Cannabis sativa L. oil-based gel, rich in cannabidiol, for therapeutic ultrasound application.

Description

Cannabis sativa L. oil-based gel, rich in cannabidiol, for therapeutic ultrasound application. Field of Invention

[001] This patent refers to a pharmaceutical product with preventive and therapeutic properties, whose active ingredient is Cannabis sativa L. extract oil, rich in cannabidiol (99%) (Institute for Research on Entheogenic and Medicinal Plants for Health and Well-being, Alto Paraíso de Goiás, GO, Brazil). The product, applied topically in gel form, is intended for the prevention or treatment of musculoskeletal injuries with inflammatory foci. The bioproduct was tested on Wistar rats, using various experimental protocols to evaluate its effectiveness in reducing musculoskeletal injuries (Costa, MM, Oliveira, CA, Silva, JF (2023). Cannabidiol and its potential anti-inflammatory effects in the musculoskeletal system. Phytotherapy Research, 37(5), 2671-2680). Fundamentals of the Invention

[002] The development of this invention aims at technological innovation, focusing on the creation of economically accessible products for the prevention and treatment of musculoskeletal injuries, minimizing adverse effects. The invention uses an oily extract of Cannabis sativa L. incorporated into a carbopol gel base, whose physicochemical properties allow it to cross tissue barriers, increasing chemical stability, solubility, and pharmacological efficacy (Rodrigues, MG, Souza, VF, Almeida, TC (2021). Carbopol as a versatile excipient in pharmaceutical formulations: Properties and applications. International Journal of Pharmaceutics, 606, 120961; Silva, PR, Costa, VH, Pereira, MG (2023). Ultrasonic-assisted transdermal delivery of cannabidiol: An innovative approach. Drug Delivery and Translational Research, 13(4), 932-943).

[003] Musculoskeletal injuries are common in sports, recreational activities, and daily life, posing a challenge for both primary care and sports and trauma medicine (Ferreira, MA, Santos, RC, Silva, ME (2020). Musculoskeletal injury treatment: A new therapeutic approach with cannabinoids. Journal of Sports Science & Medicine, 19(1), 23-34). These injuries can occur due to direct or indirect trauma, triggering Petition 870240110013, dated 12 / 25 / 2024, page 6 / 18 2 / 7 a local inflammatory response (Oliveira, JC, Ferreira, AP, Santos, FA (2022). Musculoskeletal injuries: Diagnosis and management in primary care. BMJ Open Sports & Exercise Medicine, 8(1), e001209).

[004] It is well documented that the inflammatory response is essential for the rehabilitation of injured tissues (Carvalho, DR, Ribeiro, JA, Silva, LP (2019). Inflammatory response and tissue repair mechanisms in musculoskeletal injuries. Inflammation Research, 68(4), 102-110). However, severe injuries can lead to the excessive production of reactive oxygen species (ROS), which include radicals such as superoxide and hydroxyl, as well as non-radical oxidizing agents such as hydrogen peroxide. When ROS production exceeds cellular antioxidant capacity, oxidative stress can occur, affecting lipids, proteins, and DNA (Mendes, FT, Ribeiro, LP, Silva, MR (2020). Reactive oxygen species in musculoskeletal injuries: Mechanisms and therapeutic approaches. Journal of Inflammation Research, 13, 77-91.; Silva, RT, Nogueira, CM, Souza, JP (2021). Oxidative stress and musculoskeletal injuries: Clinical implications and management strategies.Sports Medicine, 51(5), 883-897).

[005] Although the endogenous antioxidant defense system, which includes enzymes such as catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase, acts in response to oxidative stress, this defense may be insufficient to neutralize oxidative damage (Guimarães, AM, Lima, RS, Mendes, JO (2023). Oxidative stress and antioxidant defense mechanisms in skeletal muscle injuries. Free Radical Biology & Medicine, 184, 135-142). Supplementation with exogenous antioxidants, such as flavonoids, plays a crucial role in protecting against the effects of ROS (Santos, MC, Almeida, FS, Pereira, JO (2022). The role of flavonoids in oxidative stress modulation: Applications in muscle injuries. Antioxidants, 11(8), 1452).

[006] Natural substances extracted from plants, including cannabinoids, are widely studied for their anti-inflammatory and antioxidant properties. Cannabinoids include phytocannabinoids, endocannabinoids, and synthetic cannabinoids. Cannabidiol (CBD), a phytocannabinoid present in Cannabis sativa L., has proven therapeutic properties, including the reduction of acute inflammation, modulation of immune responses, and inhibition of pro-inflammatory cytokines (Costa, MM, Oliveira, CA, Silva, JF (2023). Cannabidiol and its potential anti-inflammatory effects in the musculoskeletal system. Phytotherapy Research, 37(5), 2671 Petition 870240110013, dated 12 / 25 / 2024, page 7 / 18 3 / 7 2680; Oliveira, RG, Lima, CV, Martins, FJ (2020). Cannabidiol as an alternative for inflammation modulation: A review of its mechanisms and clinical applications. Journal of Inflammation Research, 13, 979-992). Unlike non-steroidal anti-inflammatory drugs, CBD has a reduced side effect profile (Atalay, S., Jarocka-Karpowicz, I., Skrzydlewska, E. (2020). Antioxidative and anti-inflammatory properties of cannabidiol. Journal of Clinical Medicine, 9(6), 1843).

[007] Carbopol, a high molecular weight acrylic acid polymer widely used in the cosmetic, pharmaceutical and cleaning product industries, is a key component in gel formation. When dispersed in water and neutralized with substances such as triethanolamine, carbopol acquires a gel consistency, acting as a thickener (Rodrigues, MG, Souza, VF, Almeida, TC (2021). Carbopol as a versatile excipient in pharmaceutical formulations: Properties and applications. International Journal of Pharmaceutics, 606, 120961).

[008] Due to the low aqueous solubility and high lipophilicity of cannabidiol, its incorporation into carbopol is an effective solution to improve its physicochemical and pharmacological properties. The use of this combination aims to optimize tissue penetration and enhance the therapeutic effect of CBD in ultrasound treatments (Silva, PR, Costa, VH, Pereira, MG (2023). Ultrasonic-assisted transdermal delivery of cannabidiol: An innovative approach. Drug Delivery and Translational Research, 13(4), 932-943). Thus, the formulation of the gel based on Cannabis sativa oil is a viable alternative to improve its physicochemical and pharmacological properties. Several studies conducted with other secondary metabolites, such as the flavonoids naringin and rutin, have shown positive therapeutic effects, especially when complexed to be water-soluble (BR 10 2023 016640 7; BR 10 2024 016374 5).Incorporation into carbapol aims to enhance pharmacological efficacy by adapting the physicochemical properties of these products to tissues using ultrasonic waves (201810578059.7). Petition 870240110013, dated 12 / 25 / 2024, page 8 / 18 4 / 7 Brief Description of the Drawings Figure 1 shows the effect of a gel based on Cannabis sativa L. oil, rich in Cannabidiol, combined with pulsed therapeutic ultrasound on MDA levels 98 hours after gastrocnemius muscle injury. Figure 2 shows the effect of a gel based on Cannabis sativa L. oil, rich in Cannabidiol, combined with pulsed therapeutic ultrasound on SOD activity levels 98 hours after gastrocnemius muscle injury. Figure 3 shows the effect of a gel based on Cannabis sativa L. oil, rich in Cannabidiol, combined with pulsed therapeutic ultrasound on CAT activity levels 98 hours after gastrocnemius muscle injury. Description of the invention

[009] Before any experiments of the present invention are explained in detail, it should be understood that this invention is not limited to the methods and compositions described. The invention is capable of other embodiments and of being practiced in various ways. It should be understood that the descriptions employed herein are for descriptive purposes only and should not be interpreted as limiting. Preparation of inclusion complexes

[010] Cannabis sativa L. oil, rich in 99% Cannabidiol (27.6g (sunflower oil); 1.5g (CBD); 0.069g (Myrcene); 0.032g (Caryophyllene); 0.069g (D-Limonene); 0.014g (Linalool)) (Institute for Research on Entheogenic and Medicinal Plants for Health and Well-being, Alto Paraíso de Goiás, GO, Brazil, CNPJ: 49934494000170) (batch: 08 / 2023) was incorporated into carbopol hydrogel. In this way, a dispersion of the polymer in water was obtained with constant homogenization to avoid the formation of lumps. After obtaining the hydrogel, 5% of the CBD-rich oil was added and subjected to treatment. After that, a preliminary stability test of the formulation was carried out in accordance with the Cosmetic Product Stability Guide of the National Health Surveillance Agency (2004).

[011] The experimentation process for evaluating the protective activity of Cannabis sativa L. gel, rich in Cannabidiol, against musculoskeletal injury is presented in the following ways: Petition 870240110013, dated 12 / 25 / 2024, page 9 / 18 5 / 7 For the protection assessment, 40 Wistar rats weighing between 250 and 300 grams were used. All animals were kept in polyethylene cages, with a maximum of 5 rats per cage, fed commercial feed and water, and subjected to a 12-12 h light / dark cycle at a temperature of 23 ± 2°C. The project was approved by the Animal Research Ethics Committee (CEUA n° 9751050723) of the Federal University of Sergipe. Experimental Protocols

[012] The animals were allocated into five different groups. Control group (CTRL) - animals without injury; Muscle injury group (LM) - animals with injury, but without treatment; Therapeutic Pulsed Ultrasound group (UPT) - animals with injury treated with pulsed ultrasound and saline gel (0.9%); Therapeutic pulsed ultrasound plus CBD-rich gel group (UPT + CBD) - animals with injury treated with pulsed ultrasound plus incorporated cannabidiol (5%); CBD group (CBD) - animals with injury treated with cannabidiol gel. The rats were anesthetized with ketamine (90mg / kg, Ceva / São Paulo, Brazil and xylazine (25mg / kg, Ceva / São Paulo, Brazil)), the gastrocnemius muscle was shaved and injured by a single, abrupt impact using a mechanical press (FiCIDEP / RS). The trauma was caused by a metallic mass (0.459 kg) falling from a height of 18 cm onto the gastrocnemius muscle, generating a kinetic energy of 0.811 Joules.The pulsed ultrasound therapeutic treatment (Ibramed, Amparo, Brazil) was performed in pulsed mode, with an effective radiation area (ERA) of 1 cm², a duty cycle of 50%, a frequency of 1 MHz, a duration of 6 min, and an intensity of 0.8 W / cm². The area was treated with approximately 2 cm2 (Rizzi Cf., et al. Effects of low-level laser therapy (LLLT) on the nuclear factor (NF) kB signaling Pathway in traumatized muscle. Lasers Surg Med. 2006; 38:704-13) with circular movements and after the lesion focus (Saliba S, Mistry DJ, Perrin DH, Gieck J, Weltman A. Phonophoresis and the absorption of dexamethasone in the presence of an occlusive dressing. Journal of athletic training. 2007, 42(3): 349-54). The groups treated with UPT (UPT and UPT+CBD groups) received 6 minutes of application with intervals of 2, 12, 24, 48, 72, and 96 hours after the injury. A topical gel incorporating cannabidiol was administered topically.

[013] Two hours after the last application of the treatment, that is, 98 hours after the induction of the lesion, the animals were sacrificed by guillotine. The injured muscle was surgically removed and Petition 870240110013, dated 12 / 25 / 2024, page 10 / 18 6 / 7 stored at -80°C. Muscle samples were homogenized in specific buffers, according to each analysis protocol. Lipid peroxidation was analyzed by the formation of thiobarbituric acid reactive substances. Samples were mixed with 1 mL of trichloroacetic acid (10%) plus 1 mL of thiobarbituric acid (0.67%). Later, it was heated in a water bath for 30 min. TBARS were determined by absorbance at 532 nm. Results were expressed in nmol / mg of protein. Superoxide dismutase (SOD) activity was evaluated by measuring the inhibition of epinephrine auto-oxidation at absorbance at 480 nm, as described by Bannister and Calabrese (1987). Results were expressed as U SOD / mg of protein. Catalase (CAT) was measured by the hydrogen peroxide decay rate by absorbance at 240 nm, and the results were expressed as U CAT / mg of protein. Figures are attached.

[014] The results obtained were statistically analyzed using the one-way analysis of variance (ANOVA) test, followed by the Bonferroni post-test, adopting p < 0.05 as the significance level. The software used for the analysis was GraphPad Prism 6.1. The data were expressed as mean ± standard error of mean (SEM). Examples of embodiments of the invention

[015] According to the results obtained, it was observed that there was an increase in MDA formation after 98 hours of muscle injury when comparing the CTRL group (1.58 ± 0.40 nmolMDA / mg protein) to the LM group (7.17 ± 1.02 nmolMDA / mg protein, p < 0.05, n = 8). The UPT group (3.36 ± 0.19 nmolMDA / mg protein, p < 0.05); UPT+CBD (0.35 ± 0.19 nmolMDA / mg protein) and CBD (0.88 ± 0.14 nmolMDA / mg protein) showed a decrease in MDA formation levels when compared to the LM group. There was also a statistically significant difference between the UPT group (3.36 ± 0.19 nmolMDA / mg protein, p < 0.05) when compared to the UPT+CBD group (0.35 ± 0.19 nmolMDA / mg protein) and the CBD group (0.88 ± 0.14 nmolMDA / mg protein). There was no statistically significant difference between the UPT+CBD and CBD groups.Thus, the groups treated with the Cannabis sativa L oil-based gel, the UPT +CBD group and the CBD group, reduced tissue damage expressed by lipid peroxidation after LM by 95.5% and 89.1%, respectively (Figure 1). SOD activity showed a significant decrease in the UPT (2.25±0.31 U / mg protein); UPT+CBD (0.77±0.18 U / mg protein) and CBD (0.91±0.12 U / mg protein) groups when compared to the LM group (3.85±0.12 U / mg protein). The CRTL group was also statistically different. Petition 870240110013, dated 12 / 25 / 2024, page 11 / 18 7 / 7 different when compared to the LM group. CAT activity was increased when comparing the LM group (1.30 ± 0.16 CAT / mg of protein) to the CTRL group (0.78 ± 0.09 CAT / mg of protein, p < 0.05). The treatment promoted a statistically significant reduction in enzymatic activity in the UPT (0.23 ± 0.04 CAT / mg of protein, p < 0.05), UPT + CBD (0.18 ± 0.03 CAT / mg of protein, p < 0.05), and CBD (0.42 ± 0.13 CAT / mg of protein, p < 0.05) groups compared to the LM group, as shown in Figure 3. The UPT + CBD and CBD groups reduced superoxide dismutase values ​​by 80% and 76.2%, respectively (Figure 2), as well as catalase by 86.1% and 67.7% when compared to the LM group (Figure 3).

[016] In view of the above, it was possible to demonstrate that cannabidiol was a good resource used in gel form. The results indicate that the use of the gel incorporated with Cannabis sativa L. oil, rich in cannabidiol, associated with therapeutic ultrasound after musculoskeletal injury in rats is efficient in attenuating lipid peroxidation. The combination of these two resources also showed considerable effects in potentiating the antioxidant defense system, through the stimulation of CAT and SOD enzymatic activity.

Claims

CLAIMS 1. Gel based on Cannabis sativa L. oil, rich in cannabidiol, for therapeutic ultrasound application, characterized by being effective in preventing injury induced by musculoskeletal trauma.

2. Gel based on Cannabis sativa L. oil, rich in cannabidiol, for therapeutic ultrasound application, characterized by being more soluble than pure oil.

3. Gel based on Cannabis sativa L. oil, rich in cannabidiol, for therapeutic ultrasound application, characterized by being effective in the treatment of musculoskeletal injury induced by increased oxidative stress.

4. Gel based on Cannabis sativa L. oil, rich in cannabidiol, for ultrasound application, characterized by being preferentially useful in the prevention of musculoskeletal injury.

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