RS20240529A1 - Phytopreparations with high cannabidiol content for improved local inflammation, pain relief and arthritis and their obtaining procedure - Google Patents

Abstract

The invention protects the obtaining of stable and original phytopreparations based on non-psychoactive supercritical extract of hemp (Cannabis sativa L., Cannabaceae) rich in cannabidiol embedded in suitable carriers (nanoliposomes and nanoparticles of mesoporous silica). They are intended for application on the skin and allow for improved delivery and penetration of said hemp extract into the deep layers of the skin. The invention offers a safe, natural alternative in the treatment of chronic pain, skin conditions, muscle spasms, arthritis and especially in the treatment of peripheral neuropathic pain. The unique combination based on natural ingredients has technical advantages that enable improved product delivery with skin hydration effects. The resulting semi-products based on extract incorporated nano-carriers are formulated into appropriate pharmaceutical forms characterized by simple and effective application.

Description

[0001] PHYTOPREPARATIONS WITH HIGH CANNABIDIOL CONTENT FOR IMPROVED LOCAL RELIEF OF INFLAMMATION, PAIN AND ARTHRITIS AND THE PROCEDURE

[0002] OBTAINING

[0005] TECHNICAL FIELD

[0006] The invention protects medical phytopreparations based on supercritical hemp extract (Cannabis sativa L., Cannabaceae) obtained by an innovative extraction method, quantified for the content of the active non-psychogenic substance, cannabidiol (CBD), which is incorporated into specially designed carriers to provide anti-inflammatory effect and pain relief during the localized treatment of skin disorders, arthritis, peripheral neuropathy, neuropathic pain and general muscle pain of various etiologies. Nanoliposomes and nanoparticles of mesoporous silica were chosen as adequate carriers of hemp extract rich in non-psychoactive cannabinoids, based on their potential to improve the penetration of active ingredients through the skin. According to the international classification of patents, the invention can be classified into classes: A61K 8/119/107, 9/113, 9/127, 9/133, A61K 31/352, 47/24, A61P 29/02, 25/02 [2006.01].

[0009] TECHNICAL PROBLEM

[0010] The technical problem solved by the invention is obtaining stable and original phytopreparations based on supercritical hemp extract (Cannabis sativa L., Cannabaceae) embedded in carriers based on nanoparticles, intended for topical application with controlled release of active substances that enable improved delivery and penetration of cannabidiol into the deep layers of the skin. Current research and technical solutions are focused on the oral application of cannabidiol, which primarily leads to systemic absorption of cannabinoids, not local. According to the invention, phytopreparations solve the technical problem of topical absorption of cannabinoids by avoiding and overcoming the shortcomings of existing preparations for the same purpose, in addition to showing unexpectedly effective properties, because they are gentle on the skin, do not cause damage and provide an additional skin hydration effect. They represent a unique market advantage as pain patients seek natural and safer ways to treat pain, especially with the recent prevalence of the opioid crisis associated with prescription pain relievers and pain relievers. The invention offers a safe, natural alternative to the treatment of chronic pain, pathologically changed skin, muscle spasms, arthritis and especially in the treatment of peripheral neuropathic pain.

[0011] The unique combination is based on natural ingredients and has technical advantages that enable improved product delivery with skin hydration effects. Applied hemp extracts are obtained by a sustainable CO<2> supercritical extraction process, a green process (GRAS) that ensures selective extraction of cannabidiol from plant material without residues of hazardous organic solvents. The formulation according to the invention provides a higher concentration of cannabidiol compared to products available on the market.

[0012] In addition to the above, the demanding physical and chemical characteristics of cannabinoids, such as sensitivity to light, rapid degradation in various solvents and poor solubility in water, represent a technical problem. This technical problem is solved by the subject invention by incorporating it into advanced nano-systems for drug delivery.

[0013] Formulated phytopreparations for topical application according to the invention solve the technical problem of delivery of active ingredients using unique carriers, nanoliposomes and mesoporous silica nanoparticles. A surprising improvement in bioavailability is achieved, enabling increased local absorption while minimizing the risk of adverse reactions associated with systemic absorption. Numerous preparations based on hemp (i.e. cannabidiol) are available on the market: balms, creams, lotions and ointments. However, none of them use a unique system for topical delivery of cannabidiol according to the present invention. Since there is no data on the development of a suitable means for the improved delivery of the used hemp extracts, the obtained phytopreparations are innovative. The invention has undergone studies that have proven it to be safe for use on healthy human skin, with no side effects on the skin.

[0014] The technical problem that is solved by the invention is also reflected in the definition of a suitable analytical method for the identification and quantification of the active substance, cannabidiol, in extracts and prepared formulations of finished products.

[0015] The invention solves the technical problem of fully defining the technological procedure for obtaining new phytopreparations with a defined effective ratio of active components, emollients, emulsifiers and water that exhibit additional beneficial effects such as increasing the hydration level of the stratum corneum and skin elasticity.

[0018] STATE OF THE ART

[0019] The changing perception of health and well-being, which includes the increasing expectations of consumers to replace potentially harmful chemicals with natural ingredients, encourages a worldwide trend of increasing research within the food, pharmaceutical and cosmetic industries in order to design products on a natural basis. The reason for the increasing use of herbal extracts is their complex multi-component nature, suitable for simultaneous action at the level of different target sites, they are safer than competing synthetic alternatives, and the applied doses of active agents ensure the reduction of potential side effects.

[0020] Inflammation is part of the immune response that is launched against harmful stimuli, foreign objects, damaged cells, irritants and pathogens in the body. Anti-inflammatory drugs eliminate the initial cause of cell injury, dead or necrotic cells and damaged tissue from the site of the inflammatory process, triggering tissue recovery. The subject of the invention is the development of a local anti-inflammatory product based on market analysis and forecasts by GBI Research for the period from 2018 to 2026 (<u>Anti-Inflammatory Therapeutics Market: Growth, Future Prospects and Competitive Analysis, 2018-2026," https://www.credenceresearch.com/report/anti-inflammatorytherapeutics-market, available on 04/16/2018). Namely, the analysis of the anti-inflammatory therapeutics market for the specified period showed is that there is a continuing need for anti-inflammatory substances and preparations. It is estimated that the global market for anti-inflammatory therapies will reach US$ 130.6 billion by 2026, with an increase in the compound annual growth rate (CAGR) of 8.5% in the period from 2018 to 2026. The global market for herbal medicines was US$ 9.21 billion in 2018, and according to the projected value 2026 with a CAGR of 8.5% during the forecast period 2019-2026. will amount to USD 17.7 billion. Global Herbal Medicinal Products Industry Sales Forecasted to Surpass US$ 417.99 Billion by 2033 with a Notable 7.7% CAGR | FMI - PharmiWeb.com, https://www.pharmiweb.com/press-release/2023-l1-02/global-herbal-medicinal-productsindustry-sales-forecasted-to-surpass-us-41799-billion-by-2033-wit accessed 05/13/2024).

[0021] Treatment of inflammatory damage is of great importance and requires a multidisciplinary approach to recognition, diagnosis and treatment. Continuous attempts are being made to develop more effective products that are safe, easy to administer and economically viable. The persistent use of hemp over several millennia, as well as new scientific data justify further research.

[0022] Hemp (Cannabis sativa L., Cannabaceae) is an annual, dioecious plant with pronounced sexual dimorphism (male and female plants differ morphologically), (Finta-Korpel'ová Z, Berenji J. Trends and achievements in industrial hemp (Cannabis sativa L.) breeding. Bulletin for Hops, Sorghum & Medicinal Plants 2007, 39:63-75). Over 700 different varieties of hemp have been described, and it is classified as a weed. There are also monoecious varieties, developed for obtaining fibers and seeds at the same time.

[0023] The term "hemp" (C. sativa L., Cannabaceae) refers primarily to a plant that is grown as an agricultural crop and is characterized by a low content of psychoactive ∆<9>-tetrahydrocannabinol (THC, less than 0.3%). Numerous researches on the medical application of hemp have been initiated, which has led to an increase in scientific knowledge about its composition and health benefits, with great attention focused on non-psychoactive cannabinoids. (cannabidiol, CBD, cannabinol, and cannabichromene, CBCr), which represent the most promising candidates for clinical application in muscle spasms due to the lack of cognitive and psychoactive effects, with significant anti-inflammatory and neuroprotective properties. Among them, cannabidiol can make up to 40% of hemp extracts. The therapeutic effects, which are manifested in the modulation of inflammatory processes, can be explained by multiple mechanisms of action cannabinoids two known receptors for cannabinoids, the activation of which produces the effects of this group of secondary metabolites, namely CBl (mainly located in the CNS) and CB2 (mainly found in immune cells, but also detected in some other cell types, including chondrocytes, osteocytes and fibroblasts, which means that it can be considered a peripheral cannabinoid receptor). While CB1 exerts psychoactive activity, activation of CB2 results in immunomodulatory and anti-inflammatory effects. Controlling the degree of inflammation by selectively acting on CB2 receptors may be responsible for the desired anti-inflammatory activity. Considering that cannabidiol acts as a CB2 inverse agonist, it may represent the molecule of choice for anti-inflammatory effects. In addition, the anti-inflammatory properties of cannabinoids may be exerted at the cellular level due to their antioxidant capacity and their ability to affect cell wall properties. Also, cannabidiol has been shown to be a potential inhibitor of SOH-2 and LOX-5, which are involved in the metabolism of arachidonic acid, as mediator of inflammatory processes. In addition to cannabinoids, the pharmacological effects of hemp may also be mediated by other components such as the monoterpenoids myrcene, limonene, pinene and the sesquiterpenoid β-caryophyllene.

[0024] The treatment of inflammatory damage requires a multidisciplinary approach, which involves several steps, first of all recognition of the disease, then diagnosis, and finally appropriate treatment. Therefore, there is a constant need to produce more effective preparations that are easy to administer, safe, and at the same time economically viable.

[0025] The use of hemp is several millennia old, and new scientific data justify its widespread use. During the past years, researches have confirmed the therapeutic potential of cannabinoids, as the main constituents of extracts of aerial parts hemp. In particular, ∆<9>-TNS and CBD represent the most abundant phytocannabinoids in hemp plants, and today both compounds have therapeutic applications.

Recently, there are data on the encapsulation of hemp oil, terpenes and/or cannabidiol in lipid-based formulations (micelles, nano-structured lipid carriers (NLC), pronano-lipospheres (PNL), nanoemulsions) and polymeric micro- and nanoparticles for systemic (oral or parenteral) or topical (nasal, rectal, vaginal) application. When it comes to the use of nanovectors with incorporated cannabinoids, transdermal application of an ethosomal formulation with cannabidiol was shown to be effective in preventing inflammation and edema. Pain in arthritis and osteoarthritis was, in one study, alleviated by transdermal application of cannabidiol in conventional carriers.

[0027] On the market there are a large number of preparations (balms, creams, lotions and ointments) based on hemp extracts intended for application on the skin. They can be effective as analgesics and anti-inflammatory agents, but they are without a defined, scientifically based and tested action. These preparations are recommended for indications such as arthritis, muscle pain, burns, rashes, swelling and post-herpetic neuralgia (Mücke M, Phillips T, Radbruch L, Petzke F, Häuser W. Cannabis-based medicines for chronic neuropathic pain in adults. Cochrane Database Syst Rev. 2018 Mar 7;3(3):CD012182.doi: 10.1002/14651858.CD012182.pub2). In countries where the use of medical cannabis is legal, pharmaceutical companies have developed drugs containing natural or synthetic cannabinoids.

[0028] Oromucosal spray "Sativex®" with 2.7 mg of THC and 2.5 mg of cannabidiol (ratio, approximately 1:1), from C. sativa L. (www.gwpharm.com/) is the first cannabinoid herbal medicine officially approved in the world by the European Medicines Agency (EMA), intended for the treatment of multiple sclerosis and pain relief in adult cancer patients, and is registered in 27 countries outside the United States of America. For the local treatment of neuropathic pain, the company "Dr. Werner Pharma" produces "Urimil gel" and "Urimil forte heating patches" that contain five active ingredients: uridine monophosphate (UMP), amica extract (Arnica montana), devil's claw extract (Harpagophytum procumbens), methylsulfonylmethane (MSM) and magnesium chloride (https://drwerner-pharma.com/). The Spanish company "PrismaNatural" produces Apitok ointment for the treatment of local pain and neuralgia based on bee venom, menthol, devil's claw root extract (Harpagophitum procumbens), capsaicin, methyl salicylate, glucosamine and chondroitin (https://prismanatural.es/). Preparations based on cold-pressed cannabis oil (5%), menthol, chestnut extract, comfrey, camphor and eucalyptus ("Cannabis flex warming gel", "Cannabis flex cooling gel" and "Cannabis massage gel 5%") are produced by the Czech company "Palacio CZ" s.r.o. (https://www.palacio.cz/en/).

[0029] However, most topical preparations intended for the treatment of neuralgia are based on drugs as active ingredients that can often be at high risk of skin irritation and allergic reactions, which consumers recognize as potentially harmful chemicals. The company "Wörwag Pharma" from Germany produces the cream "Capsagamma Dolor Creme", intended for the local treatment of neuropathic pain with 0.05% capsaicin (https://www.woerwagpharma.rs/). However, capsaicin use is often associated with skin reactions such as irritation (Vora A, Chan S. Postherpetic Neuralgia. In Essentials of Physical Medicine and Rehabilitation, 2020. (Fourth Edition), Eds: Frontera W, Silver J, Rizzo T. Elsevier Saunders.p. 599-603). The results of a study aimed at investigating the combination of 2% amitriptyline and 1% ketamine in the treatment of patients with neuropathic pain showed that the said combination exhibits significant analgesia, but higher doses may be required. It is associated with the problem of a high risk of local skin reactions (Lynch ME, Clark AJ, Sawynok J, Sullivan MJ. Topical 2% amitriptyline and 1% ketamine in neuropathic pain syndromes: a randomized, double-blind, placebo-controlled trial. Anesthesiology. 2005; 103(l):140-6).

[0030] Also, several patents describe the local application of cannabidiol in combination with other well-known anti-inflammatory phytopreparations for the treatment of pain and wounds.

[0031] The subject of patent US 9,682,033 B2 (application WO 2016/127068) are formulations of preparations for the treatment of postherpetic neuralgia in humans. A topical pharmaceutical preparation contains one or more excipients and a therapeutically effective amount of the spiro-oxindole compound (2-8%).

[0032] Patent number US 6,528,076 B2 (WO 03/004000) formulated preparations for the treatment of neuralgia in humans. The composition for topical use for the treatment of pain contains essential oils, salicylate-based compounds; acetone; emollients and menthol.

[0033] Patent application US 2012/0264818 A1 formulates preparations for topical application (solution, spray, lotion, gel, cream or ointment) with an extract of C. sativa with at least 10% tetrahydrocannabinolic acid, intended for the local treatment of infections resistant to methicillin in order to eliminate pain, inflammation, muscle tension, muscle spasms and skin ulcers.

[0034] Patent US 10,918,686 B2 (application WO 2017/178937) protects an oil gel composition containing a combination of non-psychotropic phytocannabinoids: cannabidiol, cannabidiol acid, cannabivarin and cannabigerol in combination with an extract of olive oil Oliva europaea, essential oil of mint leaf Mentha arvensis and anhydrous colloidal silicon. The composition provides deep tissue release of cannabinoids using a pharmaceutical patch and target site release system to reduce skeletal muscle and joint pain and inflammation caused by trauma and/or arthritis/osteoarthritis.

[0035] American patent application US 2020/339487 A1 describes cannabinoid compositions for oral administration having improved bioactivity and methods of obtaining and a process for cold decarboxylation of cannabinoid acid. The composition includes dried powder of C. sativa L. and at least one catalyst (citric acid, ascorbic acid, citrus extract powder, citrus liquid extract, amino acids, Lewis acids). The composition improves the bioavailability of cannabinoids and inhibitors of the Cytochrome P450 group of enzymes in a sufficient amount to inhibit liver enzymes in vivo and enhance the bioeffects of cannabinoids.

[0036] Patent US 10,787,429 V1 protects the products and the method of mechanical extraction and purification of tetrahydrocannabinolic acid. The subject of the protection is the purification of the active component from the hemp extract, but it does not include obtaining the extract rich in cannabidiol which is applied in the present invention.

[0037] The subject of patent application CN 111 465 322 A (patent GB 2568032 B) is a method of producing phytocannabinoids by growing cultured cells of C. sativa through a specific tissue culture for use in medical treatments, but the pharmaceutical form and indication are not specified, which is the subject of protection by the present invention.

[0038] The subject of application WO 2020/121064 is an environmentally acceptable method of water extraction of cannabidiol acid from C. sativa. The maximum CBD content was 0.002%, while the SCO<2>extraction method of the present invention yields a content greater than 10%.

[0039] Patent application CA 3,060,909 protects a method of encapsulating cannabis-derived cannabinoids in nanosized phospholipid carriers/vesicles. The subject invention represents a further step in the direction of surprising uniformity and homogeneity of the preparation, as well as providing the necessary safety data for local application.

[0040] Registered patent US 6949582 (B1) protects a method for relieving analgesia and reducing inflammation using a cannabinoid thermal liniment with a composite extract of about 0.5-2.5% of a mixture of cannabinoids extracted from the female plant C. sativa L and about 97.5%-99.5% isopropyl alcohol (70%).

[0041] The subject of patent application WO 2018/148785 (Al) are cannabinoid formulations for the treatment of dermatitis and inflammatory skin diseases. The application does not specify a precise composition in terms of the content of different cannabinoids, which is solved in the present invention.

[0042] Patent EP 3 355 873 V1 (US 11,413,266 B2, application US 2018/263952 A1) protects the use of cannabidiol acid in the form of a highly purified extract of cannabis (more than 95% of the total extract) or synthetically produced for the treatment of dermatitis caused by a microbial infection. This patent does not cover phytopreparations for the treatment of conditions such as muscle stiffness, overuse and stress, it does not include a unique method supercritical extraction, does not exclude psychoactive components of cannabis such as THC or delivery methods using nanocarriers, which is a difference from the subject invention.

[0043] Patent application US 2019/224137 A1 claims protection for an anti-acne preparation and for a cannabinoid dosing regimen, but does not specify the specific chemical composition of the composition.

[0044] The process of obtaining extracts containing THC, CBD and their carboxylic acids from hemp plant mass subjected to extraction with concentrated CO<2> under supercritical and subcritical conditions (EP 1326 598 B1) has been patented.

[0045] Canadian application CA 3 062 136 A1 claimed patent protection for a product based on cannabis infusion for oral administration with controlled release. This application does not specify the cannabinoid profile or the presence of psychoactive substances, unlike the subject invention.

[0046] Patent EP 3 071 193 V1 (application AU 2016/268872 B2) protects the use of cannabinoids in the treatment of intraocular inflammation, uveitis, uveoretinitis or proliferative vitreoretinopathy and/or ocular neuropathic pain. The ocular pharmaceutical composition contains a CB2 positive allosteric modulator, a cannabimimetic agent that is a non-selective cannabinoid receptor agonist, and a carrier. The invention is specifically designed for the treatment of eye conditions, but not for topical application to the skin. The subject invention provides innovation in the application of supercritical extract and nano-systems for delivery, and action at the local level, without the risk of a systemic effect.

[0047] Cannabinoid compositions and methods of their use for immune modulation, immunoprotection and cancer treatment are the subject of protection of the invention WO 2020/163775 Al. Although it contains a nanoemulsion similar to the subject invention, this invention relates to the treatment of cancer and immune conditions, specifies a number of terpenes that must be combined, does not use supercritical extracts, nor does it provide local product delivery, while the subject invention is designed for topical application primarily for the treatment of inflamed skin and muscles.

[0048] Patent US10603301 B2 (application US 2018/0042890 A1) protects cannabis-based extracts with a CBD:THC ratio of 1:1 and formulations for topical use in skin disorders. The proposed patent specifically singles out non-psychoactive components to reduce the adverse effects associated with THC. It is indicated for skin inflammations, but not for muscle spasms or stress. Also, no supercritical carbon dioxide extraction or nanocarrier system is used.

[0049] Emulsion systems based on cannabinoids described in the application WO2020037411A1 contain an aqueous composition with a cannabinoid - they contain at least one cannabinoid in oil as a carrier, a plurality of emulsifiers for a targeted hydrophilic-lipophilic balance (HLB) and a targeted ratio of emulsifier to oil (at least 1 mg of cannabinoid in 1 mL of aqueous composition). In this patent application, no the method of extraction is specified, there is no data on the choice of non-psychoactive cannabinoids, the use of an emulsion for local application is not stated, nor has safety been tested.

[0050] Compositions based on lipid nanoparticles as cannabinoid carriers in standardized dosage forms are the subject of patent US 10,028,919 B2 (AU2019201792B2). The lipid-based composition contains nanoparticles whose composition is: therapeutic agent, phospholipid 2.5-20%, sterol 0.25-10%, lipid component other than phospholipid 2.5-20% and water. Nanoparticles with an average size of 75-500 nm, after storage for a period of 1 month, decrease by less than 20%. This patent applies lipid-based nanoparticles, but does not specify the composition of the particles containing cannabinoids for local pain relief.

[0051] The climatic influence on the yield of cannabinoids (THC and CBD) from four strains of cannabis grown in different regions obtained by the supercritical fluid extraction method was examined (Kargili, U., Aitac, E. 2021. Supercritical fluid extraction of cannabinoids (THC and CBD) from four different strains of cannabis grown in different regions. The Journal of Supercritical Fluids, 179 105410. https://doi.org/10.1016/j.supflu.2021.105410). Experiments have provided insights into productivity, but the use of such extracts for medicinal or topical application is not suggested.

[0052] The oral bioavailability of cannabinoids and the biological safety of cannabidiol nanoparticles were investigated in an animal study. The use of nanoparticles as anti-inflammatory therapy resulted in increased bioavailability of cannabinoids when taken orally (Rao, I., Li, R., Liu, S., Meng, L., Wu K., Yuan K., Liang, H., Meng, K.2022, Enhanced bioavailability and biosafety of cannabidiol nanomicelles for effective anti-inflammatory therapy. Particuologies, 69(1-9). https://doi.org/10.1016/j.partic.2021.11.010.) In contrast to the mentioned work, in the present invention the safety is unequivocally confirmed in human trials.

[0053] According to the present invention, phytopreparations contain encapsulated quantified supercritical extracts of hemp (C. sativa L., Cannabaceae) (literally proven anti-inflammatory substances) in specific nano-carriers that enable chemical stability and increased introduction of active ingredients into the skin. Qualitative and quantitative analysis enabled quality control of the developed product. Safety and efficacy studies confirm their powerful anti-inflammatory effect and pain relief for the localized treatment of skin disorders, arthritis, peripheral neuropathy, neuropathic pain and general muscle pain of various etiologies when applied locally. To the best of the inventor's knowledge, the proposed products are not known in the available literature.

[0054] DESCRIPTION OF THE INVENTION WITH IMPLEMENTATION EXAMPLES

[0056] The essence of the invention consists of formulated preparations for the local treatment of anti-inflammatory skin diseases, arthritis and peripheral neuropathic pain associated with the skin and muscles for application at the site of pain and inflammation, which are based on the application of extracts of industrial hemp (Cannabis sativa L., Cannabaceae) quantified for active non-psychogenic components, rich in cannabidiol (CBD), which are obtained using sustainable processes under high pressure.

[0057] Recently, cannabinoids have been shown to be effective in the treatment of inflammatory skin disorders, but for the present invention, a more significant application is the successful treatment of peripheral neuropathic pain. Due to the location of the target site in the deeper layers of the skin, for the treatment of the indicated indications, especially peripheral neuropathic pain, it is necessary to ensure the penetration of the active principle into the deeper layers of the skin. However, a big challenge is the transport of active substances into/through the skin, given that it is a specific skin barrier, provided by the outer layer - the stratum corneum. One of the methods for overcoming the skin barrier and penetrating deeper layers, which has been intensively researched in recent years, is the design of the so-called of colloidal nanovectors (size 10 to 1000 nm) as drug delivery systems. Taking this into account, as well as the demanding physicochemical characteristics of cannabinoids (sensitivity to light, rapid degradation in various solvents, and poor water solubility) make them suitable candidates for incorporation into advanced nano-systems for drug delivery. The aforementioned set of technical problems was the motive for formulating a nano-system with incorporated hemp extract in a concentration that proved to be effective and for which the CBD content was determined by a suitable analytical method.

[0058] In order to ensure a satisfactory anti-inflammatory effect and penetration of active substances from industrial hemp extract into the deeper layers of the stratum sogpeit, suitable nanocarriers with the potential to improve the penetration of active ingredients through the skin to the desired site of action were designed.

[0059] In one variant of the invention, active extract carriers are designed in the form of nanoliposome (NL) particles.

[0060] In another embodiment of the invention, the carriers of the active extract are designed in the form of mesoporous silica nanoparticles (MSN).

[0061] Liposomes are spherical vesicular systems composed of an internal aqueous core that encloses phospholipid bilayer membranes (lamellas) - one or more. They are made from purified soy phospholipids with fatty acids of different degrees of unsaturation and different ability to penetrate the skin. They are suitable as carriers of hydrophilic medicinal substances (which are incorporated into the aqueous phase) and hydrophobic (which are incorporated into the lipid bilayer). Because of their two-layered phospholipid structure, they show similarity to biological membranes. They are non-toxic, non-immunogenic, biodegradable and physiologically acceptable. Incorporated into the base, they enable better transport of active substances into the skin than other topical preparations. Their advantages compared to existing pharmaceutical forms for skin treatment are biocompatibility, biodegradability, prolonged release of bioactive components and a favorable effect on the barrier properties of the skin and its moisture.

[0062] Mesoporous silica nanoparticles (MSNs) are attracting increasing attention in the field of biomedicine due to unique features including high porosity, large surface area, narrow and tunable distribution of adjustable pore size 2-50 nm, colloidal stability, easy functionalization and desirable biocompatibility and abundant unsaturated hydroxyl groups on the surface. They are an ideal carrier for the storage and delivery of guest molecules and can be combined with a wide range of substances to form new materials with excellent performance. Due to the high stability and possibility of modulating drug release, they can be used to design new formulations of drugs, proteins and biomolecules with controlled release rates, fewer side effects and good therapeutic effects.

[0063] Pharmaceutical compositions in the form of nanoliposome particles and mesoporous silica nanoparticles enable improved delivery of active substances, potentiating in vitro/in vivo biopharmaceutical characteristics through assessment of physicochemical properties, stability and safety.

[0064] Selected carriers (nanoliposomes and mesoporous silica nanoparticles) are loaded with supercritical hemp extract rich in non-psychoactive cannabinoids. Extracts of industrial hemp (C. sativa L.) are obtained by a process of supercritical extraction with carbon(IV)-oxide, which extracts large amounts of CBD from plant material without creating by-products, according to the description in patent application RS-P-2020/1428. The extracts are additionally purified, with a defined CBD content (3.00 - 45.00%), and the best 13.45-22.65%, contain polyunsaturated fatty acids (PUFA) 80%, an optimal ratio of linoleic (LA) and linolenic (LNA) acids 3:1, vitamin E (100-150 mg/100 g) and do not contain tetrahydrocannabinol.

[0065] In the preparation of phytopreparations according to the invention, a commercial product extract of industrial hemp (C. sativa L.) with a defined CBD content of 20%, without tetrahydrocannabinol, obtained by the process of supercritical extraction with carbon(IV)-oxide (manufacturer "Zdrava priča", Healthy Story LLP, 27 Old Gloucester Street, London, UK, WC1N ZAH CBD oil) can be used.

[0066] Incorporation of hemp extract into nanoliposomes

[0067] Incorporation of supercritical hemp extract into liposomes was performed using raw materials containing different percentages of lecithin and/or phospholipids, namely:

[0068] - lecithin with 55% linoleic, 4% linolenic and 5.5% arachidonic acids or

[0069] -purified lecithin (from soy) with 72-78% phosphatidylcholine or

[0070] -purified lecithin (from soy) with 37% phosphatidylcholine and lysophosphatidylcholine.

[0071] Liposomes/nanoliposomes are made by the extrusion method and the homogenization method under high pressure. Nanoliposomes were prepared using a commercial raw material for liposome formation, Phosal 40 IP, which is an empty carrier for lipophilic active substances, a phospholipid formulation of phosphatidylcholine content >37% in organic sunflower oil.

[0072] A placebo sample of nanoliposomes was prepared with 10% (v/v) Phosal 40 IP and water for injection to 100% v/v.

[0073] A sample loaded with supercritical hemp extract was prepared with 01.00-55.00% v/v of the specified specific extract, 10.00-20.00% v/v Phosal 40 IP and up to 100.00%(v/v) purified water.

[0074] After measuring the ingredients, the phases are mixed using a rotor-stator homogenizer for 20-50 min, preferably 30 min at 2000-8000 rpm. In order to obtain the final emulsion, the prepared rough emulsion is passed through a high-pressure homogenizer, in a discontinuous process, in five to ten cycles, at a pressure of 500 to 1500 bar and a temperature of 25°C, in order to obtain liposomes of the desired diameter (up to 500 nm).

[0076] Incorporation of hemp extract into mesoporous silica nanoparticles Incorporation of CBD from hemp extract into the pores of mesoporous silica nanoparticles (MCM-41, Sigma Aldrich) was carried out by the impregnation method in a ratio of 2:1 to 1:2, preferably 1:1 v/v. A measured amount of mesoporous silica nanoparticles is added to a solution of a certain concentration of CBD in dimethylsulfoxide or in ethanol before encapsulation. The mixtures were shaken for 18-32 hours, preferably 24 hours at 210-450 rpm on an orbital shaker at room temperature. Mesoporous silica nanoparticles are collected by centrifugation and carefully washed 2-4 times with water to remove surface adsorption of CBD.

[0077] Efficiency of encapsulation of hemp extract in nano-systems

[0078] In order to define the qualitative and quantitative composition of hemp extract, a new HPLC method was developed, which enabled adequate separation of the peaks. The HPLC conditions are described in detail in Example 2a. CBD was detected (Figure 1), as the substance of interest. Namely, CBD is responsible for the anti-inflammatory effects of hemp extract, since it acts as an inverse agonist of the CB2 receptor (as a peripheral cannabinoid receptor), as well as SOH-2 and LOX-5 inhibitors that target the arachidonic acid pathway associated with the inflammatory response.

[0079] The loading efficiency of nanoliposomes and mesoporous silica nanoparticles with CBD was determined using the HPLC method. The concentration of CBD in the supernatant was measured after centrifugation at 3500 rpm for 30 minutes. After that, the supernatant was filtered through a regenerated cellulose syringe filter (pore size 0.2 mm) and then diluted using dimethyl sulfoxide (DMSO), i.e. with ethanol (10 ml of the mixture and 990 ml of DMSO, i.e. ethanol) before injection into the HPLC column.

[0080] Based on the obtained results, the encapsulation efficiency (EE) in mesoporous silica nanoparticles for cannabidiol dissolved in DMSO is -4.92%, while for CBD dissolved in ethanol the EE is 88.44%, while the surprisingly high efficiency of CBD encapsulation in nanoliposomes (in DMSO and in ethanol) is 99.99%.

[0082] Formulations of phytopreparations

[0083] Since the active substance of natural origin (hemp extract) is incorporated into nanocarriers made of materials of natural origin, the formulation of the product emulgel was chosen using emulsifiers and gelling agents, also of natural origin.

[0084] As modern carriers of active substances, emulgels represent emulsions (oil in water or water in oil) in which gels are incorporated. Emulsions themselves represent a system with controlled release of the active substance in that the active substance, dispersed in the internal phase, must diffuse through the external phase to reach the skin and be absorbed in it. Gels form a network that traps the molecules of the active phase, so that emulgels possess properties of both emulsions and gels, making good systems for the controlled delivery of active substances. Emulgels have numerous other advantages: easy application, good thermodynamic stability, can act as penetration enhancers.

[0085] For the preparation of emulgel-type preparations, the composition of active components and suitable excipients and their content is listed below. Emulgels with hemp extract are effective in increasing skin hydration, reducing transepidermal water loss from the skin, and after long-term use do not cause skin irritation or redness and maintain skin pH within physiological limits.

[0087] Formulations of emulsions with nanoliposome dispersion and hemp extract

[0090]

[0093] Manufacturing procedure: Hemp extract is homogenized with lecithin/phosphatidylcholine on a laboratory homogenizer (speed 2000-8000 rpm) and a primary dispersion is formed that is "passed" in several cycles (5-10) through an extruder or through a homogenizer under high pressure 500-1500 bar. The number of cycles is optimized to obtain liposomes of the desired diameter (up to 500 nm). hydroxyethylcellulose is added to the mixture of water, glycerol and preservatives and allowed to swell for 20-50 minutes, preferably for 30 minutes with occasional stirring.After complete hydration of the cellulose derivative, the aqueous phase is heated to 70°C (±2).

[0094] The components of the fat phase are melted and the temperature of the mass is adjusted to 70°C (±2).

[0095] Fat and water phase are combined with the mixer (speed 200-1000 rpm) and homogenizer (speed 1000-5000 rpm) for 5-25 min. Then the cooling of the mass begins. At a temperature of 35°C (±2), a dispersion of liposomes enriched with hemp extract is added to the mass. It is mixed while cooling, until the mass in the vessel reaches room temperature.

[0097] Formulations of emulgels with nanoparticles of mesoporous SiO<2> and hemp extract

[0100]

[0102] Manufacturing process: Analogous to the manufacturing process for liposome-based emulsions, hydroxyethyl cellulose is added to the mixture of water, glycerol and preservatives and left to swell for 30 minutes with occasional stirring. After the complete hydration of the cellulose derivative, the aqueous phase is heat to 70°C (±2). The components of the fat phase are melted and the temperature of the mass is set to 70°C (±2). The fat and water phases are combined with the mixer (speed 200-1000 rpm) and homogenizer (speed 1000-5000 rpm) for 5-25 minutes. Then the cooling of the mass begins. At a temperature of 35°C (±2), a dispersion of mesoporous SiO2 nanoparticles with hemp extract is added to the mass. It is mixed while cooling, until the mass in the vessel reaches room temperature.

[0103] Based on the conducted research, the obtained results are presented in the part of the example of feasibility of the invention. Surprisingly better results of extract encapsulation efficiency were shown by nanoliposomes (99.99%) compared to the lower values achieved by the application of mesoporous silica nanoparticles.

[0104] Dispersion of nanoliposomes, as a suitable nanocarrier with incorporated supercritical hemp extract and defined cannabinoid content was used in an in vivo study that tested their preliminary safety and efficacy in topical therapy on the skin of human volunteers.

[0105] The applicability of the composition according to the invention is based on the scientifically confirmed biological properties of the active components. It is possible to put on the market only a preparation that meets the required conditions of declared quality, efficiency and safety in accordance with the requirements of legal regulations. The safety and effect of the use of formulated phytopreparations have been tested in in vitro and in vivo tests, in accordance with the current European and national regulations - "Regulation (EC) No 1223/2009 of the European Parliament", "Regulation on cosmetic products 60/2019-4, 47/2022-3, 21/2023-97).

[0106] C. sativa extract nanoemulsions contain active, non-psychogenic cannabinoid components that act through receptors present in muscle tissue, not in the central nervous system, to which psychoactive components, such as THC, bind. Nonpsychogenic cannabinoid components extracted from S. sativa under high pressure, supercritical liquid carbon dioxide (extraction of SCO<2>) and embedded in nanoliposome carriers, are suitable for local application in the treatment of inflammatory conditions of the skin, arthritis, peripheral neuropathic pain and relief of joint and muscle pain, as well as pain of various etiologies (e.g. caused by tension, stress, overuse, minor injuries, strain, spasms and stiffness). Nanoliposomes provide surprisingly high solubilization and stabilization of CBD, while improving the penetration/delivery properties and bioavailability of active molecules through the stratum corneum of the skin, allowing for increased local absorption and minimizing the risk of adverse reactions associated with systemic absorption. By incorporating hemp extract into the pores of nanoliposomes or nanoparticles of mesoporous silica, the active components are protected for a longer period and their improved solubility and bioavailability are enabled.

[0107] The finished products were characterized from a chemical point of view, the safety profile and stability were examined, and the effectiveness of application on healthy volunteers was determined (in vivo testing). Physico-chemical properties/stability were also evaluated for a period of 12 months. Thanks to the established analytical method, non-psychogenic active substances in extracts and prepared formulations were identified and quantified. The presence of THC was not detected. The conducted tape/stripping test indicated that the entire amount of cannabidiol penetrates into the layers deeper than the stratum sogpeit, which was the goal of creating the nano-system.

[0108] The finished products have a soothing effect on painful places and accelerate the process of tissue recovery. They can be applied as:

[0109] - pharmaceutical preparations for a natural solution against pain (e.g. in sports medicine against pain in sportsmen's injuries),

[0110] - cosmetic preparations to reduce inflammation,

[0111] - veterinary preparations for the treatment of inflammation in pets/animals.

[0113] The novelty of the subject invention in relation to the available state of the art is represented by: → a new product/composition of increased bioavailability based on a supercritical non-psychoactive extract of hemp (C. sativa L.) with a quantified content of cannabinoids, which is incorporated into specially designed vesicular carriers - nanoliposomes. → new product/composition based on supercritical non-psychoactive hemp extract (C.

[0114] sativa L.) with quantified cannabinoid content, embedded in mesoporous silica nanoparticles, with increased bioavailability.

[0115] → new semi-product based on nanoliposomes and hemp extract, increased bioavailability when applied topically.

[0116] → a new intermediate product based on nanoparticles of mesoporous silica and hemp extract, increased bioavailability during topical application.

[0117] → new formulated phytopreparations based on nanoliposomes and supercritical non-psychoactive hemp extract with an effectively defined ratio of active components, emulsifiers, emollients and water for pharmaceutical, cosmetic and veterinary use.

[0118] → new formulated phytopreparations based on mesoporous silica nanoparticles and supercritical non-psychoactive hemp extract with an effectively defined ratio of active components, emulsifiers, emollients and water for pharmaceutical, cosmetic and veterinary use.

[0119] → unexpectedly effective localized application of phytopreparations that provide extracts rich in cannabidiol to penetrate into the deeper layers of the stratum corneum, improving the effectiveness for the relief and treatment of pain, inflammation, skin disorders, arthritis, peripheral neuropathy, muscle spasms, chronic pain, neuropathic pain and general muscle pain of various etiologies.

[0121] DESCRIPTION OF THE DRAWINGS

[0122] Figure 1. a) HPLC chromatograms of the investigated supercritical hemp extract and compounds identified at 225 nm, with the cannabidiol spectrum, b) HPLC chromatogram of the commercially used extract enriched with cannabidiol and c) calibration curve for cannabidiol.

[0123] Figure 2. Skin irritation test - influence of tested samples on in vivo measured skin parameters pH, TEVL, EI and both controls (under UCO occlusion and without UC occlusion); results are presented as absolute changes in mean values of the second compared to the first day and standard errors of the mean values; there were no statistically significant differences between the second and the first day for any of the tested samples and controls.

[0125] In order to facilitate the understanding of the subject matter of the present invention, examples are given below which explain the various stages in more detail, but in no way limit it.

[0128] EXAMPLES OF FEASIBILITY

[0130] Example 1: Encapsulation of hemp extract in nanoliposomes

[0131] Nanoliposomes were prepared using Phosal 40 IP (10 ml) and water for injection (up to 100 ml) to obtain a placebo sample.

[0132] A measured amount of supercritical hemp extract (10 ml) was added to 10 ml of Phosal 40 IP and the mixture was made up with purified water (up to 100 ml). After measuring the ingredients, the phases were mixed using a rotor-stator homogenizer (IKA Ultra-Turrax® T25 digital, IKA®-WerkeGmbH & Co. KG, Germany) for 20-50 min, preferably 30 min at 2000-8000 rpm. To obtain the final emulsion, the prepared coarse emulsion was passed through a high-pressure homogenizer (EmulsiFlex-C3, Avestin Inc., Canada), in a discontinuous process in five to ten cycles at a pressure of 500 to 1500 bar and a temperature of 25°C.

[0133] Example 1a: Encapsulation of hemp extract in nanoliposomes

[0134] A measured amount of 1 ml of hemp extract is added to 10 ml of Phosal 40 IP, topped up with purified water up to 100 ml and the production continues according to the procedure described in example 1.

[0136] Example 1b: Encapsulation of hemp extract in nanoliposomes

[0137] 55 ml of supercritical hemp extract was measured and added to 20 ml of Phosal 40 IP, the mixture was supplemented with purified water up to 100 m/ and production was continued according to the procedure described in example 1.

[0139] Example 2: Physico-chemical characterization of the nano-system

[0141] Droplet size analysis

[0142] For droplet size analysis, photon correlation spectroscopy (PCS) was carried out using a Zetasizer Nano ZS90 (Malvern Instruments Ltd., Worcestershire, UK). This technique ensures that the average diameter of the formed drops (Z-average diameter, Z-ave) and the polydispersity index (PDI) are determined.

[0144] Polydispersity index

[0145] Polydispersity index (PDI) is determined as a measure of particle size distribution. Before measurement, all samples were diluted with ultrapure water (1:1000, v/v) to obtain optimal scattering intensity. Measurements were performed at 25°C using a He-Ne laser with a wavelength of 633 nm, with a fixed scattering angle of 90°. All measurements were performed in triplicate.

[0147] Zeta potential

[0148] Zeta potential (ZP), as a measure of droplet surface charge and one indicator of long-term stability, was determined by measuring the electrophoretic mobility of liposomes, then converted to ZP using a Zetasizer Nano ZS90 (Malvern Instruments Ltd., Worcestershire, UK), along with built-in software. Measurements were performed immediately after diluting the samples (1:1100, v/v) with ultrapure water at 25°C.

[0150] The results of measurements of droplet size, polydispersity index and Zeta potential after 7 days, one and three months of storage are shown in Table 1.

[0151] Table 1. Average droplet diameter (Z-ave), polydispersity index (PDI) and zeta potential (ZP) of nanoliposome dispersions (placebo formulation and formulation with 10% (w/v) supercritical hemp extract) at room temperature

[0153]

[0156] Measurement of conductivity and pH value

[0158] Conductivity was measured using a HANNA Hi conductivity meter

[0159] 98311, Hanna Instruments, Woonsocket, RI. The conductance constant is set to 50

[0160] mS/cm using 0.9% (v/v) sodium chloride solution. All measurements were performed in triplicate

[0161] specimen. The results of pH value measurements are shown in Table 2.

Table 2. Measurement of pH-value and conductivity of nanoliposomal emulsion (NL) formulations (placebo and with 10% supercritical hemp extract) after production, 30 and 90 days after production

[0166]

[0169] Cannabidiol content

[0171] Content of cannabidiol in formulations based on nanoliposome and nanoparticle emulsions

[0172] of mesoporous silica was analyzed using the HPLC method, confirming that u

[0173] designed emulsions content of hemp extract 1-55% with non-psychogenic substances in

[0174] concentration 3-45%, preferably 13.44-22.65%.

[0176] Example 2a: HPLC method for determination of cannabidiol content

[0178] The determination of cannabidiol was performed using an HPLC 1200 system (Agilent

[0179] Technologies) equipped with a Phenomenex column, HydroRP 80, 150x4.6 using two

[0180] mobile phases (phase A is a 0.1 M solution of phosphoric acid, and phase B is pure acetonitrile). Speed flow rate was 1,000 mL/min, with photodiode array (PDA) detection (UV at 225 nm), always within 25 min. The best peak separation was achieved using the following combination: 78% B (0-10 min); 78-90% B (10-12 min); 90% B (12-20 min) and 90-78% B (20-25 min).

[0181] Before injection, the examined hemp extract was dissolved in a mixture of chloroform and methanol in a volume ratio of 7:3. The concentration of the extract was 0.772 mg/mL, and before injection the sample was filtered through a PTFE membrane filter.

[0182] For the CBD standard (ChemFaces) used in the study, the concentrations were 0.0125, 0.025, 0.05 and 0.1 mg/mL. The volume of injected standard solutions and tested extract was 4 μL. Identification is based on overlapping UV spectra and retention times compared to the standard used for the calibration curve. After the established overlap of the UV spectra, the identification results were confirmed by adding the test standard of the specified concentration, as well as by applying the so-called peak purity test. A peak that did not meet these requirements was not quantified. The quantification of CBD in the extract was achieved by applying an external calibration with a CBD standard, in the range of concentrations 0.0125 -0.1000 mg/mL (table 3), on the basis of which a calibration curve with R<2>= 1 was constructed.

[0183] Table 3. Peak areas at λ = 225 nm (A, expressed in mAU*s) and retention times (Rt, expressed in min), for the cannabidiol standard used to prepare the calibration curve at different concentrations (c, expressed in mg/mL)

[0186]

[0189] The HPLC chromatogram of the investigated supercritical hemp extract and the compounds identified at 225 nm, with the spectrum of CBD is shown in figure la), the HPLC chromatogram of the commercially used CBD-enriched extract in figure 1b), and the calibration curve for cannabidiol in figure 1c). It was calculated that the content of cannabidiol in the examined supercritical hemp extract is 13.45%.

[0191] Bearing in mind the presented results of physico-chemical characterization, which indicated only minor changes in the evaluated parameters, the preliminary physicochemical stability of the tested samples was observed.

[0192] Example 3: Impregnation of MSN with hemp extract

[0193] Example 3al: Impregnation of MSN with hemp extract using DMSO

[0194] Introduction of cannabidiol from hemp extract inside the pores (MCM-41, Sigma Aldrich) was carried out by impregnation method in the ratio 1:1 v/v. Prepare a solution of supercritical hemp extract in dimethyl sulfoxide (concentration 13.34 mg/mL cannabidiol). A measured amount of MSN (200 mg) was added to 15 mL of the prepared dimethyl sulfoxide solution from cannabidiol (CBD concentration before encapsulation, c1). The mixture was shaken for 24 hours at 210 rpm on an IKA® KS 260 basic orbital shaker (IKA® Werke GmbH & Somranu KG Staufen, Germany) at room temperature. MSNs are collected by centrifugation and carefully washed 3 times with water to remove surface adsorption of CBD.

[0196] Example 3aII:

[0197] Cannabidiol from hemp extract in DMSO was incorporated into the pores of the MSN by the method of impregnation in a ratio of 2:1 v/v in an identical manner, described in the previous paragraph.

[0199] Example 3aIII:

[0200] According to the procedure described in the first paragraph of example 3a, cannabidiol from hemp extract in dimethylsulfoxide was incorporated into the pores of MSN by the impregnation method in a 1:2 v/v ratio.

[0202] Example 3bl: Impregnation of MSN with hemp extract using ethanol

[0203] Introduction of cannabidiol from hemp extract into the pores of mesoporous silica nanoparticles (MCM-41, Sigma Aldrich) was carried out by the method of impregnation in the ratio 1:1 v/v. A solution of supercritical hemp extract in ethanol (concentration 11.76 mg/mL cannabidiol) is prepared. A measured amount of MSN (175 mg) is added to 15 mL ethanol, which contains 11.63 mg/mL CBD (CBD concentration before encapsulation, c1). The mixture is then treated in the same way as in Example 3a.

[0205] Example 3bII:

[0206] According to the procedure described in the first paragraph of example 3b, cannabidiol from hemp extract in ethanol was incorporated into the pores of the MSN by the impregnation method in a 1:2 v/v ratio.

[0207] Example 3bIII:

[0208] Cannabidiol from hemp extract in ethanol was incorporated into the pores of the MSN using the impregnation method in a ratio of 2:1 v/v in the manner described in the first paragraph of example 3b.

[0209] Example 4: Encapsulation efficiency of hemp extract in nano-systems

[0211] The loading efficiency of nanoliposomes and mesoporous silica nanoparticles with cannabidiol was determined using the results of HPLC analysis of the samples (HPLC conditions are listed in Example 2a). The concentration of cannabidiol in the supernatant was measured after centrifugation (LLG UNICFUGE5, Lab Logistic Group) at 3500 rpm for 30 minutes.

[0212] After that, the supernatant was filtered through a regenerated cellulose syringe filter (pore size 0.2 mm, Premium Syringe Filters) and then diluted using dimethyl sulfoxide (DMSO), ie. of ethanol (10 ml of the mixture and 990 ml of DMSO, i.e. ethanol) before injection into the HPLC column.

[0214] Example 4a: Encapsulation efficiency (EE) of cannabidiol in nanoliposomes

[0215] The content of cannabidiol (in DMSO and in ethanol) after encapsulation (s2) in nanoliposomes was calculated using the t linear regression equation derived from the calibration curve. The calculation of the concentration of cannabidiol in the analyzed samples, Ca, was performed using the expression:

[0218]

[0220] where cs is the concentration of cannabidiol in the CBD standard sample, Pa - the area under the peak corresponding to the tested substance, Ps - the area under the peak corresponding to the standard.

[0222] Standard CBD: Retention time 4.797 min, Cs=0.025 mg/ml, Ps=301.04916.

[0223] Theoretical amount of cannabidiol

[0224] Supercritical extract incorporated: 1.345 w%, density (m=87.34 mg, V=100 μl) ρ=873.4 mg/ml Amount of CBD: V=2 ml, mu=0.8734g/ml·2 ml=1.75g; m=1.345/100·1.75=0.0235; g=23.5 mg of CBD. Incorporated commercial extract 1: 2.1 w%, density: (m=96.48 mg, V=100 μl) ρ =964.8 mg/ml Amount of CBD: V=2 ml, mu=0.9648 g/ml·2 ml=1.93g; m=2.1/100·1.93=0.0405, g=40.5 mg of CBD. Incorporated commercial extract 2: 2.26 w%, density: (m=94.82 mg, V=100 μl) ρ =948.2 mg/ml Amount of CBD: V=2 ml, mu=0.9482g/ml·2 ml =1.896g; m=2.26/100·1.896=0.0428; g=42.8 mg of CBD.

[0225] Encapsulation efficiency

[0226] Incorporated supercritical extract

[0227] Pa=31.74543; Ca=0.0026 mg/ml; V=0.3 ml; Ma=0.0026-0.3=0.00078 mg - the same amount is found in 2 ml of supernatant. After 2x dilution: Ma=0.00078-2=0.00156 mg, which is of the total CBD:

[0228] 100 = 0.0066%. Encapsulation efficiency EE% = 100 % - 0.0066 % = 99.9934%.

[0230] an commercial extract 1

[0232]

a=0.00813 mg/ml; V=0.3 ml. Diluted 2x: Ma=0.008130.3-2=0.00488 mg; and that's from

[0233]

0.012%.

[0234] Encapsulation efficiency = 100% - 0.012% = 99.988%.

[0235] Incorporated commercial extract 2

[0236] Pa=84.82533; Ca=0.00704 mg/ml; V=0.3 ml. Diluted 2x; Ma=0.00704·0.3·2=0.00423 mg, which is

[0237] of the total

100 = 0.0099%

[0239] Encapsulation efficiency = 100% - 0.0099% = 99.9901%.

[0240] The encapsulation efficiency in nanoliposomes for cannabidiol dissolved in DMSO and in ethanol is 99.99%.

[0242] Example 4bl: Encapsulation efficiency (EE) of cannabidiol in MSNs in DMSO

[0243] The content of cannabidiol in DMSO after encapsulation (s2) in mesoporous silica nanoparticles was calculated using the equation / linear regression derived from the calibration curve using the following calculation:

[0246]

[0248] c2(mg/mL)=(1691.60-2.2377)/l 2016=0.14059 mg/mL (∙ 100)=14.06 mg/mL.

[0249] The encapsulation efficiency (EE) of cannabidiol in DMSO was calculated according to the following equation:

[0250] EE (%)=(c<1>-s<2>)/s<1>·100

[0251] where c1 and c2 are the concentrations of cannabidiol in DMSO before and after encapsulation, respectively

[0252] EE (%)=(13.40 mg/mL-14.06 mg/mL)/13.40 mg/mL ∙100= - 4.92%.

[0254] Example 4bII: Encapsulation Efficiency (EE) of Cannabidiol in MSNs in Ethanol

[0255] The content of cannabidiol in ethanol was calculated using the t linear regression equation derived from the calibration curve using the following calculation:

[0258]

[0261] c2(mg/mL)=(11309.08-2.2377)/12016=0.94 mg/mL(∙ 100)=94.098 mg/mL.

[0262] The encapsulation efficiency (EE) in ethanol was calculated according to the following equation:

[0263] EE (%)=((c<1>-c<2>)/ c<1>) ∙ 100

[0264] where c1 and c2 are the concentrations of CBD in ethanol before and after encapsulation, respectively

[0265] EE (%)=(94.098 mg/mL-10.87 mg/mL)/94.098 mg/mL∙100=88.44%.

[0267] The encapsulation efficiency in MNS for cannabidiol dissolved in DMSO is -4.92%, while for cannabidiol dissolved in ethanol the encapsulation efficiency is 88.44%.

[0268] Example 5. Method of obtaining emulgel with nanoliposome dispersion

[0271]

[0274] Preparation method:

[0275] Hemp extract is homogenized with lecithin/phosphatidylcholine on a laboratory homogenizer (speed 5000-8000 rpm) and a primary dispersion is formed which is "passed" in five to ten cycles through an extruder or through a homogenizer under high pressure. The number of cycles is optimized to obtain liposomes of the desired diameter (up to 500 nm).

[0276] Add hydroxyethyl cellulose to the mixture of water, glycerol and preservatives and let it swell for 30 min with occasional stirring. After complete hydration of the cellulose derivative, the aqueous phase is heated to 70°C (±2).

[0277] The components of the fat phase are melted and the temperature of the mass is adjusted to 70°C (±2).

[0278] Fat and water phases are combined with the mixer (speed 200-1000 rpm) and homogenizer (speed 1000-5000 rpm) for 5-25 minutes. Then the cooling of the mass begins. At a temperature of 35°C (±2), a dispersion of liposomes enriched with hemp extract is added to the mass. It is mixed while cooling, until the mass in the vessel reaches room temperature.

[0280] Example 6. Procedure for obtaining emulgels using MSN with hemp extract Composition of emulgel formulations with mesoporous silica nanoparticles

[0283]

[0286] Production procedure: Analogous to the procedure for the production of emulsifiers based on liposome dispersion, hydroxyethyl cellulose is added to the mixture of water, glycerol and preservatives and allowed to swell for 30 min with occasional stirring. After complete hydration of the cellulose derivative, the aqueous phase is heated to 70°C (±2).

[0287] The components of the fat phase are melted and the temperature of the mass is adjusted to 70°C (±2). The fat and water phases are combined with the mixer (speed 200-1000 rpm) and homogenizer (speed 1000-5000 rpm) for 5-25 minutes. Then the cooling of the mass begins. At a temperature of 35°C (±2), a dispersion of mesoporous SiO2 nanoparticles with hemp extract is added to the mass.

[0288] It is mixed while cooling, until the mass in the vessel reaches room temperature.

[0290] Example 7. In vivo skin bioengineering techniques

[0292] A nanoliposome formulation loaded with supercritical hemp extract and their corresponding placebo formulation were used in an in vivo study testing their preliminary safety and efficacy in topical therapy on the skin of human volunteers.

[0293] In vivo skin bioengineering techniques provide information on erythema index (EI), transepidermal water loss (TEWL), skin pH in a 24-hour study on normal skin under occlusion for a preliminary safety profile.

[0294] The safety profile of the tested samples - liposomal dispersion (L) and liposomal dispersion with incorporated hemp extract (LH) was evaluated in vivo by measuring the relevant biophysical parameters of human skin: TEWL, EI and skin pH after the end of the 24-hour occlusive treatment. The in vivo skin irritation potential can be assessed by analyzing important parameters related to skin health and function (ie skin pH, TEWL and EI) after a single application of a particular formulation under occlusion for 24 hours, on the skin of healthy volunteers. Because an increase in TEWL is observed after application of a skin irritant, TEWL measurements are often used to support mildness claims of topical formulations (cosmetic or dermopharmaceutical).

[0295] The parameters were measured before (baseline values on the first day of the experiment) and 60 min after the end of the 24-hour occlusive treatment (on the second day of the experiment). Ten healthy volunteers (24.3 ± 2.1 years) were recruited. The flexor side of the left forearm was treated with samples L and LH, and additional sites were left as an untreated control under occlusion (UCO) and an untreated control without occlusion (UC) on the right forearm. Samples were applied in amounts of 0.016 g/cm<2>, stretched vigorously with a rubber glove and immediately covered with Parafilm® application followed by cotton adhesive tapes.

[0296] In vivo skin performance data are presented as mean/-standard error of the mean (SEM). The measured parameters (pH, TEWL and EI) were expressed as absolute changes from the initial value (D value) of the second compared to the first day in the safety study. In vivo effects of samples were compared with each other and related to untreated controls, under and without occlusion (UCO and UC, respectively), using one-way ANOVA, followed by Tukey's t-test where appropriate. Differences were accepted as statistically significant at p<0.05. Statistical analysis was performed using the commercial statistical software SPSS for Windows 17.0. All other data are presented as mean/- standard deviation.

[0297] Figure 2 shows the results of the skin irritation test showing the influence of the tested samples on the in vivo measured skin parameters pH, TEVL, EI and both controls (under UCO occlusion and without UC occlusion). Results are presented as absolute changes in mean values from the second day compared to the first day and standard errors of the means. There were no statistically significant differences between the second and the first day for any of the tested samples and controls.

[0298] The obtained results shown in Figure 2 confirmed the satisfactory safety profile of all tested samples, as no skin irritation or a significant increase in TEWL or EI or a significant change in pH that could indicate a damaged skin barrier function was recorded.

[0299] Skin irritation is a complex biological event, usually described by a clinical response (edema, dryness, erythema) to chemical or physical stimuli that cause inflammation at the site of contact after single or repeated exposure. Exposure to surfactants often causes skin dehydration and irritant contact dermatitis (ICD), which is a non-immunological local inflammatory reaction; sodium lauryl sulfate (SLS) is often used for this purpose.

[0300] Irritant contact dermatitis following the application of sodium lauryl sulfate is characterized by erythema, increased transepidermal water loss (TEWL), dryness and scaling of the skin. Although originally developed to assess the susceptibility of substances to induce ICD, the SLS test is often used to examine the protective and anti-inflammatory properties of topical preparations.

[0302] Non-invasive in vivo model of evaluation of efficacy in the treatment of irritated skin

[0303] The non-invasive in vivo model for evaluating the effectiveness of samples in the treatment of human skin with induced irritant contact dermatitis, irritated by sodium lauryl sulfate (SLS), included the assessment of the state of hydration of the stratum corneum by measuring electrical capacitance (EC), TEWL and EI, and the results are shown in Table 4.

[0304] Nine healthy volunteers (24.8+2.1 years) participated in the evaluation of the effectiveness of the samples in the treatment of skin irritated by sodium lauryl sulfate (SLS). Experimental irritation with SLS under 8-hour occlusion was performed in accordance with published guidelines. A total of 200 mL of 8% SLS aqueous solution (purity 99%, Merck, Germany) was placed on each of the three filter papers. Filter papers were subsequently placed on the test sites of the volar aspects of the forearms using a cardboard ruler with four rectangular blanks. The filter papers were then immediately covered with Parafilm® and Sensifix® cotton adhesive tape. The test site next to the left wrist represented the induced UCO, and the rectangular empty space next to the right wrist was covered without induced irritation and served as the UC. Volunteers removed Sensifix®, Parafilm® and filter paper after 8 h. After irritation, a 7-day treatment of the test sites was carried out. The tested samples, liposomal dispersion (L) on the right hand and liposomal dispersion with incorporated hemp extract (LH) on the left hand, were applied twice a day (morning and evening).

[0306]

[0308] The following parameters were measured: EC, TEWL and EI, before the irritation test ("primary", baseline values), 24 h after removal of the occlusion ("secondary", baseline values) and after 7 days treatment. The last application of the sample was performed 12 h before the measurement. Corneometer® CM825 was used to measure skin EC as an indicator of skin hydration. TEWL was measured using Tewameter® TM210, EI using Mexameter® MH18 (all devices manufactured by Courage Khazaka Electronic GmbH, Germany). The results of stratum corneum (SC) electrical capacitance (EC) measurement as a measure of SC hydration state, TEWL and EI measurements in SLS-induced ICD human skin in a non-invasive in vivo model are shown in Table 4.

[0309] Occlusive application of 8% SLS led to a significant decrease in skin hydration levels compared to baseline/baseline values.

[0310] Treatment with a liposomal dispersion sample incorporated with hemp extract (LH) resulted in a significant improvement in skin hydration (parameter EC) compared to baseline/baseline values after only 4 days of administration. On the other hand, there was no statistical difference in the increase in hydration levels between the samples after 7 days of treatment. The liposomal dispersion sample (L) was not as effective. The level of hydration for untreated, irritated skin (UCO) after 7 days remained without a significant increase compared to the level after irritation. However, a significant improvement in the level of hydration of the irritated skin was noticeable after 2 days of treatment with the liposomal dispersion sample incorporated with hemp extract (LH). This indicates that the sample regained its skin hydration potential within a short period of time after the start of application and can be attributed to the hemp extract.

[0311] As expected, TEWL, a measure of skin water loss, was significantly increased after SLS-induced irritation in control (UCO) and treated sites compared to baseline/baseline values, indicating that the skin barrier was disrupted after SLS application. That increase in control sites (UCO) remained after 7 days, indicating that untreated skin was unable to repair itself in just one week without any treatment, while treated skin repaired in both test sites. However, at the LH-treated skin site, the decrease in TEWL as a measure of the activity of the sample in terms of skin barrier recovery was significant compared to the post-irritation value. In fact, the results according to the invention show that the hemp extract sample is more effective in repairing the skin barrier than the liposome dispersion alone. It is known that chemical irritants (such as SLS), in addition to disrupting the barrier function of the SC, have a direct effect on skin cells. The latter includes several mechanisms of initiation of the inflammatory cascade either by damaging the keratinocyte membrane or by provoking the rise of reactive oxygen species (ROS) to a non-physiological level. Erythema is considered the main feature of an acute inflammatory reaction.

[0312] The EI parameter, as a direct measure of erythema, was increased after SLS-induced irritation at treated and control sites (UCO) compared to baseline/baseline values.

[0313] A significant recovery of EI to baseline values was observed only after treatment with sample L. However, a significant reduction in erythema index (EI) after irritation was noted only at this test site.

[0314] The invention has undergone studies demonstrating safe and natural pain relief and treatment in healthy human skin, especially with the recent prevalence of the opioid crisis associated with prescription painkillers. This invention offers a safe, natural alternative to such drugs and enables local instead of systemic absorption of the active ingredients. As an effective, safe and simple pain relief solution it has the potential to improve the productivity of people experiencing work-related pain in a number of industries and occupations, e.g. office workers and students who type for long hours, musicians who want to practice longer without feeling discomfort, athletes due to sports injuries. The invention has numerous biopharmaceutical applications in the treatment of chronic pain, skin conditions, muscle spasms, arthritis and neuropathic diseases, as it offers a uniquely affordable and effective option for patients seeking natural and environmentally sustainable treatments.

Claims (12)

1. PATENT CLAIMS 1. Composition for topical application in the treatment of peripheral neuropathic pain and general muscle pain of various etiologies, arthritis and inflammatory skin diseases based on medicinal herbs, characterized by the fact that it contains 01.00% - 55.00% of a specific extract of industrial hemp, Cannabis sativa L., characterized by the presence of a fraction with non-psychogenic substances in a concentration of 3-45%, primarily 13.44-22.65% which is incorporated into nanocarriers such as nanoliposomes or mesoporous silica nanoparticles. 2. The process of encapsulating hemp extract into nanoliposomes, indicated by the fact that it is performed by mixing 10-20% of the raw material for the formation of liposomes, an empty carrier based on: - lecithin with 55% linoleic, 4% linolenic and 5.5% arachidonic acid or - purified soy lecithin with 72-78% phosphatidylcholine or - purified soy lecithin with 37% phosphatidylcholine and lysophosphatidylcholine, and 1-55% hemp extract, make up to 100% with ultrapure water, the mixture is homogenized for 20-50 min, preferably 30 min at 2000-8000 rpm, and then the prepared rough emulsion is passed through the extrusion method through a homogenizer under high pressure from 500 to 1500 bar in a discontinuous process in five to ten cycles at a temperature of 25°C. 3. The method of encapsulation of hemp extract into nanoparticles of mesoporous silica, characterized by the fact that it is carried out by the impregnation method, by adding nanoparticles of mesoporous silica to a solution of hemp extract in dimethylsulfoxide or in ethanol, the mixture is subjected to shaking at 210-450 rpm for 18-32 hours, preferably 24 hours, after which the nanoparticles of mesoporous Silica with cannabidiol is collected by centrifugation and washed three to four times with water. 4. The method according to claim 3, characterized in that the mass ratio of hemp extract and mesoporous silica nanoparticles is in the range of 2:1 to 1:2, preferably 1:1. 5. Nanoliposomes with encapsulated hemp extract according to claim 2, characterized by the fact that the dispersion of encapsulated nanoliposomes with hemp extract is characterized by medium particle diameter size 241.53 nm to 253.32 nm, pH value 4.62 and encapsulation efficiency 99.99%. 6. Nanoparticles of mesoporous silica with encapsulated hemp extract according to claims 3 and 4, characterized by the fact that the dispersion of encapsulated nanoparticles with hemp extract in ethanol is characterized by an encapsulation efficiency of up to 88.44%. 7. Phytopreparations according to claims 1 to 6, indicated by the fact that the composition incorporated into the appropriate substrates can be in the form of emulgel. 8. Phytopreparations in the form of emulgels according to claims 1 to 7<,>indicated by the fact that they contain 1.00-55.00%, primarily 13.44-22.65% of the active component of hemp extract (Cannabis sativa L.) incorporated into nano-carriers such as nanoliposomes or mesoporous silica nanoparticles and common pharmaceutical additives for the formation of a pharmaceutical-technological form, namely components of the oil phase and aqueous phase components. 9. Composition according to claim 1 for local application in the treatment of pain in inflammatory skin disorders, arthritis, peripheral neuropathic pain and general muscle pain of various etiologies, without systemic absorption of cannabinoids. 10. Composition according to claim 9 for use as a pharmaceutical product. 11. Composition according to claim 9 for use as a cosmetic product. 12. Composition according to claim 9 for use as a veterinary product.