)

14. (canceled)

15. (canceled)

16. The method according to claim 11 wherein solution of step b) comprises a hyaluronic acid salt in a concentration from 0.5 to 3% (w/w), preferably from 0.5% to 2.5% (w/w).

17. (canceled)

18. (canceled)

19. The method according to claim 11 wherein the acidic aqueous solution of step b) is selected from a buffer consisting of acetic acid, in a concentration from 0.2% to 0.9%, and sodium acetate, in a concentration from 0.2% to 0.9%, or an aqueous solution consisting of sea water, in a concentration from 0.1 to 10%, preferable from 3 to 4% (w/v), with the pH adjusted by the addition of HCl.

20. The method according to claim 11 wherein in step c), the concentration of the cannabinoid in the solvent (w/v) is from 0.1 to 50%, preferably from 10 to 40% and more preferably from 15 to 30%.

21. The method according to claim 11 wherein in step d) the solution of cannabinoids is added to the solution of step a) in a ratio (v/v) from 0.04:1 to 0.2:1, preferably from 0.04:1 to 0.1:1.

22. (canceled)

23. The method according to claim 11 wherein in step e) the solution obtained in step b) is added to solution obtained in step d) in a ratio (v/v) from 0.5:1 to 1:0.5, preferably from 0.7:1 to 1:0.7, more preferably from 0.8:1 to 1:0.8.

24. The method according to claim 11 wherein in step f) the solution is shaken for a period from 30 s to 5 min, preferably from 30 s to 90 s, followed by mechanical or magnetic stirring for a period from 12 to 48 h, preferably from 12 to 36, and more preferably from 18 to 30.

25. A nutraceutical, pharmaceutical or cosmetic composition comprising the composition of any of claim 1.

26. Pharmaceutical composition according to claim 25 for use in the treatment of skin conditions selected from acne and atopic dermatitis.

27. Pharmaceutical composition according to claim 25 for use in the treatment of eye disorders selected from diabetic retina.

28. A non-therapeutic use of the cosmetic composition, according to claim 25, for relaxing, soothing and moisturizing effects on the skin.

29. Pharmaceutical composition according to claim 25 for use in the control of the body weight or for the improvement of the well-being of the human body.

30. (canceled)

31. (canceled)

32. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0046] FIG. 1. Configuration of the polyelectrolite complex. Phytocannabinoids (balls) are placed in hydrophobic patches (black regions) formed between the protonated amine (+) of chitosan (hollow regions) and carboxylate anions () of hyaluronic acid (dotted regions); (=) interactions between pytocannabinoids and hydrophobic patches.

DETAILED DESCRIPTION OF THE INVENTION

[0047] In response to the needs of the state of the art, the authors of the invention have performed new methods for solubilization of cannabinoids in aqueous media through their incorporation in polymer submicroparticles, preferably in the presence of sea water. The subsequent product is suitable for pharmaceutical, cosmetic and nutraceutical applications.

[0048] In a first aspect, the present invention refers to a composition comprising cannabinoid loaded polymer submicroparticles soluble in aqueous media wherein said submicroparticles comprise: [0049] a polyelectrolyte complex formed by: [0050] a positive charged polymer selected from chitosan or a derivative thereof, [0051] a negative charged polymer selected from hyaluronic acid or a derivative thereof, [0052] a cannabinoid, or a derivative thereof, or a mixture of cannabinoids, or derivatives thereof, and [0053] water,
being the size of the submicroparticles from 600 to 750 nm, the weight ratio of positive charged polymer/negative charged polymer from 2.6:1 to 3.8:1, and the weight ratio polymers/cannabinoid from 100:1 to 1:100, preferably from 10:1 to 1:10 and most preferably from 5:1 to 1:5, and wherein the cannabinoids are arranged in hydrophobic patches formed between the positive charged polymer and the negative charged polymer chains.

[0054] In a particular embodiment, the pH of the composition ranges from 4 to 7.8, preferably from 4.5 to 5.2.

[0055] Submicroparticles are formed due to polyelectrolyte complexation. In this process, electrostatic interaction occurs between the protonated amines of the chitosan and the carboxylate anions of the hyaluronic acid and therefore promotes the formation of the particles (nucleation). The increase in the size of the particles proceeds via aggregation due to interaction of patches of the polymer chains of different charge (protonated amines of the chitosan and carboxylate anions of hyaluronic acid).

[0056] The internalization of the CHI/HA particles of the present invention in the cell proceeds via recognition of the hyaluronic acid chains by the CD44 protein receptor of the cell surface.

[0057] Once molecular recognition occurs, vesicles containing the CHI/HA particles are formed. Such vesicles get entrapped into endosomes which later disrupt and liberate the cannabinoids in the cytoplasm due to the endosomolytic activity of the chitosan chains.

[0058] Cannabinoids are dibenzopyrane or benzochromen derivatives and in all cases hydrophobic molecules. The incorporation of the cannabinoids into the submicroparticles takes place in the hydrophobic patches formed between the chitosan and hyaluronic acid chains (see FIG. 1). CHI and HA possess a hydrophobic patch of eight or nine CH units, stretching along three neighboring sugar units and present on alternate sides of tapelike helices. Such helices interact between neighbouring CHI and HA chains and create hydrophobic pockets where cannabinoids accommodate.

[0059] In a preferred embodiment, cannabinoids are a phytocannabinoid or a derivative thereof, or a mixture of phytocannabinoids, or derivatives thereof.

[0060] Phytocannabinoids can be obtained not only from natural sources but also from chemical synthesis, biochemical synthesis or from genetically modified microorganism. Accordingly, phytocannabinoids used in the formulation of the present invention can be natural or synthetic.

[0061] Phytocannabinoids can be selected, among others, from cannabigerolic acid, cannabigerolic acid monomethylether, cannabigerol, cannabigerolmonomethylether, cannabigerovarinic acid, cannabigerovarin, cannabichromenic acid, cannabichromene, cannabichromevarinic acid, cannabichromevarin, cannabidiolic acid, cannabidiol, cannabidiol monomethylether, cannabidiol C4, cannabidivarinic acid, cannabidivarin, cannabidioreol, delta-9-(trans)-tetrahydrocannabinolic acid A, delta-9-(trans)-tetrahydrocannabinolic acid B, delta-9-(trans)-tetrahydrocannabinol, delta-9-(trans)-tetrahydrocannabinolic acid C4, delta-9-(trans)-tetrahydrocannabinol-C4, delta-9-(trans)-tetrahydrocannabivarinic acid, delta-9-(trans)-tetrahydrocannabivarin, delta-9-(trans)-tetrahydrocannabiorcolic acid, delta-9-(trans)-tetrahydrocannabiorcol, delta-8-(trans)-tetrahydrocannabinolic acid, delta-8-(trans)-tetrahydrocannabinol, cannabicyclolic acid, cannabicyclol, cannabicyclovarin, cannabielsoic acid A, cannabielsoic acid B, cannabielsoin, cannabinolic acid, cannabinol, cannabinol methylether, cannabinol-C4, cannabivarin, cannabiorcol, cannabinodiol, cannabinodivarin, ()-cannabitriol, (+)-cannabitriol, (+)-9,10-dihydroxy-delta 6a(10a)-tetrahydrocannabinol, ()-10-ethoxy-9-dihydroxy-delta 6a(10a)-tetrahydrocannabinol, (+)-8,9-dihydroxy-delta 6a(10a)-tetrahydrocannabinol, cannabidiolic acid tetrahydrocannabitriol ester and mixtures thereof.

[0062] In a particular embodiment, the phytocannabinoids are selected from cannabidiol, cannabigerol or a mixture thereof. In another particular embodiment a cannabis sativa extract can be used as phytocannabinoids source. The cannabis sativa extract comes from any part of the cannabis sativa plant including flower, leaf, stem and seeds.

[0063] The polymers used in the formulations of the present invention can be natural or synthetic polymers.

[0064] In a preferred embodiment the negative charged polymer is a hyaluronic acid salt of low molecular weight (M), where M0.75.Math.10.sup.6 Da, or a hyaluronic acid salt of high molecular weight (M), where M2.2.Math.10.sup.6 Da, or a mixture thereof, more preferably the hyaluronic acid salt is of low molecular weight, where 0.5.Math.10.sup.6 DaM0.75.Math.10.sup.6 Da or a hyaluronic acid salt of high molecular weight (M), where 1.9.Math.10.sup.6M2.2.Math.10.sup.6 Da or a mixture thereof. In a most preferred embodiment, the hyaluronic acid salt is of a molecular weight from 0.5 to 0.75 MDa.

[0065] Said low molecular or high molecular weight salts are of the same nature. In a most preferred embodiment, they hyaluronic acid salt is sodium hyaluronate.

[0066] In a preferred embodiment the positive charged polymer is chitosan of a molecular weight from 0.7 to 1 MDa.

[0067] In a preferred embodiment the degree of deacetylation of chitosan is >20%, in a more preferred embodiment the degree of deacetylation is>50% and most preferably the degree of acetylation is >70%.

[0068] In a preferred embodiment, the water component of the composition of the invention is sea water. The incorporation of sea water in the formulation increases the cannabinoid encapsulation comparing to regular water.

[0069] Additionally, sea water enhances the benefits of cannabinoids for the treatment of skin conditions such as acne, atopic dermatitis and psoriasis and for the management of eye disorders.

[0070] In the composition of the invention, the size of the CHI and HA chains are similar in molecular weight to each other which, together with the presence of the cations of seawater that acts as a counterion of the excess of negative charges of HA with respect to the positive ones of CHI, results in the formation of submicroparticles with a homogeneous particle size distribution with a Polydispersity Index (PDI) from 0.25 to 0.9, preferably from 0.35 to 0.5.

[0071] In a second aspect, the present invention refers to a method of producing the composition comprising cannabinoid loaded polymer submicroparticles soluble in aqueous media, said method comprising the steps of: [0072] a) Obtaining a solution of the positive charged polymer dissolved in an acidic aqueous solution, said solution consisting of acetic acid and sodium acetate, with pH adjusted at 4-4.5, [0073] b) Obtaining a solution of the negative charged polymer dissolved in an acidic aqueous solution, said solution selected from a buffer, consisting of an acetic acid and sodium acetate, or water, with pH adjusted at 4-4.5, [0074] c) Obtaining a solution of a cannabinoid, or a derivative thereof, or a mixture of cannabinoids, or derivatives thereof, by dissolving said cannabinoids in a solvent selected from ethanol, methanol and 1-isopropanol, [0075] d) Addition of the solution obtained in c) to the solution obtained in a), [0076] e) Addition of the solution obtained in b) to the solution obtained in d), and [0077] f) Shaking and mechanical or magnetic stirring the solution obtained in e) until the solvent is evaporated.

[0078] The positive charged polymer is selected from chitosan or a derivative thereof and the negative charged polymer is selected from hyaluronic acid or a derivative thereof.

[0079] In a particular embodiment, the solution of step a) comprises chitosan in a concentration 2-6% (w/w), preferably 2.5-5% (w/w) with a molecular weight from 0.7 to 1 MDa. Preferably, the acidic aqueous solution consists of acetic acid 0.45% and sodium acetate 0.55% and the pH is adjusted with a suitable solution of HCl 5M and/or NaOH 5M.

[0080] In another particular embodiment, the solution of step b) comprises a hyaluronic acid salt, in a concentration from 0.5 to 3% (w/w), preferably 0.5-2.5% (w/w). Preferably, the hyaluronic acid salt is sodium hyaluronate of a molecular weight from 0.5 to 0.75 MDa.

[0081] In step b), the acidic aqueous solution is preferably selected from: [0082] a buffer consisting of acetic acid, in a concentration from 0.2% to 0.9%, and sodium acetate, in a concentration from 0.2% to 0.9%, or [0083] an aqueous solution consisting of sea water, in a concentration from 0.1 to 10%, preferable from 3 to 4% (w/v), with the pH adjusted by the addition of HCl.

[0084] In step c) the solution of cannabinoids is dissolved in a proper solvent, preferably selected from the group consisting of methanol, ethanol, 1-propanol and mixtures thereof. The concentration of the cannabinoid in the solvent (w/v) is 0.1-50%, preferably 10-40% and more preferably 15-30%.

[0085] In step d) the solution of cannabinoids is added to the solution of step a) in a ratio (v/v) 0.04:1-0.2:1, preferably 0.04:1-0.1:1.

[0086] Preferably, the cannabinoids are phytocannabinoids selected from cannabidiol, cannabigerol or a mixture thereof.

[0087] In step e) the solution obtained in step b) is added to solution obtained in step d) in a ratio (v/v) 0.5:1-1:0.5, preferably 0.7:1-1:0.7, more preferably 0.8:1-1:0.8.

[0088] In step f) the solution thus obtained is shaken for a period from 30 s to 5 min, preferably from 30 s to 90 s, followed by mechanical or magnetic stirring for a period from 12 h to 48 h, preferably from 12 h to 36 h, and more preferably from 18 h to 30 h.

[0089] The method provides homogeneously distributed submicroparticles that contain cannabinoids.

[0090] The methods of the present invention afford scalability so that the fabrication method can be performed at industrial level (manufacturing).

[0091] The compositions of the invention are suitable for pharmaceutical, cosmetic and nutraceutical applications.

[0092] Therefore, in another aspect, the invention refers to a pharmaceutical composition comprising the cannabinoid formulations of the present invention and their uses in different pharmaceutical or medical applications. In particular, the present invention refers to the use of this pharmaceutical composition in the treatment of skin conditions such as acne, atopic dermatitis and psoriasis and for the treatment of eye disorders.

[0093] The presence of sea water in the composition of the invention enhances the benefits of cannabinoids on the skin and also on eye disorders.

[0094] The present invention also refers to a cosmetic composition comprising the phytocannabinoid formulations of the present invention and its use for cosmetic applications. Specifically, the cosmetic composition of the invention affords compositions with relaxing, soothing and moisturizing effects on the skin.

[0095] The present invention also refers to a nutraceutical composition comprising the composition of the present invention and its use for nutraceutical applications.

[0096] Specifically, nutraceutical compositions of the invention are useful for relaxation, calming and moisturization of the skin and for the management of the body weight and for the improvement of the well-being of the human body.

[0097] The composition of the invention can be formulated for topical, systemic or oral administration. For example, compositions can be formulated as a solid ingredient, topical gel/serum, eye/nasal drop and oral suspension.

[0098] Suitable compositions for topical administration can be formulated as gels, ointments, creams, lotions, drops, etc.

[0099] The systemic administration of the composition includes delivering the phytocannabinoid composition by injection, wherein the injection is intravenous, intra-articular, intramuscular, intradermal, intraspinal, intraperitoneal, subcutaneous, a bolus or a continuous administration. The composition of the invention can also be administered by intranasal administration.

[0100] The composition of the invention can also be administered by oral administration, such edible gels in the case of nutraceutical compositions.

[0101] The compositions of the invention can also be administered including in a medical device. Specifically, the composition of the invention can be drawn into a syringe for a water-based injection medium.

[0102] In particular embodiments, the resulting formulation can be liophilized in order to obtain a solid product that can be included as an ingredient in the formulation of pharmaceutical, cosmetic and nutraceutical product.

[0103] Submicroparticles can be also embedded in a matrix of hydroxypropylmethylcellulose (HPMC) in order to generate different final products, such as serum and eye drops.

[0104] Resulting formulations can be optionally sterilized, as in the case of injectable formulations. Sterilization process may be performed by steam sterilization, in an autoclave at a temperature ranging from 120 C. to 140 C. In particular, the sterilization can be performed at 121 for 15 to 20 minutes, preferably 15 min, to obtain F0>15 (sterilizing value). Dry-heat is also employed to achieve sterilization.

EXAMPLES

Analytical Techniques

Viscosity

[0105] The viscosity of the product was obtained at 25 C. using cone-and-plate geometry of 40 mm diameter and a truncation (gap) of 115 m, at a shear rate of 1 s.sup.1. Formulations made from this product were measured at a shear rate of 1s.sup.1, except for the drops, which were measured using a shear rate of 66 s.sup.1.

pH

[0106] pH of the product is determined after calibrating the pH meter with a minimum sensitivity of 95%.

High Performance Liquid Cromatography (HPLC)

[0107] This technique was used to determine the total content of phytocannabinoids (CBD) in the formulations and the amount of CBD encapsulated in the chitosan:hyaluronic acid submicroparticles.

[0108] The analysis of the total amount of CBD in the system was performed by direct dilution of the samples in methanol followed by filtration through disposable 0.22 m PVDF filters and subsequent injection in the chromatography equipment. In addition, for the determination of the encapsulated CBD, the product was filtered through disposable 0.22 m PVDF filters, subsequently diluted in methanol and refiltered through these PVDF filters.

[0109] A HPLC-DAD analytical method according to Table 1 was developed in order to quantify the CBD concentration in the formulations.

TABLE-US-00001 TABLE 1 Chromatographic method for the quantification of CBD. System HPLC (1260 series Agilent Technologies) Column Zorbax Eclipse XCB-C18 (150 4.6 mm, 5 m particle, Agilent) Mobile- Channel A: Ammonium formate 10 mM (pH 3.6, with formic Phase acid) Channel B: Acetonitrile Gradient 0-4 min 52-80% of B; 4-9.5 min 80% of B at 1 ml/min (post- run = 2 min) Detector DAD (210, 228 and 270 nm)

[0110] In this method a stock solution of 1000 mg/L of CBD in methanol was prepared from which 0.5, 1, 3, 5, 7.5, 10, 20 and 30 mg/L standard dilutions of CBD in methanol were made.

Dynamic Light Scattering (DLS)

[0111] DLS measurements were performed by diluting 200 L of the samples in 1000 L of water and filtering them using PES syringe filters of 0.45 m followed by analysis in the DLS equipment at 173 measurement angle. Attenuator value, measurement position and count number were employed as measurement quality indicators (table 2). In addition, zeta potential was measured at 40 V voltage.

TABLE-US-00002 TABLE 2 DLS analysis method parameters for the characterization of vehiculized CBD. Dispersant Water Temperature 25 C. Viscosity 0.8872 cP RI 1.330 Dielectric constant 78.5 Fitting model Smoluchowski Equilibration time 120 s Cell time Capillary cell DTS1070 Measurement angle 173.sup. Number of measurements per 3 sample

Preparation of Artificial-Sea Water (SW)

[0112] Artificial Sea Water was prepared as follows:

[0113] 1 L of artificial SW was prepared by dissolving Sodium chloride (24.53 g), Magnesium chloride (5.20g), Sodium sulfate (4.09 g), Calcium chloride (1.16 g), Potassium chloride (0.695 g), Sodium bicarbonate (0.201 g), Potassium Bromide (0.101 g), Boric acid (0.027 g), Strontium chloride (0.0025 g) and Sodium Fluoride (0.003 g) in purified water (988.968 g). The solution was kept under magnetic stirring until the complete dissolution of the reactants was reached. Artificial Sea Water was prepared according to ASTM D1141-1 98.

Example 1. CHI:HA-CBD Submicroparticle Solution Without Sea Water (CHI:HA1.25:0.38 With 1.3 mg CBD)

[0114] 0.5 g of Chitosan (MW 0.70 MDa) was dissolved in 20 mL acetic acid and sodium acetate buffer (pH 4.22) for 12 hours to obtain a chitosan solution of 2.5% (v/w). The pH of the solution was adjusted to 4.5 by the addition of appropriate volumes of HCl 5 M. 0.150 g of low molecular weight hyaluronic acid (MW 0.66 MDa) was added via spatula to 20 mL acetic acid and sodium acetate buffer (pH 4.22). The resulting solution was stirred mechanically for 12 h until complete solution of the polymer was reached. Following a solution of phytocannabinoid (1.3 mg of CBD) in 130 microL of ethanol absolute was added to 2 mL of the 2.5% chitosan solution. 2 mL of the previously prepared 0.75% (w/v) HA solution were added to the CHI and CBD solution while it was kept under magnetic stirring. The resulting solution was shaken for 60 seconds followed by mechanical stirring for 30 minutes. Finally, the solution was kept under magnetic stirring for 24 hours to ensure evaporation of ethanol.

Example 2. CHI:HA-CBD Submicroparticle Solution (CHI:HA1.25:0.38 With 1.3 mg CBD)

[0115] The experiment of example 1 was repeated using sea water for the solubilization of hyaluronic acid instead of using acetic acid/sodium acetate buffer. The protocol was adapted as follows:

[0116] 0.5 g of Chitosan (MW 0.70 MDa) was dissolved in 20 mL acetic acid and sodium acetate buffer (pH 4.39) for 12 hours to obtain a chitosan solution of 2.5% (v/w). The pH of the solution was adjusted to 4.5 by the addition of appropriate volumes of HCl 5 M. 0.150 g of low molecular weight hyaluronic acid (MW 0.66 MDa) was added via spatula to 20 mL of sea water (pH 4.03, adjusted with HCl). The resulting solution was stirred mechanically for 12 h until complete solution of the polymer was reached. Following a solution of phytocannabinoid (1.3 mg of CBD) in 130 microL of ethanol absolute was added to 2 mL of the 2.5% chitosan solution. 2 mL of the previously prepared 0.75% (w/v) HA solution were added to the CHI and CBD solution while it was kept under magnetic stirring. The resulting solution was shaken for 60 seconds followed by mechanical stirring for 30 minutes. Finally, the solution was kept under magnetic stirring for 24 hours to ensure evaporation of ethanol.

Example 3. CHI:HA-CBD Submicroparticle Solution (CHI:HA1.25:0.38 With 20 mg CBD)

[0117] The experiment of example 2 was repeated increasing the CBD % of the formulation from 0.032% (w/v) to 0.5% (w/v). The protocol was adapted as follows:

[0118] 0.5 g of Chitosan (MW 0.70 MDa) was dissolved in 20 mL acetic acid and sodium acetate buffer (pH 4.39) for 12 hours to obtain a chitosan solution of 2.5% (v/w). The pH of the solution was adjusted to 4.5 by the addition of appropriate volumes of HCl 5 M. 0.150 g of low molecular weight hyaluronic acid (MW 0.66 MDa) was added via spatula to 20 mL of sea water (pH 4.03 (adjusted with HCl)). The resulting solution was stirred mechanically for 12 h until complete solution of the polymer was reached. Following a solution of phytocannabinoid (20 mg of CBD) in 92.6 microL of ethanol absolute was added to 2 mL of the 2.5% chitosan solution. 2 mL of the previously prepared 0.75% (w/v) HA solution were added to the CHI and CBD solution while it was kept under magnetic stirring. The resulting solution was shaken for 60 seconds followed by mechanical stirring for 30 minutes. Finally, the solution was kept under magnetic stirring for 24 hours to ensure evaporation of ethanol.

Example 4. CHI: HA-CBD Submicroparticle Solution Without Sea Water (CHI:HA2.5:0.75, No Sea Water)

[0119] The experiment of example 1 was repeated doubling the polymer concentration and increasing the CBD concentration from 0.032% (w/v) to 0.98% (w/v). The protocol was adapted as follows:

[0120] 1 g of Chitosan (MW 0.70 MDa) was dissolved in 20 mL acetic acid and sodium acetate buffer (pH 4.39) for 12 hours to obtain a chitosan solution of 5% (v/w). The pH of the solution was adjusted to 4.5 by the addition of appropriate volumes of HCl 5 M. 0.3 g of low molecular weight hyaluronic acid (MW 0.66 MDa) was added via spatula to 20 mL of acetic acid and sodium acetate buffer (pH 4.39). The resulting solution was stirred mechanically for 12 h until complete solution of the polymer was reached. Following, a solution of phytocannabinoid (39 mg of CBD) in 177 microL of ethanol absolute was added to 2 mL of the 5% chitosan solution. 2 mL of the previously prepared 1.5% (w/v) HA solution were added to the CHI and CBD solution while it was kept under magnetic stirring. The resulting solution was shaken for 60 seconds followed by mechanical stirring for 30 minutes. Finally, the solution was kept under magnetic stirring for 24 hours to ensure evaporation of ethanol.

Example 5. CHI:HA-CBD Submicroparticle Solution (CHI:HA2.5:0.75)

[0121] The experiment of example 4 was repeated using sea water for the solubilization of hyaluronic acid instead of acetic acid/sodium acetate buffer. The protocol was adapted as follows:

[0122] 1 g of Chitosan (MW 0.70 MDa) was dissolved in 20 mL acetic acid and sodium acetate buffer (pH 4.39) for 12 hours to obtain a chitosan solution of 5% (v/w). The pH of the solution was adjusted to 4.5 by the addition of appropriate volumes of HCl 5 M. 0.3 g of low molecular weight hyaluronic acid (MW 0.66 MDa) was added via spatula to 20 mL of sea water (pH 4.03 (adjusted with HCl)). The resulting solution was stirred mechanically for 12 h until complete solution of the polymer was reached. The pH of the solution was adjusted to 4.5 by the addition of appropriate volumes of HCl 5 M. Following, a solution of phytocannabinoid (39 mg of CBD) in 177 microL of ethanol absolute was added to 2 mL of the 5% chitosan solution. 2 mL of the previously prepared 1.5% (w/v) HA solution were added to the CHI and CBD solution while it was kept under magnetic stirring. The resulting solution was shaken for 60 seconds followed by mechanical stirring for 30 minutes. Finally, the solution was kept under magnetic stirring for 24 hours to ensure evaporation of ethanol.

Example 6. CHI:HA-CBD Submicroparticle Solution (CHI:HA2.5:0.25)

[0123] The experiment of example 5 was repeated using a 0.5% (w/v) HA solution instead of 1.5% (w/v). The protocol was adapted as follows:

[0124] 1 g of Chitosan (MW 0.70 MDa) was dissolved in 20 mL acetic acid and sodium acetate buffer (pH 4.39) for 12 hours to obtain a chitosan solution of 5% (v/w). The pH of the solution was adjusted to 4.5 by the addition of appropriate volumes of HCl 5 M. 0.1 g of low molecular weight hyaluronic acid (MW 0.66 MDa) was added via spatula to 20 mL of sea water (pH 4.4 (adjusted with HCl)). The resulting solution was stirred mechanically for 12 h until complete solution of the polymer was reached. The pH of the solution was adjusted to 4.5 by the addition of appropriate volumes of HCl 5 M. Following, a solution of phytocannabinoid (39 mg of CBD) in 177microL of ethanol absolute was added to 2 mL of the 5% chitosan solution. 2 mL of the previously prepared 0.5% (w/v) HA solution were added to the CHI and CBD solution while it was kept under magnetic stirring. The resulting solution was shaken for 60 seconds followed by mechanical stirring for 30 minutes. Finally, the solution was kept under magnetic stirring for 24 hours to ensure evaporation of ethanol.

Example 7. CHI:HA-CBD Submicroparticle Solution (CHI:HA2.5:0.5)

[0125] The experiment of example 5 was repeated using a 1% (w/v) HA solution instead of 1.5% (w/v). The protocol was adapted as follows:

[0126] 1 g of Chitosan (MW 0.70 MDa) was dissolved in 20 mL acetic acid and sodium acetate buffer (pH 4.39) for 12 hours to obtain a chitosan solution of 5% (v/w). The pH of the solution was adjusted to 4.5 by the addition of appropriate volumes of HCl 5 M. 0.2 g of low molecular weight hyaluronic acid (MW 0.66 MDa) was added via spatula to 20 mL of sea water (pH 4.54 (adjusted with HCl)). The resulting solution was stirred mechanically for 12 h until complete solution of the polymer was reached. The pH of the solution was adjusted to 4.5 by the addition of appropriate volumes of HCl 5 M. Following, a solution of phytocannabinoid (39 mg of CBD) in 177 microL of ethanol absolute was added to 2 mL of the 5% chitosan solution. 2 mL of the previously prepared 1% (w/v) HA solution were added to the CHI and CBD solution while it was kept under magnetic stirring. The resulting solution was shaken for 60 seconds followed by mechanical stirring for 30 minutes. Finally, the solution was kept under magnetic stirring for 24 hours to ensure evaporation of ethanol.

Example 8. CHI:HA-CBD Submicroparticle Solution (CHI:HA2.5:1)

[0127] The experiment of example 5 was repeated using a 2% (w/v) HA solution instead of 1.5% (w/v). The protocol was adapted as follows:

[0128] 1 g of Chitosan (MW 0.70 MDa) was dissolved in 20 mL acetic acid and sodium acetate buffer (pH 4.39) for 12 hours to obtain a chitosan solution of 5% (v/w). The pH of the solution was adjusted to 4.5 by the addition of appropriate volumes of HCl 5 M. 0.4 g of low molecular weight hyaluronic acid (MW 0.66 MDa) was added via spatula to 20 mL of sea water (pH 4.31 (adjusted with HCl)). The resulting solution was stirred mechanically for 12 h until complete solution of the polymer was reached. The pH of the solution was adjusted to 4.5 by the addition of appropriate volumes of HCl 5 M. Following, a solution of phytocannabinoid (39 mg of CBD) in 177 microL of ethanol absolute was added to 2 mL of the 5% chitosan solution. 2 mL of the previously prepared 2% (w/v) HA solution were added to the CHI and CBD solution while it was kept under magnetic stirring. The resulting solution was shaken for 60 seconds followed by mechanical stirring for 30 minutes. Finally, the solution was kept under magnetic stirring for 24 hours to ensure evaporation of ethanol.

Example 9. CHI:HA-CBD Submicroparticle Solution (CHI: HA2.5:1.25) The experiment of example 5 was repeated using a 2.5% (w/v) HA solution instead of 1.5% (w/v). The protocol was adapted as follows:

[0129] 1 g of Chitosan (MW 0.70 MDa) was dissolved in 20 mL acetic acid and sodium acetate buffer (pH 4.39) for 12 hours to obtain a chitosan solution of 5% (v/w). The pH of the solution was adjusted to 4.5 by the addition of appropriate volumes of HCl 5 M. 0.5 g of low molecular weight hyaluronic acid (MW 0.66 MDa) was added via spatula to 20 mL of sea water (pH 4 (adjusted with HCl)). The resulting solution was stirred mechanically for 12 h until complete solution of the polymer was reached. The pH of the solution was adjusted to 4.5 by the addition of appropriate volumes of HCl 5 M. Following, a solution of phytocannabinoid (39 mg of CBD) in 177 microL of ethanol absolute was added to 2 mL of the 5% chitosan solution. 2 mL of the previously prepared 2.5% (w/v) HA solution were added to the CHI and CBD solution while it was kept under magnetic stirring. The resulting solution was shaken for 60 seconds followed by mechanical stirring for 30 minutes. Finally, the solution was kept under magnetic stirring for 24 hours to ensure evaporation of ethanol.

Example 10. Solid Ingredient

[0130] The solution resulting from the experiment 3 was lyophilized in order to obtain submicroparticles of CHI:HA loaded with CBD as a solid ingredient. 0.0962 g of the solid ingredient was reconstituted in 1 mL of type II water and the pH, particle size, zeta potential, PDI and CBD loading (total and encapsulated) were analyzed.

Example 11. Topical Gel/Serum

[0131] The solution resulting from example 5 was modified by adding some ingredients in order to obtain a cosmetic product (serum) containing CBD loaded CHI:HA submicroparticles. The serum was prepared as follows:

[0132] 0.5 g of glycerin (5% w/v) and 0.2 g of propyleneglycol (2% w/v) were added to 4 mL of the NP solution resulting from example 5 and it was kept under magnetic stirring for 10 minutes. 0.07 g (0.7% w/v) of high molecular weight hydroxypropylmethylcellulose (HPMC) (Benecel K100M) was added via spatula to the previous solution under magnetic stirring. Finally type II water was added (q.s. 10 mL) and the resulting mixture was shaken mechanically until the complete dissolution of the reactants was reached.

Example 12. Eye Drops/Nasal Drops

[0133] The solution resulting from example 5 was modified in order to obtain an eye drop/nasal drop formulation containing CBD loaded CHI:HA submicroparticles. The eye or nasal drops were prepared as follows:

[0134] 0.04 g (0.2% w/v) of high molecular weight hydroxypropylmethylcellulose (HPMC) (Benecel K100M) was added to 8 mL of the submicroparticle solution resulting from example 5 and it was kept under magnetic stirring. Then type II water was added (q.s. 20 mL) and the resulting mixture was shaken mechanically until the complete dissolution of the polymer was reached.

Example 13. Oral Suspension

[0135] The solution resulting from example 5 was modified in order to obtain an oral suspension formulation containing CBD loaded CHI:HA submicroparticles. The oral suspension was prepared as follows:

[0136] 0.2 g of glycerin (2% w/v) was added to 4 mL of the submicroparticle solution resulting from example 5 and it was kept under magnetic stirring for 10 minutes. 0.015 g of potassium sorbate (0.15% w/v), 0.2g of xylitol (2% w/v) and 0.2g of mannitol (2% w/v) were added via spatula to the previous solution under magnetic stirring and it was kept in agitation until the complete dissolution of the ingredients. Finally, 0.02 g (0.2% w/v) of xanthan gum and purified water (q.s 10 mL) were added to the previous solution and the resulting mixture was shaken mechanically until the complete dissolution of the reactants was reached.

Example 14

[0137]

TABLE-US-00003 TABLE 3 Physicochemical characterization of CHI:HA-CBD NP formulations Total Cannabinoid (encapsulated + Encapsulated Ratio non-encapsulated) Cannabinoid Method of Size of Composition CHI:HA % (HPLC) % (HPLC) synthesis pH particles (nm) PDI Example 1 1.25:0.38 CBD 0.026% 0.023% Method 4.37 638.64 0.29 NO SW example 1 Example 2 1.25:0.38 CBD 0.033% 0.029% Method 4.52 692.56 0.35 example 2 Example 3 1.25:0.38 CBD 0.21% 0.11% Method 4.61 629.36 0.35 example 3 Example 4 2.5:0.75 CBD 0.74% 0.40% Method 4.41 734.17 0.86 NO SW example 4 Example 5 2.5:0.75 CBD 0.74% 0.57% Method 5.19 613.27 0.50 example 5 Example 6 2.5:0.2 CBD 0.034% 0.017% Method 4.15 Aggregates 0.97 example 6 Example 7 2.5:0.5 CBD 0.041% 0.037% Method 4.15 Aggregates 1.00 example 7 Example 8 2.5:1.sup. CBD 0.075% 0.062% Method 4.06 Aggregates 1.00 example 8 Example 9 2.5:1.25 CBD 0.059% 0.041% Method 4.11 Aggregates 1.00 example 9

Example 15

[0138]

TABLE-US-00004 TABLE 4 Physicochemical characterization of final products containing CBD loaded CHI:HA submicroparticles Total Cannabinoid (encapsulated + Encapsulated non-encapsulated) Cannabinoid Method of Size of Composition % (HPLC) % (HPLC) synthesis pH particles (nm) (cP) Example 10 CBD 0.73% 0.30% Method 5.48 327.97 NA example 10 Example 11 CBD 0.10% 0.040% Method 5.11 536.32 2585 example 11 Example 12 CBD 0.18% 0.063% Method 4.99 1209.63 40 example 12 Example 13 CBD 0.15% 0.090% Method 5.23 577.20 NA example 13

[0139] Data from examples 14 and 15 show that cannabinoids are incorporated into CHI:HA particles formulated in aqueous media with and without sea water.

[0140] Incorporating sea water into the formulation produces an increase of the amount of encapsulated cannabinoids (example 2 vs example 1; example 5 vs example 4).

[0141] The ratios CHI:HA 2.5:0.75 and 1.25:0.38 (examples 1-5) are optimal for the formation of submicroparticles. When ratios out of this range were used, aggregates were formed and no submicroparticles were obtained, as shown in examples 6, 7, 8 and 9.

[0142] The ratio 2.5:0.75 maximizes the amount of total cannabinoid found in the formulation.