Use of a formulation, use of a combination, method for the prevention or treatment of inflammatory conditions causing orofacial pain, and method for the prevention or treatment of conditions related to muscle recovery

Abstract

The present invention relates to the antinociceptive effect and anti-inflammatory mechanism of using a formulation based on Alpinia zerumbet essential oil (Fb-EOAz) in the prevention or treatment of orofacial mechanical hyperalgesia and orofacial thermal hyperalgesia, in the prevention of muscle injury, and in muscle recovery. The invention also relates to a method for the prevention or treatment of inflammatory conditions causing orofacial pain, for the prevention of muscle injury, and for muscle recovery.

Description

[0001] USE OF A FORMULATION, USE OF A COMBINATION, METHOD FOR PREVENTING OR TREATING INFLAMMATORY OROFACIAL PAIN CONDITIONS AND METHOD FOR PREVENTING OR TREATING CONDITIONS RELATED TO MUSCLE RECOVERY FIELD OF THE INVENTION

[0002]

[0001] The present invention relates to the antinociceptive effect and anti-inflammatory mechanism of using a formulation based on Alpinia zerumbet essential oil (EOAz) in the prevention or treatment of orofacial mechanical hyperalgesia and orofacial thermal hyperalgesia, in the prevention of muscle injury and in muscle recovery. The invention also relates to a method of preventing or treating inflammatory orofacial pain conditions, in the prevention of muscle injury and in muscle recovery.

[0003] FUNDAMENTALS OF THE INVENTION

[0004]

[0002] Pain involving the head, face, neck, or intraoral structures is known as orofacial pain, which can have different etiologies and can be acute or chronic. Acute orofacial pain includes pain from surgical and non-surgical procedures, and both require effective pain control during treatment, and often pain control in the postoperative period as well.

[0005]

[0003] It is estimated that pain after oral surgery affects more than 90% of patients, being considered most intense 5-6 hours after the procedure is completed and reaching a peak during the first postoperative day.

[0006]

[0004] The pharmacological treatment of acute orofacial pain includes the use of non-steroidal anti-inflammatory drugs (NSAIDs, mainly ibuprofen), non-opioid analgesics (paracetamol and dipyrone) and opioid analgesics, which may be prescribed alone or in combination.

[0007]

[0005] The prescription of these medications varies by region, with opioids being widely prescribed for acute dental pain in the US, but not in Brazil, where NSAIDs in combination with paracetamol or dipyrone are frequently used.

[0008]

[0006] These strategies generally provide satisfactory pain relief, but are contraindicated for some patients and are associated with risks and side effects. For example, paracetamol can cause severe hepatotoxicity, dipyrone can induce agranulocytosis, and NSAIDs increase the risk of bleeding and can cause gastrointestinal complications, kidney disorders, and cardiovascular toxicity.

[0009]

[0007] Thus, the search for effective and safer therapeutic options, which can be used alone or in combination with currently available therapies, is clearly necessary. In this sense, the study of essential oils has gained attention for the treatment of various medical conditions.

[0010]

[0008] Alpinia zerumbet (Pers.) BL Burtt & RM Sm, also known as shell ginger, is a perennial species belonging to the Zingiberaceae family, originating from the East Indies, but later introduced to Brazil, where it is commonly found in the northeast region.

[0011]

[0009] Various parts of Alpinia zerumbet have been used for medicinal purposes and for the extraction of essential oils. In Brazil, the medicinal use of Alpinia zerumbet by the population includes the treatment of rheumatism, viral infections, cardiovascular diseases, high blood pressure, and gastric lesions.

[0012]

[0010] Similarly, there is an extensive list of biological and pharmacological activities of Alpinia zerumbet essential oil, which vary according to the part of the plant used (i.e., leaves, rhizomes, fruits, seeds, and flowers), probably due to differences in chemical components and / or the accumulation of active compounds in each part. However, many of these indications lack scientific validation, and high-quality, rigorous preclinical and clinical studies are necessary.

[0013]

[0011] The document by Melo, LA, "Effectiveness of topical use of Alpinia zerumbet essential oil in the treatment of patients with fibromyalgia", Federal University of Sergipe, August 20, 2014, evaluates the therapeutic effects of Alpinia zerumbet essential oil (EOAz) in the treatment of patients with fibromyalgia through the mechanism related to the increased responsiveness of nociceptive neurons, which can be regulated through calcium influx (calcium channel).

[0014]

[0012] The state of the art therefore shows the growing importance of phytomedicine. More particularly, there is a pressing need to search for safer alternative pharmacological treatments for the prevention or treatment of acute orofacial pain, which have good efficacy, as well as fewer limitations, risks and side effects.

[0015]

[0013] In addition to orofacial pain conditions, it has also become necessary to evolve the approach to conditions associated with muscle injuries and states of fatigue resulting from intense physical exertion. In this context, it becomes desirable to promote muscle recovery, in order to avoid the aggravation of muscle injuries, and / or when these occur, to allow the complete recovery of muscle fibers in the shortest possible time, ensuring the recovery of strength and maintenance of functional balance.

[0016]

[0014] From a safety standpoint, herbal remedies with anti-inflammatory and analgesic properties are increasingly valued for presenting a lower risk of side effects compared to non-steroidal anti-inflammatory drugs and other conventional medications, especially in populations under constant toxicological surveillance, such as professional athletes. This scenario highlights the need to develop interventions aimed at muscle recovery based on natural compounds with properties capable of helping to reduce oxidative stress, pain, and recovery time.

[0017] SUMMARY OF THE INVENTION

[0018]

[0015] A first embodiment of the present invention relates to the use of a formulation based on Alpinia zerumbet essential oil (Fb-EOAz) in the preparation of a medicament for the prevention or treatment of inflammatory orofacial pain conditions.

[0019]

[0016] A second embodiment of the present invention relates to the use of a formulation based on Alpinia zerumbet essential oil (Fb-EOAz) in the preparation of a medicament for the prevention or treatment of conditions related to muscle recovery.

[0020]

[0017] A third embodiment consists of using a combination of Alpinia zerumbet essential oil (EOAz) with at least one additional vegetable oil in the preparation of a medicine for the prevention or treatment of inflammatory orofacial pain conditions.

[0021]

[0018] A fourth embodiment consists of using a combination of Alpinia zerumbet essential oil (EOAz) with at least one additional vegetable oil in the preparation of a medicine for the prevention or treatment of conditions related to muscle recovery.

[0022]

[0019] A fifth embodiment of the present invention relates to a method of preventing or treating inflammatory orofacial pain conditions.

[0023]

[0020] A sixth embodiment of the invention relates to the method of preventing or treating conditions related to muscle recovery. BRIEF DESCRIPTION OF THE FIGURES

[0024]

[0021] Figures 1A-1D evaluate the effect of EOAz and Fb-EOAz on orofacial thermal hyperalgesia in a postoperative pain model.

[0025]

[0022] Figures 2A-2B show the effect of EOAz and Fb-EOAz on orofacial mechanical hyperalgesia in a postoperative pain model.

[0026]

[0023] Figures 3A-3D evaluate the effect of EOAz and Fb-EOAz on LPS-induced myofascial pain and inflammatory parameters in vitro.

[0027]

[0024] Figures 4A-4H show the effect of EOAz and Fb-EOAz in the open field test.

[0028] DETAILED DESCRIPTION OF THE INVENTION

[0029]

[0025] The terms "orofacial thermal hyperalgesia", "orofacial heat hyperalgesia", "thermal hyperalgesia" and "heat hyperalgesia" are interchangeable and should be understood as a condition that causes increased sensitivity to pain in the face and mouth area, including to heat.

[0030]

[0026] The terms "orofacial mechanical hyperalgesia" and "mechanical hyperalgesia" are interchangeable and should be understood as a condition that causes an increased sensation of pain or discomfort in the face and mouth region, caused by injuries to soft tissues or peripheral nerves.

[0031]

[0027] The terms "Alpinia zerumbet essential oil-based formulation", "Fb-EOAz" and "Alpinia extract-based formulation" are interchangeable and should be understood as a formulation comprising Alpinia zerumbet essential oil.

[0032]

[0028] Worldwide, more than 300 million surgeries are performed annually, with pain being the most prevalent postoperative symptom. Despite advances in medical research, the treatment of acute postoperative pain has several limitations and potential side effects, and the preclinical search for new alternatives for pain control is clearly justified.

[0033]

[0029] The present invention relates to a novel medical use of a formulation based on Alpinia zerumbet essential oil extract (EOAz) in the prevention or treatment of inflammatory orofacial pain conditions, in the prevention of muscle injury and in muscle recovery.

[0034]

[0030] The aforementioned new medical use relates to the antinociceptive effect and anti-inflammatory mechanism of a formulation based on Alpinia zerumbet essential oil in combination with at least one additional vegetable oil. Preferably, the vegetable oil is an oil comprising terpenes. Even more preferably, the vegetable oil is olive oil.

[0035]

[0031] Specifically, the inventors of the present invention have found that the advantages of said formulation based on Alpinia zerumbet essential oil combined with another vegetable oil in the prevention or treatment of inflammatory orofacial pain conditions, in the prevention of muscle injury and in muscle recovery are related to the anti-hyperalgesic effect against mechanical and thermal hyperalgesia in the postoperative pain model and against lipopolysaccharide (LPS)-induced thermal muscle hyperalgesia, in addition to in vitro anti-inflammatory effects at a concentration that does not interfere with cell viability.

[0036]

[0032] Specifically, the formulation based on the combination of Alpinia zerumbet essential oil (Fb-EOAz) showed an anti-inflammatory activity corroborated by the fact that it reduced, in vitro, the release of nitric oxide (NO) and interleukin-6 (IL-6) by macrophages stimulated with LPS.

[0037]

[0033] Mechanical and heat hyperalgesia are frequently associated with postoperative pain, but it is well established that they are driven by distinct mechanisms.

[0034] In this sense, the effect of a formulation based on Alpinia zerumbet essential oil (EOAz) on both mechanical and thermal hyperalgesia indicates that the formulation may target a common signaling pathway for both sensory alterations, or that it has multiple mechanisms or targets.

[0038]

[0035] There are many proposed mechanisms of action to support the anti-inflammatory effects of Alpinia zerumbet, but it is worth noting that the biological and pharmacological activities of extracts and essential oils prepared from different parts of the plant show great variation, probably due to differences between the chemical components and / or the accumulation of active compounds in the individual parts.

[0039]

[0036] Furthermore, the present invention also relates a potential anti-inflammatory effect of EOAz and Fb-EOAz on the inflammatory response induced by lipopolysaccharides (LPS), a component of the cell wall of gram-negative bacteria.

[0040]

[0037] LPS is considered an exogenous ligand of Toll-like receptor 4 (TLR-4), whose activation leads to stimulation of nuclear factor kappa B (NF-κB).

[0041]

[0038] This pathway is responsible for regulating the transcription of many pro-inflammatory cytokines that contribute to the development of inflammatory hyperalgesia, as well as stimulating the release of several direct-acting hyperalgesic mediators.

[0042]

[0039] Furthermore, TLR4 / NFKB signaling is activated not only in response to LPS, but contributes to the development of thermal and mechanical hyperalgesia in postoperative pain models.

[0043]

[0040] Furthermore, the present invention is also related to the use of Fb-EOAz in an approach to reduce LPS-induced hyperalgesia after injection into the masseter muscle, which is a quadrangular muscle of the face that elevates the mandible and closes the mouth, and is therefore one of the muscles responsible for mastication.

[0044]

[0041] The inventors surprisingly found that the use of a formulation based on the combination of Alpinia zerumbet essential oil and at least one additional vegetable oil provides effects on LPS-induced hyperalgesia and in the postoperative pain model, thus exhibiting anti-inflammatory properties.

[0045]

[0042] Additionally, the use of a formulation based on a combination of Alpinia zerumbet essential oil and at least one additional vegetable oil also provides prevention of muscle injuries and muscle recovery.

[0046]

[0043] Specifically, it is known that the essential oil of Alpinia zerumbet exhibits activity associated with the modulation of neuromuscular processes related to muscle contraction and relaxation.

[0047]

[0044] In sports contexts, training microcycles often include high-intensity sessions capable of inducing ionic imbalances and neuromuscular fatigue, impairing performance and recovery. Interventions that promote muscle relaxation, reduce oxidative stress, and aid in calcium homeostasis are needed. 2+ They can be beneficial for maintaining performance over extended periods of training or competition.

[0048]

[0045] Thus, EOAz allows for faster and more complete recovery of muscle fibers, enabling a more effective (in terms of recovery time) and safer (in terms of muscle fiber healing) return to activities.

[0049]

[0046] In one embodiment, the present invention relates to the use of a formulation based on Alpinia zerumbet essential oil (Fb-EOAz) in the preparation of a medicament for the prevention or treatment of inflammatory conditions of orofacial pain and muscle recovery.

[0050]

[0047] For this purpose, a quantity of the said formulation is associated with an adequate quantity of at least one pharmaceutically acceptable vehicle, such as mineral oil, vegetable oil, physiological saline, cetostearyl alcohol base, olive oil, non-ionic self-emulsifying wax, phosphoric esters and suitable emollients.

[0051]

[0048] The Alpinia zerumbet essential oil-based formulation contains 0.08 mL / mL of Alpinia zerumbet essential oil.

[0052]

[0049] More specifically, the aforementioned formulation based on Alpinia zerumbet essential oil comprises mainly, but not exclusively, 1,8-cineole and terpene 4-ol, substances that are obtained through the extraction and purification of the essential oil of the plant species of the genus Alpinia and whose association acts as an active principle.

[0053]

[0050] Preferably, the said formulation comprises at least one additional vegetable oil. More preferably, the vegetable oil is an oil comprising terpenes. Even more preferably, the said additional vegetable oil is olive oil.

[0054]

[0051] The medicine, now obtained, has its presentation form as an oily liquid, such as emulsions or lotions for direct application, or also, in liquid form with dosage in drops for inhalation, specific gelatin capsules for dermal application, controlled-release transdermal pellets and nanoparticles, in addition to the form in cream, gel and lotion.

[0055]

[0052] Specifically, the use of the Alpinia zerumbet essential oil-based formulation showed an influence on the release of nitric oxide (NO), a marker for estimating the anti-inflammatory activity of different compounds.

[0056]

[0053] Additionally, the use of the Alpinia zerumbet essential oil-based formulation also showed an influence on the perception of recovery status (PSR) in athletes, demonstrating beneficial potential for the prevention and / or treatment of conditions related to muscle recovery.

[0057]

[0054] In another embodiment, the present invention relates to the use of a combination of Alpinia zerumbet essential oil (EOAz) with at least one additional vegetable oil in the preparation of a medicament for the prevention or treatment of inflammatory conditions of orofacial pain and muscle recovery. Preferably, the vegetable oil is an oil comprising terpenes, and said additional vegetable oil is olive oil.

[0058]

[0055] The medicine, now obtained, has its presentation form as an oily liquid, such as emulsions or lotions for direct application, or also, in liquid form with dosage in drops for inhalation, and specific gelatin capsules for dermal application, in addition to the form in cream, gel and lotion.

[0059]

[0056] In a further embodiment, the present invention relates to a method of preventing or treating inflammatory orofacial pain conditions by administering to a patient in need thereof a therapeutically effective amount of a formulation based on Alpinia zerumbet essential oil (Fb-EOAz).

[0060]

[0057] Preferably, the said formulation comprises at least one additional vegetable oil. More preferably, the additional vegetable oil is olive oil.

[0061]

[0058] In another embodiment, the present invention relates to a method of preventing or treating conditions related to muscle recovery by administering to a patient in need thereof a therapeutically effective amount of a formulation based on Alpinia zerumbet essential oil (Fb-EOAz).

[0062]

[0059] Preferably, the said formulation comprises at least one additional vegetable oil. More preferably, the additional vegetable oil is olive oil.

[0063]

[0060] The Alpinia zerumbet essential oil present in the aforementioned formulation was previously subjected to tests to assess its toxicity, so that the result of such analyses implies the use of said oil for the production of a medicine that can be used for administration by various routes, including inhalation.

[0064] EXAMPLES

[0065]

[0061] The following examples, described in detail, serve to illustrate embodiments of the present invention without, however, limiting the scope of protection thereof.

[0066] EXAMPLE 1

[0067] MATERIALS AND METHODS

[0068] • Animals

[0069]

[0062] The experiments were conducted on 218 adult male Wistar rats (Rattus norvegicus) weighing 200 to 250 g. The number of animals per group was determined by means of a pilot study and the G*Power software (V. 3. 1.9. 7, Heinrich-Heine- Universität Düsseldorf, Düsseldorf, NRW, Germany). The rats were housed four per cage in a climate-controlled room at 22±2 °C in a 12-hour light / dark cycle with food and water ad libitum and wood shavings changed on alternate days. The animals were provided by the vivarium of the Federal University of Paraná, and all protocols were previously approved by the Research Ethics Committee for the Use of Animals in the Biological Sciences Sector of the Federal University of Paraná (CEUA / BIO-UFPR #1588), all in accordance with the Brazilian guidelines of the National Council for the Control of Animal Experimentation (CONCEA) and the guidelines of Animal Research: Reporting of In Vivo Experiments (ARRIVE).

[0070] • Medications and reagents

[0071]

[0063] The study used: Alpinia zerumbet essential oil (EOAz, obtained by steam distillation of the stem and leaves in Pernambuco, a northeastern state in Brazil, before the flowering period), a formulation based on Alpinia zerumbet essential oil (Fb-EOAz, containing 0.08 mL / mL of Alpinia zerumbet essential oil), Vehicle (Fb-Vehicle), Avocado oil (inert oil), Ketamine hydrochloride, Xylazine hydrochloride, Isoflurane, Sterile saline solution 0.9%, Pseudomonas aeruginosa lipopolysaccharide (LPS) (L9143), ELISA kit for interleukin (IL) assay (DuoSet, Mouse IL-6, R&D System, Minneapolis, MN, USA), MTT reduction assay and Reagent de Griess.

[0072] • Orofacial postoperative pain model

[0073]

[0064] Initially, the animal was anesthetized with intraperitoneal (ip) injection of ketamine and xylazine (50 mg / kg and 10 mg / kg, respectively). For the buccal mucosal incision, the animal was positioned laterally for the insertion of an intraoral device, which kept its mouth open throughout the procedure. Using a #5 scalpel blade, an intraoral incision (2 mm x 10 mm) was made on the right side of the animals' mucosa. After the incision, the mucosa was sutured with 5-0 silk thread with only one stitch in the middle of the incision. The sham animals (simulated procedure - control group) underwent the same procedures, but the incision and suturing were not performed. After surgery, the animals were monitored in a heated room until complete recovery from anesthesia.

[0074] • LPS-induced myofascial pain

[0075]

[0065] The animals were anesthetized via inhalation with halothane (1.5 L / min) and a small area of ​​the facial region was shaved, preserving the vibrissae. Then, the animals received an injection of LPS (10 pg / 50 pL) or vehicle (0.9% NaCl, 50 pL) directly into the masseter muscle. The injections were repeated on two consecutive days. This method of anesthesia allows for rapid recovery of the animals, so that behavioral assessments can be made.

[0076] • Assessment of orofacial hyperalgesia due to heat

[0077]

[0066] Facial heat hyperalgesia was assessed in rats as follows: first, the rats were habituated to the restraint method to avoid stress during the test. The sensory threshold to facial heat was assessed by bringing a radiant heat source (approximately 50°C) to within 1 cm of the surface of the whisker pad area of ​​the right vibrissa. The latency of the response to exhibit head withdrawal or vigorous snout movement was recorded. A cutoff time of 20 seconds was established to avoid tissue damage.

[0078] • Assessment of orofacial mechanical hyperalgesia

[0079]

[0067] This assessment was carried out as follows: first, the rats were kept individually in acrylic boxes (30 cm 3) for at least 2 hours for habituation. The baseline mechanical threshold was measured using a graduated series of 8 von Frey filaments ranging from 0.04 to 8.0 g in ascending order (Semmes-Weinstein monofilaments, Stoelting, Wood Dale, IL - USA). Each filament was applied near the center of the right vibrissa pad, to the point of bending, three times on the same side until it evoked two positive responses, including rapid head withdrawal, facial cleaning, or abrupt fight-or-flight reactions against the filament. Only rats that did not react to the application of the 8 g filament at baseline were included in this study, to avoid nonspecific responses. Open Field Test (OFT)

[0080]

[0068] This test consisted of placing the rats in the center of an arena (50 cm long, 50 cm wide and 40 cm high) with closed side walls and a floor divided into nine quadrants. The rats' behavior was recorded for 5 minutes to evaluate subsequent behaviors using Any-maze 7.48 software (Stoelting Co. Wood Dale, IL, USA) for Windows, from which the total distance traveled, average speed, time spent by the animals in the central zone of the field, trajectory graph and average heat graph of the groups throughout the duration of the tests were extracted.

[0081] • Experimental design

[0082] EXPERIMENT 1: Effect of EOAz and Fb-EOAz in the orofacial postoperative pain model

[0083]

[0069] First, the baseline response to heat and mechanical stimulation was assessed. Then, the rats underwent trichotomy in the facial region to be stimulated, followed by a sham procedure (sham procedure - control group) or incision. Trichotomy was performed in the masseter muscle region, on the same side as the incision, to facilitate the application of treatments and ensure penetration. On days 1, 2, and 3 after the surgical procedure, the rats received topical treatment (60 µL applied to the trichotomized facial region with a micropipette) of EOAz, Fb-EOAz, Fb-Vehicle, or avocado oil. For the assessment of heat hyperalgesia, the animals were treated once a day (08:00), twice a day (08:00 and 12:00) or three times a day (08:00, 12:00 and 16:00) and for the assessment of mechanical hyperalgesia, they were treated three times a day (08:00, 12:00 and 16:00).The animals' thermal and mechanical thresholds were evaluated daily, 30 minutes after the last application, until the fourth day after surgery. EXPERIMENT 2: Effect of EOAz and Fb-EOAz on the Open Field Test (OFT).

[0084]

[0070] On day 3 after the surgical procedure, after the third application of the different compounds (EOAz, Fb-EOAz, Fb-Vehicle or avocado oil) the animals were transferred to the open field arena. The animals' behavior was recorded for 5 minutes for later analysis.

[0085] EXPERIMENT 3: Effect of EOAz and Fb-EOAz on LPS-induced myofascial pain

[0086]

[0071] After injection of LPS or vehicle (0.9% NaCl) into the masseter muscle, the animals received topical treatment (60 µL applied to the shaved facial area with a micropipette) of EOAz, Fb-EOAz, Fb-Vehicle or avocado oil, 3 times a day (08:00, 12:00 and 16:00) for 3 days. The animals' thermal threshold was evaluated daily, before the application of LPS or vehicle on days 1 and 2, and 30 minutes after the treatments on days 1, 2 and 3.

[0087] EXPERIMENT 4: In vitro effects of EOAz and Fb-EOAz on LPS-stimulated macrophages

[0088]

[0072] The protocols were based as follows: immortalized mouse macrophages (RAW 264.7) were cultured in RPMI medium supplemented with 10% Fetal Bovine Serum (FBS), sodium bicarbonate (1,500 mg / L), penicillin (100 U / mL), streptomycin (100 pg / mL), and gentamicin (10 pg / mL). The cells were maintained under a humidified atmosphere with 5% CO2 at 37 °C. Nitric oxide (NO) release was determined in the cell culture supernatants. The cells were seeded in 96-well cell culture plates (1x10 5Cells were divided into 1-well plates and incubated for 24 h. Then, the cells were treated with either Fb-EOAz (4 and 40 pg / mL) or Fb-Vehicle, and incubated for 1 h (37 °C and 5% CO2). The inflammatory response was stimulated with 0.5 pg / well of LPS from E. coli serotype (O111:B4). The plates were then incubated for 24 h at 37 °C and 5% CO2. A positive control was performed with cells treated only with LPS, and unstimulated cells were used as a negative control. NO production was determined by the presence of nitrites in the cell culture supernatant using Griess reagent, following the manufacturer's instructions (Griess Reagent System, Promega Corporation, Madison, Wisconsin, USA). Results were expressed as nitrite concentration (pM). Cell viability was assessed using the MTT reduction assay (Sigma-Aldrich, St. Louis, MO, USA).In summary, the wells were washed with phosphate-buffered saline (PBS; pH 7.4) and 200 pL of RPMI with MTT sodium salt (0.5 mg / ml) were added. The plates were incubated for 4 h (37 °C and 5% CO2) for MTT reduction. Optical density was measured using a microplate reader set to a wavelength of 570 nm. Results were expressed as a percentage of viable cells. For quantification of interleukin 6 (IL-6) levels, RAW 264.7 cells were seeded in 24-well cell culture plates (3x10⁻⁶). 5Cells were treated with Fb-EOAz (4 and 40 pg / mL) or Fb-Vehicle and after 1 h, 2.5 pg / mL of LPS from E. coli serotype (O111:B4) were added and the cells were incubated for another 24 h. The supernatants were collected and the IL-6 level was measured. IL-6 quantification was performed using an ELISA kit, according to the protocols provided by the manufacturer (DuoSet, Mouse IL-6, R&D System, Minneapolis, MN, USA). The result was expressed in pg / mL.

[0089] Statistical analysis

[0090]

[0073] The normality of the data was tested using the Shapiro-Wilk test. Two-way repeated measures ANOVA with Bonferroni post-hoc test was used for the assessment of thermal and mechanical orofacial hyperalgesia, and Kruskal-Wallis tests with Dunn-Bonferroni post-hoc test were used for the assessment of the open field. Statistical analyses were performed using JASP software for Windows (JASP Team, version 0.19.1), graphs were created using GraphPad Prism 10.3.0 for Windows (GraphPad Software, Boston, Massachusetts, USA), and p-values ​​< 0.05 were considered statistically significant.

[0091] RESULTS

[0092] Influence of EOAz and Fb-EOAz on thermal and mechanical hyperalgesia in a postoperative orofacial pain model.

[0093] Experiment 1: Effect of EOAz and Fb-EOAz on orofacial heat hyperalgesia in a postoperative pain model.

[0094]

[0074] The development of thermal orofacial hyperalgesia after intraoral incision was evaluated with treatments administered topically once, twice, and three times a day.

[0095]

[0075] Latency to heat response was assessed in sham and incision rats before (BL) and once daily after treatments. (Figure 1A) Sham rats received avocado oil, Fb-Vehicle, EOAz, or Fb-EOAz 3 times daily for 3 days. (Figure 1B) Incision rats received Fb-Vehicle, EOAz, or Fb-EOAz 1 time daily; (Figure 1C) Incision rats received Fb-Vehicle, EOAz, or Fb-EOAz 2 times daily; (Figure 1D) Incision rats received Fb-Vehicle, EOAz, or Fb-EOAz 3 times daily. All treatments were repeated for 3 days, and after the last treatment, heat hyperalgesia was assessed daily. Data are expressed as mean with Standard Error of the Mean (SEM) (n=6). p<0.05 when compared with: groups *Sham+ Fb-Vehicle; #Incision+Fb-EOAz and & Incision+EOAz. Two-way ANOVA followed by Bonferroni post-hoc test.

[0096]

[0076] There was no significant difference between the sham-operated groups that received the different treatments [F(3, 20) = 0.803, p = 0.507, ω 2 = 0.000; Figure IA]. The incision caused significant orofacial heat hyperalgesia, as there was a significant difference on days 2, 3, and 4 when comparing the Sham+Fb-Vehicle group with the Incision+Fb-Vehicle group (Day 2: Mean Difference (MD) = -2.750, Standard Error (SE) = 0.470, p < 0.001; Day 3: MD = -3.000, SE = 0.386, p < 0.001; Day 4: MD = -1.750, SE = 0.413, p = 0.002). However, a single daily treatment with EOAz and Fb-EOAz did not show an anti-hyperalgesic effect [F(3, 20) = 18.253, p < 0.001, ω 2 = 0.381; Figure 1B ].

[0097]

[0077] Figure 1C illustrates the development of orofacial hyperalgesia to heat after incision [F(3,20) = 5.578, p = 0.006, ω 2=0.141; Figure 1C] when comparing the Sham+Fb-Vehicle group with the Incision+Fb-Vehicle group (Day 2: MD=-1.583, SE=0.493, p=0.026;

[0098] Day 3: MD=-2.417, SE=0.620, p=0.005; Day 4: MD=-1.750, SE=0.378, p<0.001). Treatment with EOAz performed twice daily on days 2 and 3 caused a significant reduction in orofacial hyperalgesia to heat when compared to the Incision+Fb-Vehicle group (MD=-1.500, SE=0.378, p=0.005).

[0099]

[0078] Similarly, the development of orofacial hyperalgesia to heat is depicted in Figure 1D [F(3, 20) = 8.707, p < 0.001, ω 2= 0.216; Figure 1D], when comparing the Sham+Fb-Vehicle group with the Incision+Fb-Vehicle group (Day 2: MD=2.417, SE=0.452, p<0.001; Day 3: MD=2.833, SE=0.456, p<0.001; Day 4: MD=1.417, SE=0.452, p=0.031). Both treatments, EOAz and FB-EOAz, applied topically 3 times a day, caused a significant reduction in orofacial hyperalgesia to heat. On day 2, there was a statistically significant difference when comparing the Incision+Fb-Vehicle group with the Incision+EOAz group (MD=-1.500, SE=0.452, p=0.021), and on day 3, when comparing the Incision+Fb-Vehicle group with the Incision+Fb-EOAz group (MD=-3.167, SE=0.456, p<0.001) and the Incision+EOAz group (MD=-1.500, SE=0.456, p=0.022).

[0100] Experiment 2: Effect of EOAz and Fb-EOAz on orofacial mechanical hyperalgesia in a postoperative pain model.

[0101]

[0079] The assessment of orofacial mechanical hyperalgesia was performed with treatments administered three times a day for 3 consecutive days.

[0080] Orofacial mechanical hyperalgesia was assessed in sham and incision rats before (BL) and once a day after treatments. (Figure 2A) Sham rats received avocado oil, Fb-Vehicle, EOAz, or Fb-EOAz 3 times a day for 3 days. (Figure 2B) Incision rats received Fb-Vehicle, EOAz, or Fb-EOAz 3 times a day for 3 days, and after the last treatment, mechanical hyperalgesia was assessed. Data are expressed as mean with SEM (n=8). p<0.05 when compared with: *Sham+Fb-Vehicle groups; #Incision+Fb-EOAz and &Incision+EOAz. Two-way ANOVA followed by Bonferroni post-hoc test.

[0102]

[0081] Figure 2A illustrates that there was no significant difference between the sham-operated groups that received the different treatments [F(3, 28) = 1.087, p = 0.371, w 2= 0.002]. In Figure 2B, the comparison between the Sham+Fb-Vehicle group and the Incision+Fb-Vehicle group showed that the intraoral incision induced a significant reduction in the mechanical threshold on day 2 (MD=5.880, SE=1.274, p<0.001), on day 3 (MD=6.775, SE=1.127, p<0.001), and on day 4 (MD=5.025, SE=1.405, p=0.008). On day 3, Fb-EOAz caused a significant reduction in mechanical hyperalgesia when compared to the Incision+Fb-Vehicle group (MD=-4.150, SE=1.127, p=0.006).

[0103] Experiment 3: Effect of EOAz and Fb-EOAz on LPS-induced myofascial pain and inflammatory parameters in vitro

[0104]

[0082] Latency to heat response was assessed before (BL) and once daily after treatments. (Figure 3A) Rats received LPS or vehicle injection into the masseter muscle followed by topical treatment with Fb-Vehicle, EOAz, or Fb-EOAz, 3 times daily for 3 days. (Figure 3B) Cell viability of RAW 264.7 cells exposed to LPS alone or combined with Fb-Vehicle, EOAz, Fb-EOAz (4 pg / mL), or Fb-EOAz (40 pg / mL). (Figure 3C) Nitric oxide (NO) release by LPS-stimulated RAW 264.7 cells exposed to Fb-Vehicle, Fb-EOAz (4 pg / mL), or Fb-EOAz (40 pg / mL). (Figure 3D) IL-6 release in RAW 264.7 macrophages stimulated with LPS and treated with Fb-Vehicle, Fb-EOAz (4 pg / mL) or Fb-EOAz (40 pg / mL). Data are expressed as: (A) mean with SEM (n=5-6); (BD) mean with SD (n=5-7). p<0.05 when compared with: (A) *Vehicle+Fb-Vehicle; #LPS+Fb-EOAz. Two-way ANOVA followed by Bonferroni post-hoc test; (BD) *Control; & LPS. ANOVA followed by Tukey post-hoc test.

[0105]

[0083] Figure 3A illustrates that LPS induced orofacial thermal hyperalgesia on day 3 (Vehicle+Fb-Vehicle compared to LPS+Fb-Vehicle, MD=-1.700, SE=0.437, p=0.006). Repeated treatment with Fb-EOAz caused a significant reduction in LPS-induced orofacial thermal hyperalgesia on day 3 (MD=-1.450, SE=0.437, p=0.022).

[0106]

[0084] For the in vitro evaluation of the effect in cell culture using RAW264.7 macrophages, the formazan reduction test was necessary to investigate whether the treatment concentrations could interfere with cell viability and, consequently, with the subsequent evaluation of nitric oxide dosage. In cell viability, the EOAz group was the only one that showed a statistically significant difference compared to the control (p<0.0001, Figure 3B), indicating cytotoxicity. Therefore, the influence of EOAz on NO and IL-6 levels was not investigated, since the interpretation of the results would be compromised by its cytotoxic effect. Fb-vehicle and Fb-EOAz (4 and 40 pg / mL) did not alter cell viability compared to the control group (Figure 3B), but both treatments significantly reduced NO release compared to the LPS group (Fb-vehicle: p<0.0001; Fb-EOAz 4 µg / mL: p=0.0006; Fb-EOAz 40 µg / mL: p<0.0001).0001; Figure 3C), that is, preventing the activation of TLR4 / NFKB signaling. Similarly, both treatments significantly reduced IL-6 release by LPS-stimulated macrophages, but Fb-EOAz caused a concentration-dependent reduction in IL-6 levels in LPS-stimulated macrophages, reinforcing the idea of ​​a potential anti-inflammatory effect (Fb-vehicle: p<0.0001; Fb-EOAz 4 µg / mL: p=0.0098; Fb-EOAz 40 µg / mL: p<0.0001; Figure 3D).

[0107] Experiment 4: Effect of EOAz and Fb-EOAz in the open field test

[0085] On day 3 after the surgical procedure and after the third application of the different compounds, the rats were tested in an open field ("Open Field Test - OFT"). The sham rats (sham procedure) received avocado oil, Fb-Vehicle, EOAz or Fb-EOAz.

[0108]

[0086] Panels A and E are representative reports of the single rat's track graph recorded during 5-minute test sessions, sham and incision rats, respectively, in which the following were evaluated: (B, F) Total distance traveled, (C, G) average speed, and (D, H) time in the central zone. Rats subjected to intraoral incision received Fb-Vehicle, EOAz, or FB-EOAz. Data are expressed as minimum and maximum values ​​(whiskers), 1 o and 3 o Quartile and median (n=6). Statistical difference when p<0.05. Kruskal-Wallis followed by the Dunn-Bonferroni post-hoc test.

[0109]

[0087] Figure 4A is a representative report of the track graph of a single sham rat recorded during the 5-minute test sessions. For the sham group, there was no statistically significant difference in the assessment of distance traveled [H(3)=1.887; p=0.596; η rank 2 =0.000; Figure 4B] and at average speed [H(3)=1.718; p=0.633; classification η 2=0.000; Figure 4C] of sham rats that received avocado oil, Fb-Vehicle, EOAz, or Fb-EOAz. However, there was a significant difference in time in the central zone [H(3) =11.347; p=0.010; η classification 2 =0.417; Figure 4D] between the Sham+EOAz group and the Sham+Avocado Oil group (z=-3.266; p=0.001, p bonf =0.0065).

[0110]

[0088] Figure 4E is a representative report of the track graph of a single-incision rat recorded during the 5-minute test sessions. For the incision group, there was no difference between the Sham+Fb-Vehicle and Incision+Fb-Vehicle groups, considering all parameters, and there was no influence of any treatment on the distance traveled [H (3) =7.620; p=0.055; η classification 2 = 0.231; Figure 4F], average velocity [H (3) = 7.411; p = 0.060; classification η 2 = 0.221; Figure 4G] and time in the central zone [H (3) = 2.576; p = 0.462; classification η 2 =0.000; Figure 4H].

[0111]

[0089] The effect of the different treatments was evaluated in the open field test. The locomotor behavior of the rats, assessed by the distance traveled and the average speed, was not influenced by the surgical procedure (sham procedure or incision procedure) or by any of the treatments, indicating that EOAz and Fb-EOAz do not cause hyperlocomotion or have a potential sedative effect.

[0112]

[0090] Similarly, the time spent by the animals in the center of the arena was not different between the sham and incision rats, although the latter showed a high intergroup variation.

[0113]

[0091] Compared to the inert oil (i.e., avocado oil), EOAz caused a significant reduction in time spent in the center of the arena, suggesting a potential anxiogenic effect. Despite this, this result indicates another advantage of the Alpinia zerumbet essential oil-based formulation (Fb-EOAz) compared to EOAz, since the Alpinia oil-based formulation did not alter any of the parameters evaluated in the OFT.

[0114] EXAMPLE 2

[0115] Study Design and General Plan

[0116]

[0092] A randomized, double-blind, crossover, placebo-controlled clinical study was conducted to evaluate the safety, tolerability, and functional effects of the topical use of an Alpinia zerumbet essential oil-based formulation (Fb-EOAz) as an aid in muscle recovery after intense physical exertion in individuals undergoing a regular routine of high-intensity sports training.

[0093] The study was conducted in accordance with the Guidelines of the International Conference on Harmonisation of Good Clinical Practice (ICH-GCP), Americas Document, as well as all applicable ethical and regulatory requirements.

[0117] A formulation based on essential oil extracted from Alpinia zerumbet (Fb-EOAz), which includes vegetable oil, butylated hydroxytoluene, and vitamin E as excipients, and is presented in 15 mL spray bottles, was evaluated. Each 1 mL of the solution contains 0.08 mL of essential oil, equivalent to 17.5 mg of sabinene, its analytical marker. A spray jet releases approximately 0.2 mL, corresponding to about 3.5 mg of sabinene.

[0118] • Treatments Administered

[0119]

[0094] Fb-OAz was applied topically to muscle groups in the lower limbs, preferably after training or competition sessions and after showering, in a regimen of repeated applications over several consecutive days. This regimen proved compatible with the participants' actual sports routine and allowed for the evaluation of the continued use of the formulation under practical conditions.

[0120] RESULTS

[0121]

[0095] Muscle recovery was assessed using the subjective perceived recovery scale (PSR), used in sports contexts to estimate the functional readiness status of individuals before subsequent training or competition sessions. The PSR consists of a quick and non-invasive assessment, in which the athlete assigns a score on a continuous scale from 0 to 10, with higher values ​​indicating a better perceived recovery status.

[0122]

[0096] In the present example, each participant recorded their perception of recovery before the start of daily activities, on the days before and after the main effort, allowing for the analysis of the evolution of functional recovery over the evaluated period.

[0123]

[0097] The subjective perception of effort (RPE) was also used as a parameter for evaluating the workload associated with the physical activities performed, which estimates the degree of physical exertion perceived during the execution of a given activity, integrating peripheral and central signals related to effort.

[0124]

[0098] In the present example, each athlete recorded their RPE at the end of daily activities, also using a continuous scale from 0 to 10, in which higher values ​​indicate greater perceived effort, allowing for the analysis of the response to effort over the days evaluated, including periods before and after the main physical effort.

[0125]

[0099] Muscle function and performance were assessed using physical tests designed to measure functional parameters related to muscle strength and power, performed at defined times after the main physical exertion. These tests were used to verify whether the use of the evaluated formulation interfered with the functional capacity of the participants, as well as to analyze the muscle response during the recovery period following high-intensity activities.

[0126]

[0100] The clinical evaluation consisted of an intra-individual comparative analysis of perceived recovery, measured using the Perceived Recovery Scale (PSR), between periods of use of Fb-EOAz and the control formulation. Evaluations were performed on multiple days, including periods before and after the main physical exertion.

[0127]

[0101] The analysis of the results indicated that, on the four days evaluated, the average PSR scale scores associated with Fb-EOAz were higher than those observed with the control formulation, including in the combined analysis of the period, in which an average of 5.02 was observed for Fb-EOAz compared to 4.86 for the control formulation. Additionally, the qualitative analysis demonstrated that, on three of the four days evaluated, more participants perceived their recovery as superior during the use of Fb-EOAz compared to the period with the control formulation.

[0128]

[0102] Consistently throughout the evaluated days, the average values ​​of perceived recovery were higher during the periods of use of Fb-EOAz when compared to the control formulation, indicating a favorable trend associated with the use of the formulation.

[0129]

[0103] The subjective perception of effort (RPE) was evaluated comparatively between the periods of use of Fb-EOAz and the control formulation, considering intra-individual variations throughout the days evaluated.

[0130]

[0104] The analysis indicated favorable signals associated with the use of Fb-EOAz in specific follow-up periods. In particular, on the day following the main physical exertion (D+1), the qualitative analysis indicated a greater number of participants who perceived less exertion during this period compared to the use of the control formulation.

[0131]

[0105] Thus, a favorable trend was observed associated with the use of the Fb-EOAz formulation, suggesting a possible benefit in the acute post-exercise recovery phase.

[0132]

[0106] Performance in countermovement jump tests was assessed as an additional functional parameter over the days evaluated, and no overall impairment of muscle function associated with the use of Fb-EOAz was observed.

[0133]

[0107] Efficacy assessment was performed considering only athletes who completed both periods of the clinical trial. Overall, a favorable trend associated with the use of Fb-EOAz was observed throughout the evaluated days and in the combined analysis.

[0108] The incidence of adverse events observed during the study was low and consistent with the context of the participants' usual sports practice. No treatment-emergent adverse events were considered related to the use of the evaluated formulation, and the changes observed in laboratory tests were classified as not clinically relevant and not associated with the treatment. Overall, Fb-EOAz showed a good safety profile and was well tolerated throughout the period of use.

[0134]

[0109] Consistently throughout the evaluated days, as well as in the combined analysis of the experimental period, the average values ​​of perceived recovery were higher during the use of the Fb-EOAz formulation compared to the control formulation, indicating a favorable trend associated with the use of the formulation, without observed functional impairment.

[0135]

[0110] It is understood that when a range of parameters is provided, all whole numbers and ranges within that range, and tenths and hundredths, are also provided by the modalities. For example, "5-10%" includes 5%, 6%, 7%, 8%, 9% and 10%; 5.0%, 5.1%, 5.2%.... 9.8%, 9.9% and 10.0%; and 5.00%, 5.01%, 5.02%.... 9.98%, 9.99% and 10.00%, as well as, for example, 6-9%, 5.1%-9.9% and 5.01%-9.99%. Similarly, when a list is presented, unless otherwise indicated, it should be understood that each individual element of that list and each combination of components of that list is a separate modality. For example, "1, 2, 3, 4 and 5" encompasses, among numerous modalities, 1; 2; 3; 1 and 2; 3 and 5; 1, 3 and 5; 1, 2, 4 and 5.

[0136]

[0111] It should be understood that the embodiments described above are merely illustrative and that various modifications may be made to them by a person skilled in the art without departing from the scope of the present invention. Consequently, the present invention should not be considered limited to the exemplary embodiments described in this application. Furthermore, the present disclosure may include matter not claimed at present, but which may be claimed in the future in combination with or separately from the features claimed herein.

Claims

CLAIMS 1. Use of a formulation based on Alpinia zerumbet essential oil (Fb-EOAz) characterized by being in the preparation of a medicine for the prevention or treatment of inflammatory orofacial pain conditions.

2. Use of a formulation based on Alpinia zerumbet essential oil (Fb-EOAz) characterized by being in the preparation of a medicine for the prevention or treatment of conditions related to muscle recovery.

3. Use according to claim 1 or 2, characterized in that said formulation comprises at least one additional vegetable oil.

4. Use according to claim 3, characterized in that said additional vegetable oil is an oil comprising terpenes, preferably olive oil.

5. Use of a combination of Alpinia zerumbet essential oil (EOAz) with at least one additional vegetable oil, characterized by being in the preparation of a medicine for the prevention or treatment of inflammatory orofacial pain conditions.

6. Use of a combination of Alpinia zerumbet essential oil (EOAz) with at least one additional vegetable oil, characterized by being in the preparation of a medicine for the prevention or treatment of conditions related to muscle recovery.

7. Use according to claim 5 or 6 characterized in that said additional vegetable oil is an oil comprising terpenes, preferably olive oil.

8. Use according to any one of claims 1 to 4, characterized in that said formulation is in the form of an oily liquid, gelatin capsules, controlled-release transdermal pellets or nanoparticles.

9. Use according to claim 8, characterized in that the oily liquid is for the inhalation of oil droplets from Alpinia zerumbet.

10. A method for preventing or treating inflammatory orofacial pain conditions, characterized by administering a therapeutically effective amount of a formulation based on Alpinia zerumbet essential oil (EOAz) to a patient in need thereof.

11. A method for preventing or treating conditions related to muscle recovery, characterized by administering a therapeutically effective amount of a formulation based on Alpinia zerumbet essential oil (EOAz) to a patient in need thereof.

12. Method according to claim 10 or 11, characterized in that said formulation comprises at least one additional vegetable oil.

13. Method according to claim 12, characterized in that the additional vegetable oil is olive oil.

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