WO2011015692A2

WO2011015692A2 - Use of maslinic acid for treating nociceptive, inflammatory and neurogenic pain

Info

Publication number: WO2011015692A2
Application number: PCT/ES2010/000356
Authority: WO
Inventors: Francisco Rafael NIETO LÓPEZ; José Manuel BAEYENS CABRERA; Andrés GARCÍA-GRANADOS LÓPEZ DE HIERRO; José Manuel ENTRENA FERNÁNDEZ; Enrique José COBOS DEL MORAL; Antonio MARTÍNEZ RODRÍGUEZ; Andrés PARRA SÁNCHEZ; Antonio RIVAS SÁNCHEZ
Original assignee: Universidad De Granada
Priority date: 2009-07-29
Filing date: 2010-07-29
Publication date: 2011-02-10
Also published as: WO2011015692A3; ES2333638B2; ES2333638A1

Abstract

Use of maslinic acid for treating nociceptive, inflammatory and neurogenic pain. The present invention relates to the use of maslinic acid and any of the derivatives thereof for treating painful pathological processes of a (1) nociceptive, (2) inflammatory or (3) neurogenic nature by any galenically acceptable means and very especially topically, including compositions that contain maslinic acid, any of the derivates thereof, or natural, synthetic or semi-synthetic mixtures rich in maslinic acid or the derivatives thereof.

Description

USE OF MASLINIC ACID FOR THE TREATMENT OF PAIN OF NOCICEPTIVE NATURE. INFLAMMATORY AND NEUROGENIC

The present invention relates to the use of maslinic acid and any of its derivatives for the treatment of painful pathological processes, of a (1) nociceptive, (2) inflammatory or (3) neurogenic nature, by any means that are galenically acceptable and especially by route topical, including compositions containing maslinic acid, any of its derivatives, or natural, synthetic or semi-synthetic mixtures rich in maslinic or its derivatives.

STATE OF THE TECHNIQUE Oleanolic acid (3-betahydroxy-28-carboxy-lean-12-ene) is a triterpenic acid ubiquitously distributed in the plant kingdom. Thus, the phytochemical database of the United States Department of Agriculture collects its presence in almost a hundred plants, among which is the Olea europaea, as well as a series of proven biological activities (antiabortive, anticariogenic, antifertility, antihepatotoxic , anti-inflammatory, antisarcomic, cancer preventive, cardiotonic, diuretic, hepatoprotective and uterotonic). There are some references that demonstrate the efficacy of oleanolic acid in experimental models of somatic and visceral nociceptive pain in mice [Maia et al., Pharmacol Res. (2006) 54: 282-286; Maia et al., Biol Pharm BuII. (2006) 29: 82-5]. Likewise, the results obtained in a pilot clinical trial [Lukaczer et al., Phytother Res. (2005) 19: 864-869], also suggest some efficacy of oleanolic acid in reducing pain in patients with arthritis, although in this case Oleanolic acid was administered in tablets, in combination with an extract of rosemary (Rosmarinus officinalis) and reduced iso-alpha acids from hops (Humulus lupulus). In these tablets the proportion of each of the components, including oleanolic acid, is unknown.

Maslinic acid (2-alpha, 3-betadihydroxy-28-carboxylean-12-ene), also called crataegolic acid, is a much less distributed acid in nature, having been detected in a dozen plants. Is known its activity as an antihistamine and anticancer, although its shortage means that it has not been studied extensively. The isolation of oleanolic and maslinic acids from the waxes of the surface of the fruit of the Olea europaea has been described [Bianchi.G., Pozzi.N. And Vlahov, G. Phytochemistry (1994) 37, 205-207] by means of the methanolic extraction of olives previously washed with chloroform. The separation of this type of acids has been described by high speed countercurrent chromatography (HSCCC) [Du, QZ, Xiong, XP and Ito, Y .; Journal of Liquid Chromatography (1995) 18, 1997-2004]. H

Maslinic acid

The University of Granada, in its patent application ES2111498 describes a process of industrial use of oleanolic and maslinic acids contained in the by-products of the milling of the olive. This procedure allows obtaining a suitable material for many applications. There is currently a large number of patents in which the maslinic acid acts as an active component: antitumor agent US2003153538 or WO0252956; apoptosis inducer WO03057224; abnormal food WO203039270 and WO03011267; external agent for skin and bleach US2003133958; treatment of vascular lesions WO02078685.

In some publications it is suggested that this triterpene may also have an anti-inflammatory and antioxidant activity by inhibiting the release of pro-inflammatory cytokines IL-6 and TNFα and the production of NO and hydrogen peroxide (ROS) in murine peritoneal macrophages stimulated with LPS [Márquez-Martín et al., Free Radie. Res. (2006) 40: 295-302]. However, the same authors do not find a clear effect of maslinic acid on the production of pro-inflammatory cytokines in human peripheral blood mononuclear cells [Márquez-Martín et al., Cytokine (2006) 36: 11-17]. In any case, what these discrepancies show is that the answers are not homogeneous and therefore not predictable, which makes the study necessary in each model and / or type of tissue.

There are no bibliographical references that relate maslinic acid to pain treatments.

Currently the treatment of pain is of great importance in medicine since it is necessary to find new therapies more effective and / or better tolerated than those currently available to combat it. This is evident in the large number of scientific papers in basic and clinical research on new strategies for the treatment of pain that have appeared in recent years.

Among the different definitions of pain, the most accepted is the one proposed by the International Association for the Study of Pain (International

Association for the Study of Pain (IASP) that defines pain as "an unpleasant sensory and emotional experience with actual or potential tissue damage or described in terms of such damage." Although pain is always subjective, it is possible to classify it. The main subtypes of pain that this invention deals with are: (1) nociceptive pain (that is, pain caused by direct activation of nociceptors [2008 IASP Pain

Terminology, http://www.iasp-pain.org]), (2) inflammatory pain (ie, pain caused by tissue injury with inflammation [Woolf, Br. J. Anaesth. 75: 169-

176, 1995]) and (3) neurogenic pain (ie pain caused by a primary lesion, dysfunction or transient alteration in the peripheral or central nervous system [1994 IASP Pain Terminology, http://www.iasp-pain.org ]).

The pain (1) of nociceptive type usually responds well to treatment with non-steroidal anti-inflammatory analgesics (NSAIDs) and with opioid analgesics, according to the WHO ladder [Krakowski et al., Br. J. Cancer

(2003) 89, S67-S72]. However, both types of drugs produce with side effects frequency For example, NSAIDs can cause gastrointestinal disorders and lesions, reduction of renal function with retention of water, sodium and potassium and hypersensitivity reactions among others. Opioid analgesics often cause nausea and vomiting, constipation, respiratory depression, as well as tolerance and dependence among other side effects.

Pain (2) of the inflammatory type responds to treatments with NSAIDs, although as mentioned previously these drugs frequently produce side effects [Muñir et al., Med Clin North Am (2007) 91: 97-111]. Likewise, steroidal anti-inflammatories, also called corticosteroids, are effective for the treatment of inflammation [Stephens et al., Am. Fam. Physician (2008) 78: 971-976], but can cause the suppression of endogenous secretion of steroids, peptide ulcer, congestive heart failure, hypertension, diabetes, osteoporosis and glaucoma among other side effects.

Neurogenic pain (3) responds to the administration of some antiepileptics (such as gabapentin, pregabalin or carbamazepine) and tricyclic antidepressants (such as amitriptyline) [Jackson, Pain Pract. (2006) 6: 27-33], but more than a third of patients do not respond to any treatment, so that new effective drugs are necessary for their treatment. EXPLANATION OF THE INVENTION

The present invention describes the use of maslinic acid or any of its derivatives for the symptomatic treatment of any kind of pain clinically ascribable to type (1) nociceptive, and / or type (2) inflammatory and / or type (3) neurogenic.

In this invention, the efficacy of the treatment is demonstrated even by topical administration. It is very important to highlight this fact since it allows to quickly and efficiently locate high concentrations of active product or its biologically acceptable salts, so that a rapid effect on the pain experienced is achieved.

In the present invention, and as a consequence of the results obtained, shown in the examples section, it is shown that:

The subcutaneous administration of maslinic acid inhibits pain induced by chemical stimuli in the mouse, in this case, the second phase of pain in the 5% formalin test, in which a phenomenon of inflammation located in Ia occurs. leg injected with formalin (see section 6.1 and table 1 in the examples section).

Topical administration of maslinic acid also inhibited the inflammatory pain of the second phase of the formalin test. The analgesic effect was due to a local action because the application of the maslinic in one leg did not produce analgesia in the contralateral leg (see section 6.2 and table 2 in the examples section).

Subcutaneous administration of maslinic acid was also effective in a neurogenic pain model induced by the administration of capsaicin.

[Baumann et al., J. Neurophysiol. (1991) 66: 212-227]. In this model, maslinic acid inhibited mechanical allodynia to a punctiform stimulus induced by intraplantar administration of capsaicin in the mouse (see section 6.3 and table 3 in the examples section). Since this is a model of neurogenic pain caused by central sensitization [lannetti et al., Proc. Nati

Acad. Sci. USA (2005) 102: 18195-18200] These data indicate that the maslinic is able to act in the central nervous system to exert its effects.

It is interesting to note that the administration of the rofecoxib COX-2 inhibitor was not able to inhibit the neurogenic pain (mechanical hypersensitivity) induced by capsaicin (see section 6.7 and table 7 in the examples section). Since as demonstrated in the examples (section 6.3), maslinic acid is capable of doing so, the analgesic effect of maslinic acid must be due to a mechanism different from that of COX-2 inhibition. Maslinic acid administered subcutaneously in mice also achieved inhibit somatic nociceptive pain induced thermal cutaneous stimulation (Ia model hot plate at 50 ⁰ C) and visceral nociceptive pain induced by intraperitoneal administration of acetic acid 0.6 % in the mouse (see sections 6.4 and 6.5, and tables 4 and 5 in the examples section).

Maslinic acid (administered subcutaneously at the highest dose evaluated in pain models) had no appreciable side effect when observing the spontaneous behavior of the animal and also did not alter the motor coordination of the animals when they were subjected to the Rotarod test (see section 6.6 and table 6 in the examples section). This indicates that the antinociceptive and antialodynic action of maslinic acid is not due to a nonspecific inhibition of the central nervous system. Preferably, a natural active ingredient is used in which the maslinic acid is accompanied by smaller amounts of oleanolic acid, all in a pharmaceutically acceptable carrier. In any case, maslinic acid, or any of its derivatives, or the combination thereof or the combination thereof with oleanoic acid, constitutes more than 66% of this natural active ingredient and preferably more than 85% thereof.

If oleanoic acid is included in the mixture of active ingredients, the ratio between maslinic acid and oleanolic acid is between 2: 1 and 7: 1. That is, maslinic acid, its salts, pharmaceutically acceptable derivatives or its prodrugs, will be formulated in an appropriate pharmaceutical composition, in the therapeutically effective amount, together with one or more pharmaceutically acceptable carriers, adjuvants or excipients.

Specifically, for topical administration the maslinic acid, its derivatives, or the mixture of maslinic acid and oleanolic acid described above as active ingredient, is in a proportion between 0.5% and 10% of the total composition, preferably between 0.5% and 5% and even more preferably between 1% and 3%. In any case, maslinic acid or some of its derivatives, derived from industrial by-products of plants of the genus Olea, will preferably be used.

This composition can be administered in different ways, including, but not limited to, orally, orally, rectally, intramuscularly, topically, subcutaneously, intraarticularly or by inhalation.

Preferably it is administered topically, orally, subcutaneously or through joint infiltrations. In another preferred embodiment, said composition is presented in a form adapted to topical administration. In each case the composition will be adapted to the type of administration used, therefore, the composition described above can be presented in the form of tablets, dragees, capsules, solutions, emulsions, creams, body milk, ointments, inhalants, aerosols, suppositories or any other form of clinically permitted administration and in a therapeutically effective amount. More preferably in the form of solution, emulsion, cream, body milk or ointment.

By a pharmaceutically acceptable derivative is meant any pharmaceutically acceptable salt or any other compound that, after administration, is capable of providing (directly or indirectly) the maslinic acid.

In the sense used in this description, the expression "pharmaceutically acceptable vehicle" refers to those substances, or combination of substances, known in the pharmaceutical sector, used in the preparation of pharmaceutical administration forms and includes solids or liquids, solvents, surfactants , etc.

Throughout the description and the claims, the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For experts in the field, other objects, advantages and characteristics of the invention will be derived partly from the description and partly from the practice of the invention. The following examples and tables are provided by way of illustration, and are not intended to be limiting of the present invention.

EXAMPLES

Next, the invention will be illustrated by tests carried out by the inventors, which show, among other aspects, the activity and efficacy of maslinic acid and demonstrate what has been described in general in the previous sections.

In a generic way for all the examples, the experimental animals, compounds, materials and methods used and results obtained are described:

1. Experimental animals

Female mice of the strain CD-1 (Charles River Spain, Barcelona) of 25-30 g weight were used, with at least 8 animals in each experimental group. The animals were housed in makrolon boxes and were kept in a room at 22 ± 1 ° C of room temperature, with air change every 20 minutes and with a light-dark rhythm of 12 hours (light from 8 to 20 hours). The animals had food and water "ad libitum" until the time of the experiment. The animals were managed according to the community directive of November 24, 1986 (86/609 / EEC)

2. Compounds, drugs and solutions

The chemical compounds that were used were the algae substances capsaicin, formalin (38% formaldehyde) and acetic acid, and the drugs maslinic acid (in its sodium salt form), rofecoxib, baclofen and gabapentin. For the systemic treatment three types of solvent were used: a) dimethylsulfoxide (DMSO), in which the capsaicin was dissolved; b) sterile saline, which was used to dilute the solutions of capsaicin in DMSO (until obtaining a concentration of 1 μg of capsaicin in 1% DMSO), of formalin (until obtaining a concentration of 5%) and of acetic acid ( up to a concentration of 0.6%); as well as to dissolve rofecoxib, baclofen and gabapentin; and c) a solution of tween 80 at 2% in ultrapure water, in which the maslinic acid was dissolved in its sodium salt form.

For the evaluation of the local effect of maslinic acid, a topical treatment was used by formation of the salt of the maslinic acid with triethanolamine in propylene glycol, which was vehiculized in a carbopol gel reaching a final proportion of maslinic acid of 1%. Experiences were also carried out in control animals treated with the propylene glycol, triethanolamine and carbopol hydrogel base (without maslinic acid).

The capsaicin stock solution in pure DMSO was prepared at a concentration of 5 mg / ml which was fractionated and frozen (for a maximum of 5 days) at -20 ° C in aliquots. Each day of the experiment, the corresponding aliquot was thawed and diluted in saline at the concentration of 0.05 μg / μl. The formalin solution was prepared on the same day of the experiment and diluted to 5% concentration in saline. Likewise, the acetic acid solution was prepared on the same day as the experiment, diluting the acetic acid to the 0.6% concentration in saline.

The maslinic acid solution was prepared daily before the beginning of the experiments at the concentration necessary to administer the dose studied, dissolving it in a solution of tween 80 to 2% in ultrapure water. Baclofen, rofecoxib and gabapentin were administered and prepared following the same procedure, but in this case the solvent was saline. 3. Material and instruments Insulin syringes were used for the administration of the drugs subcutaneously. For the intraperitoneal administration of acetic acid the same syringes were used, but provided with a 30 ^1/2 G needle. For the intraplantar injection of capsaicin a Hamilton syringe of 100 μl model 1710 TLL provided with a 30 ¹ needle was used. ² G. For the topical application of the maslinic acid in hydrogel and the base hydrogel in the back leg of the animal, an aluminum spatula was used.

For the measurement of the lick response induced by the formalin, digital chronometers with an accuracy of 0.01 seconds, 1-liter transparent glass beakers with a diameter of 12 cm and 14 cm high were used in which introduced the animal to observe its behavior and mirrors of 20 x 20 cm placed behind the vessel to facilitate the vision of the injected leg.

To measure the contortions induced by the acetic acid solution, makrolon boxes were used, with the floor covered with wood chips. Likewise, 20 x 20 cm mirrors placed behind the makrolon box were used to facilitate the vision of the contortions.

For the evaluation of mechanical allodynia induced by capsaicin, an apparatus called Dynamic Plantar Aesthesiometer model 37400 (Ugo Basile, Italy) was used. The apparatus consists of a cabin to keep the animals during the study and an electronic unit that allows the stimulation of the animal's leg by means of a filament (electronically controlling the applied force and the speed with which it is applied) and the recording of the data (latency time from the application of the stimulus to the removal of the leg). A commercial hot plate (Letica, Spain) surrounded by transparent methacrylate walls 16 cm high and connected to a thermostated water bath (JP Selecta, Spain) was used to perform the hot plate test. A probe for surface temperature measurement connected to an electronic thermometer (Fisher Bioblock Scientific, Spain) was used to verify that the surface temperature of the plate was adequate. To check if maslinic acid produces alterations in motor coordination, an apparatus called Accelerating Rotarod (Cibertec, Madrid, Spain) was used. The apparatus consists of a rotating cylinder divided into 5 compartments, separated by opaque plastic walls, where the mice are located. The device has a motor that allows to apply a constant acceleration (from 4 to 40 rpm in 5 minutes) and automatically time the time it takes to fall from the cylinder each mouse. 4. Experimental procedures

4.1. Administration of drugs and solutions:

For the intraplantar injection, the procedure previously described was used [Cendán et al., Psychopharmacology (Berl.) (2005) 182: 485-493]: the needle of the Hamilton syringe was introduced in the right hind leg of the animal, from the part after the plant in the direction of the fingers, the 20 μl of capsaicin or formalin solution (or their respective solvents) were injected in a time of approximately 5 seconds and the needle was slowly extracted, with the precaution of pressing on the place where the needle penetrated so that no liquid was lost. The acetic acid solution was injected intraperitoneally in a volume of 10 ml / kg as previously described [Del Pozo et al., Eur. J. Pharmacol. (1987) 137: 155-160]. To assess the systemic effect of the drugs under study, these were administered subcutaneously in the interscapular area, in a volume of 5 ml / kg following standard procedures [Del Pozo et al., Eur. J. Pharmacol. (1987) 137: 155-160]. The animals of the control groups received the same volume of the solvents by the same route of administration (tween 80 to 2% in water for the maslinic or saline for rofecoxib, baclofen and gabapentin).

For the cutaneous application of the hydrogel with 1% maslinic acid or its solvent

(base hydrogel) an amount of 0.1 ml of hydrogel was extended per tae the hind leg, from the ankle to the toes, following the experimental procedure previously described [Li et al., Anesthesiology (2007) 107: 486- 494]. Depending on the objective of the experiment, the hydrogel was applied in the right hind leg, which was later to be injected with formalin (ipsilateral leg) or, in another experimental group, in the left hind leg (leg-contralateral), that is to say in the leg Not treated with formalin. In this case, it was to verify that the effect of morelinic acid when applied topically is local and not systemic.

4.2. Model of the formalin: The procedure previously described was used [Cendán et al., Psychopharmacology (Berl.) (2005) 182: 485-493]. The animals remained at least 30 min in the room where the experiment was performed, before it began. After the time of habitation to the room, they received an injection of maslinic acid (or its solvent) by s.c. and they were returned to the makrolón boxes. In the experiments in which the maslinic acid hydrogel or its solvent was applied, each animal was placed directly in a beaker, to prevent the chips from sticking on the leg smeared with the hydrogel. 30 minutes after administration s.c. of maslinic acid or its solvent (2% solution of tween 80 in saline) and 15 minutes after the ipsilateral topical application of maslinic acid (administered in the form of hydrogel) or its solvent (base hydrogel), the animals received in the leg right back an intraplantar injection of formalin. To verify that the hydrogel effect of maslinic acid is local, experiments were also carried out in which the hydrogel was applied in the contralateral leg (left leg) to the injected with formalin.

Immediately after the injection of the formalin, each mouse was placed under an inverted 1 liter beaker, and the time the animals licked / nibbled the injected leg was evaluated for 50 min (divided into 10 periods of 5 min each one). As previously described [Cendán et al., Psychopharmacology (Berl.) (2005) 182: 485-493] the licking / nibbling time observed during the period of 0-5 min after the formalin injection was considered indicative of the first phase of pain, while the licking / nibbling time observed between 10 and 50 min after the injection was considered indicative of the second phase of pain. 4.3. Capsaicin-induced mechanical allodynia model:

The experimental procedure used was that described previously [Entrena et al., Pain (2009) 143: 252-261]. To prevent exploratory mouse behaviors from interfering with the results obtained, each animal was placed in the corresponding compartment of the apparatus 2 hours before administering the drugs (habituation time). Then each animal was taken out of its compartment, received the product under study by s.c. in the interscapular area and immediately afterwards it was returned to its compartment. Maslinic acid or its solvent was administered 30 minutes before the intraplantar injection of capsaicin. After 15 minutes from the injection of capsaicin or its solvent (time in which maximum sensitization by capsaicin is achieved) each mouse was exposed to a mechanical stimulus of 0.5 g by means of a rigid filament of 0.5 mm in diameter. The time elapsed from the stimulation of the leg until the animal Ia removed was automatically recorded by the apparatus. The maximum exposure time to the mechanical stimulus was 50 seconds in each of the determinations. 3 measurements of the latency of withdrawal of the leg were made in each animal, leaving a minimum interval of 5 seconds between each of the measures. The average of the three measures was considered the response latency of the animal.

4.4. Model of acetic acid induced contortions: For its realization a previously described method was used [Del Pozo et al., Eur. J. Pharmacol. (1987) 137: 155-160]. The animals remained at least 30 min in the room where the experiment was performed, before it began. After this time they received an injection of maslinic acid (or its solvent) by sc route and were returned to the makrolon boxes. 30 minutes after administration of the maslinic acid, the animals received an intraperitoneal injection of 0.6% acetic acid. Immediately after the injection of the acetic acid, each mouse was placed in another smaller makrolon box with the floor covered with wood chips, and the number of contortions that the animal performed during 30 min (divided into 6 periods of 5 min) was evaluated each). It was considered that the mouse made a contortion, when bowing the body stretched at least one of the hind legs completely. 4.5. Hot plate model:

The experimental methodology used was previously described [Del Pozo et al., Gen. Pharmacol. (1990) 21: 681-685]. The water bath thermostat was adjusted, so that the temperature of the surface of the plate was 5O ⁰ C. The animals remained at least 30 min in the room where the experiment was performed, before starting the experiment. After this time they received an injection of maslinic acid (or its solvent) by sc route and were returned to the makrolon boxes. 30 minutes after the administration of the drug or its solvent, the animals were placed on the plate at this temperature and by means of a stopwatch the time taken to lick any of the hind legs was measured, what we call the latency time of the lick of the Ia rear leg (LPT). A cutting time of 60 seconds was imposed to prevent damage to the animal.

4.6. Rotarod Model:

The methodology used was previously described [Nieto et al., Pain (2008) 137: 520-531]. The animals remained at least 30 min in the room where the experiment was performed, before it began. The experimental protocol consists of two phases that are carried out in two consecutive days: on the first day, the learning phase is carried out, since it is necessary for the animals to learn to stay on top of the cylinder and run in a certain direction. Therefore, on the first day, the animals are placed on top of the rotating cylinder at a progressively increasing speed with a constant acceleration of 4-40 rpm in 5 min and the time taken to fall from the cylinder is timed. This procedure is repeated 3 times leaving a time interval of 30 minutes between each one. With this it is achieved, that the animals get used to the apparatus and reach an average latency of fall of about 200 seconds. Afterwards, the animals are returned to the makrolón boxes.

The next day, the evaluation phase is carried out: first it is verified that the animals continue to have an average latency of 200 sec, when they are subjected to a constant acceleration of 4-40 rpm in 5 minutes. They then receive an sc injection of maslinic acid, baclofen, gabapentin or its solvents and are returned to the makrolón boxes; 30 minutes later they are placed again on top of the cylinder to subject them to the same acceleration, and their fall latency is checked. This same procedure was repeated at 60, 90, 120 and 180 minutes after subcutaneous administration. The cutting time was 300 seconds, which occurs just when the cylinder reaches the maximum acceleration of 40 rpm.

5. Statistical analysis The statistical comparisons of the means of the values obtained in the different treatment groups or of the percentages of analgesia, were performed by means of an analysis of the variance (ANOVA) of one way or two ways followed by a test of Bonferroni In cases where there were only two groups, statistical comparisons were made using the Student's t-test for unpaired data. All comparisons were made using the SigmaStat version 2.03 program (Systat Software Inc., San Jose, California, USA). In all cases it was considered that there were significant differences when the P value was less than 0.05.

For the calculation of the percentage of analgesia in the models of mechanical allodynia induced by capsaicin and hot plate, the following formula was used:% Analgesia = [TLT - TLC / TC - TLC] x 100; where TLT is the latency time of mice treated with maslinic acid, TLC is the latency time of mice belonging to the control group (solvent treated) and CT is the cut-off time (maximum latency time: 50 sec for allodynia model and 60 sec for hot plate model). To calculate the percentage of analgesia in the formalin model, the following formula was used:% Analgesia = [(TLC - TLT) / TLC] x 100; where TLT is the licking time of mice treated with maslinic acid, and TLC is the licking time of mice belonging to the control group (solvent treated). For the calculation of the percentage of analgesia in the acetic acid contortion model, the following formula was used:% Analgesia = [(NCC - NCT) / NCC] x 100; where NCT is the number of contortions of mice treated with maslinic acid, and NCC is the number of contortions of mice belonging to the control group (solvent treated).

6. Results 6.1. Effect of the systemic administration of maslinic acid in the formalin test: Maslinic acid (64-512 mg / kg), administered subcutaneously, did not produce statistically significant changes in the first phase (0-5 min) of induced pain by the formalin (data not shown), but was able to reduce the pain of the second phase (10-50 min) (Table 1). Statistically significant differences were found with maslinic acid doses 256 and 512 mg / kg with respect to the control group (Table 1).

Table 1: Effect of subcutaneous administration of maslinic acid, in the second phase of formalin-induced pain

Treatment Dose Licking time Percentage of

(mg / kg; sc) (seconds) "analgesia ^b

Solvent - 273.73 ± 29.03 O ± 10.49

Ac. Maslinic 64 196.38 ± 34.13 29.03 ± 12.34

128 175.85 ± 31.93 36.46 ± 11.54

256 108.85 ± 24.63 ^** 60.70 ± 8.90 ^***

512 145.73 ± 14.90 ^* 47.34 ± 5.38 ^* Effect of subcutaneous administration of different doses of maslinic acid (64, 128, 256 and 512 mg / kg) and its solvent (2% TweenδO in water ultrapure), in the second phase of pain induced by intraplantar injection (i. pl.) of formalin (5%). Formalin injection was performed 30 min after administration of maslinic acid or its solvent. Each value represents the mean ± ESM of the values obtained in at least 12 animals. Statistically significant differences between the groups treated with maslinic acid and its solvent: * P <0.05; ** P <0.01; *** P <0.001 (one-way ANOVA followed by the Bonferroni test).

Cumulative licking / nibbling time in the second phase (10-50 min) of pain after administration (i. Pl.) Of 5% formalin (see materials and methods section for detailed explanation).

Percentage of analgesia calculated using the formula:% Analgesia = [(TLC - TLT) / TLC] x 100; where TLT is the licking time of mice treated with maslinic acid, and TLC is the licking time of mice belonging to the control group (solvent treated).

6.2. Effect of the local administration of maslinic acid in the formalin test: Maslinic acid 1% in hydrogel applied directly to the leg 15 minutes before the injection i.pl. of formalin did not modify the first phase (0-5 min) of the painful response induced by capsaicin (data not shown); On the other hand, it was able to inhibit the second phase of pain induced by formalin (Table 2). In this second phase, statistically significant differences were found, with respect to the control group (Table 2). On the contrary, when 1% maslinic acid in hydrogel was applied in the contralateral leg to which it would be injected with formalin, it had no effect on the pain induced by the irritant (Table 2).

These data indicate that the locally applied maslinic acid achieves sufficient concentration in the ipsilateral leg to inhibit the response to the formalin, but is not absorbed sufficiently to exert systemic effects (and therefore an analgesic effect in the contralateral leg). Table 2: Effect of topical administration of 1% maslinic acid in the form of hydrogel, in the second phase of formalin induced pain.

Treated leg Topical treatment Licking time Percentage of

(seconds) "analgesia ⁰

Ipsilateral Hydrogel-Control 282.27 ± 43.18 O ± 15.30

Hydrogel- AM 1% 149, 13 ± 17, 16 ^** '^# 47, 17 ± 6.26 ^** ' ^

Contralateral Hydrogel-Control 246.55 ± 30.14 0 ± 12.23

Hydrogel- AM 1% 250.30 ± 20.73 -1.52 ± 8.41

Effect of topical, ipsilateral and contralateral administration of 1% maslinic acid in the form of hydrogel (AM-1%) or base hydrogel without maslinic acid (Control), in the second phase of pain induced by intraplantar injection (i pl.) of formalin (5%). Formalin injection was performed 15 min after topical cutaneous application of maslinic acid or its solvent in the same leg injected with formalin (ipsilateral) or in the non-injected (contralateral) leg. Each value represents the mean ± ESM of the values obtained in at least 12 animals. Statistically significant differences between the group treated with ipsilateral maslinic acid and the group treated with contralateral maslinic acid: ** P <0.01; Statistically significant differences between the group treated with ipsilateral maslinic acid and the group treated with the ipsilateral base hydrogel: # P <0.01 ## P <0.01 (two-way ANOVA followed by the Bonferroni test)

a Cumulative licking / nibbling time in the second phase (10-50 min) of pain after administration (i. pl.) of 5% formalin (see materials and methods section for detailed explanation).

Percentage of analgesia calculated using the formula:% Analgesia = [(TLC - TLT) / TLC] x 100; where TLT is the licking time of mice treated with maslinic acid, and TLC is the licking time of mice belonging to the control group (solvent treated). 6.3 Effect of the systemic administration of maslinic acid on mechanical allodynia induced by capsaicin:

Subcutaneous administration of maslinic acid was able to inhibit mechanical allodynia to a punctiform stimulus induced by capsaicin (Table 3). Statistically significant differences were found between the withdrawal latencies of the leg of the control group and those obtained in the groups treated with maslinic acid at the doses of 64, 256 and 512 mg / kg. Table 3: Effect of subcutaneous administration of maslinic acid on mechanical allodynia induced by capsaicin

Treatment Dose Latericia time Percentage of

(mg / kg; sc) (seconds) "analgesia ⁰

Solvent - 16.68 ± 2.21 0.58 ± 4.41

Ac. Maslinic 64 28.56 ± 3.67 ^* 36.04 ± 7.33 ^***

128 27.08 ± 3.02 31.62 ± 6.05 ^*

256 34.26 ± 3.91 ^*** 53.05 ± 7.82 ^***

512 36.18 ± 4.00 ^*** 58.76 ± 8.0 (T ^*

Antialodinic effect of subcutaneous administration (s.c.) of different doses of maslinic acid (64,

128, 256 and 512 mg / kg) and its solvent, in mechanical allodynia induced by intraplantar injection of capsaicin (1 μg). Capsaicin injection was performed 30 min after subcutaneous application of maslinic acid or its solvent. Each value represents the mean ± ESM of the values obtained in at least 8 animals. Statistically significant differences between the groups treated with maslinic acid and its solvent: * P <0.05; ** P <0.01; *** P <0.001 (one-way ANOVA followed by the Bonferroni test).

"Latency time of withdrawal of the back leg sensitized with capsaicin after ipsilateral stimulation with a punctate stimulus (0.5 mm in diameter) applied with a force of 0.5 grams

(see the materials and methods section for an explanation in detail).

Percentage of analgesia calculated using the formula:% Analgesia = [TLT - TLC / TC - TLC] x

100; where TLT is the latency time of mice treated with maslinic acid, TLC is the latency time of mice belonging to the control group (treated with solvent) and CT is the cut-off time (50 s).

6.4 Effect of the systemic administration of maslinic acid in the test of acetic acid induced contortions:

The subcutaneous administration of maslinic acid was able to reduce the number of contortions induced by intraperitoneal administration of acetic acid to 0.6% (Table 4). Statistically differences were found. significant, with respect to the control group, in the number of contortions shown by the groups of animals treated with all doses of maslinic that were used (64, 128 and 256 mg / kg). Table 4: Effect of subcutaneous administration of maslinic acid in the test of acetic acid induced contortions

Treatment Dose Number Percentage of

(mg / kg; sc) contortions "analgesia ⁶

Solvent - 50.17 ± 4.84 0.00 ± 9.66

Ac. Maslinic 64 24.67 ± 5.34 ^** 50.83 ± 10.64 ^**

128 30.08 ± 4.71 ^* 40.04 ± 9.38 ^*

256 19.58 ± 4.14 ^*** 60.97 ± 8.25 ^***

Effect of subcutaneous administration of different doses of maslinic acid (64, 128, 256 mg / kg) and its solvent, on pain induced by intraperitoneal injection (i.p.) of acetic acid (0.6%). Acetic acid injection was performed 30 min after administration of maslinic acid or its solvent. Each value represents the mean ± ESM of the values obtained in 12 animals per group.

Statistically significant differences between the groups treated with maslinic acid and its solvent: * P <0.05; ** P <0.01; *** P <0.001 (one-way ANOVA followed by the Bonferroni test).

Number of accumulated contortions (0-30 min) after administration (i.p.) of acetic acid (see materials and methods section for detailed explanation).

Percentage of analgesia calculated using the formula:% Analgesia = [(NCC - NCT) / NCC] x 100; where NCT is the number of contortions of mice treated with maslinic acid, and NCC is the number of contortions of mice belonging to the control group (solvent treated).

6.5 Effect of the systemic administration of maslinic acid in the hot plate test of Ia at 50 ⁰ C:

Subcutaneous administration of maslinic acid to increase the latency of either licking of the hind legs, when the animal was placed on hot plate at 5O Ia ⁰ C (Table 5). Statistically significant differences were found with respect to the control group in the licking times obtained in the groups treated with the doses of 128, 256 and 512 mg / kg of maslinic acid (Table 5).

Table 5: Effect of subcutaneous administration of maslinic acid in the hot plate test

Treatment Dose Latency time Percentage of

(mg / kg; sc) (seconds) "analgesia ⁶ Solvent 13.68 ± 0.79 0.0 ± l, 72

Ac. Maslinic 64 16.12 ± l, 01 5.28 ± 2.19

128 25.40 ± 2.89 ^* 25.30 ± 6.25 ^* 256 23.80 ± 2.69 ^* 21.85 ± 5.8f 512 20.30 ± l, 29 14.29 ± 2.79

Effect of subcutaneous administration of different doses of maslinic acid (64, 128, 256 and 512 mg / kg) or its solvent, in the latency time of the back leg lick in the hot plate test at 5O ⁰ C. Each value represents the mean ± ESM of the values obtained in at least 8 animals. Statistically significant differences between the groups treated with maslinic acid and its solvent: *** P <0.01 (one-way ANOVA followed by the Bonferroni test).

a Latency latency time of any of the hind legs when the animal is placed on the plate at 50 ⁰ C (see materials and methods for a more detailed explanation).

Percentage of analgesia calculated using the formula:% Analgesia = [TLT - TLC / CT— TLC] x 100; where TLT is the latency time of mice treated with maslinic acid, TLC is the latency time of mice belonging to the control group (treated with solvent) and CT is the cut-off time (60 s).

6.6 Effect of maslinic acid on the Rotarod test:

The highest dose of maslinic acid that was used systemically in the experiments in which its analgesic or antialodinic effect was evaluated (512 mg / kg; sc) had no effect on motor coordination when the animals were subjected to the test of Rotarod (Table 6). On the contrary, the control drugs that were used, gabapentin (60 mg / kg, sc) and baclofen (8 mg / kg, sc), did clearly alter the motor coordination of the animals (Table 6), which is consistent with the data. previously described in the literature with both controls [Hong-Ju et al., Bioorg Med Chem Lett (2004) 14: 2537-2541; Patel et al., Pain (2001) 90: 217-226].

Table 6: Verification of the absence of side effects of maslinic acid administration in the rotarod model (comparison with baclofen and gabapentin)

Rotarod latency time (sec)

Time since subcutaneous injection (min)

Treatment 0 30 | 60 90 120 180

TW-80 203 ± 34 218 ± 32 211 ± 22 225 ± 26 194 ± 23 199 ± 23 Salt 187 ± 22 202 ± 26 193 ± 22 206 ± 19 195 ± 20 173 ± 25 AM-512 200 ± 24 210 ± 34 204 ± 31 204 ± 32 208 ± 35 184 ± 35

** tiü

Bac-8 181 ± 25 _{99 ± 18} **. ## 59 ± 13 ^** , ## 83 ± 14 ^M 106Í21 ^* '** 146 ± 26

Gab-60 175 ± 31 138 ± 10 102 ± 19 ^* , # 98Í18 ^* '** 118 ± 16 ^# 112 ± 13 ^#

Effect on the latex time of Rotarod fall from subcutaneous administration of maslinic acid 512 mg / kg (AM-512), baclofen 8 mg / kg (Bac-8), gabapentin 60 mg / kg (Gab-60) and its Respective Solvents: Tween 80 2% in water (TW-80), for maslinic acid, and saline (Salt), for baclofen and gabapentin. The results represent the latency time of fall of the Rotarod when the animals are exposed to the apparatus in the evaluation phase of the experimental procedure (see materials and methods for a more detailed explanation). Each value represents the mean ± ESM of the values obtained in at least 8 animals. Statistically significant differences between the groups treated with any of the drugs and their solvent: ** P <0.01, * P <0.05; Statistically significant differences between post-treatment measures (30, 60, 90, 120 and 180 minutes) and the baseline measure of each group (time 0): ## P <0.01, # P <0.05 (Two-way ANOVA followed by Bonferroni test).

6.7 Effect of the COX-2 inhibitor rofecoxib on mechanical allodynia induced by capsaicin

As described in experience 3, maslinic acid is capable of inhibiting mechanical hypersensitivity induced by capsaicin. In order to rule out that the cause of the efficacy of maslinic acid is due to its possible action as a COX-2 inhibitor, the effect of the COX-2 inhibitor rofecoxib in this model has been evaluated. We have found that both doses of rofecoxib (8 and 32 mg / kg) were unable to significantly modify the mechanical hypersensitivity induced by capsaicin (Table 7). These data agree with others previously described in the literature that demonstrate that the selective inhibitors of COX-2 celecoxib and rofecoxib and the inhibitor of COX1 and COX-2 ibuprofen had no effect on mechanical allodynia induced by capsaicin [Joshi et al. , Neuroscience (2006) 143: 587-596; Entrena et al., Psychopharmacology (2009) 205: 21-23], or in other models of neurogenic pain [Broom et al., Neuroscience (2004) 124: 891-900].

Therefore, it can be concluded that the analgesic action of maslinic acid described in experiment 3 cannot be explained by a possible inhibitory effect of COX-2 Table 7: Effect of subcutaneous administration of the COX-2 inhibitor rofecoxib on allodynia capsaicin-induced mechanics Treatment Dose Latency time Percentage of (mg / kg; sc) (seconds) "analgesia ⁰

Solvent 7.41 ± 0.94 l, 58 ± 0.91 Rofecoxib 8 mg / kg 7.99 ± l, 20 1.15 ± 2.81

32 mg / kg 8.56 ± 0.70 2.72 ± 1.64

Effect of subcutaneous administration (s.c.) of different doses of rofecoxib (8 and 32 mg / kg) and its solvent, in mechanical allodynia induced by intraplantar injection of capsaicin (1 μg). Capsaicin injection was performed 30 min after subcutaneous application of rofecoxib or its solvent. Each value represents the mean ± ESM of the values obtained in at least 8 animals. No statistically significant differences were found between the groups treated with rofecoxib and its solvent (one-way ANOVA).

"Latency time of withdrawal of the back leg sensitized with capsaicin after ipsilateral stimulation with a punctate stimulus (0.5 mm in diameter) applied with a force of 0.5 grams (see the materials and methods section for a detailed explanation ).

Percentage of analgesia calculated using the formula:% Analgesia = [TLT - TLC / TC - TLC] x 100; where TLT is the latency time of mice treated with maslinic acid, TLC is the latency time of mice belonging to the control group (treated with solvent) and CT is the cut-off time (50 s).

Claims

1. Use of maslinic acid or any of its derivatives for the elaboration of a medication for the treatment of painful pathologies of nociceptive nature (somatic or visceral), inflammatory or neurogenic

2. Use of maslinic acid or any of its derivatives according to any of claims 1 for the preparation of a medicament for the treatment of painful pathologies selected from the list comprising: cancerous pain, postoperative pain, rheumatologic pain, neurogenic pain, neuropathic pain , post-traumatic pain (including pain associated with sports injuries), drug-induced pain (antineoplastic, antiretroviral, etc.), pain caused by visceral pathologies

3. Use of glycollated or analogous extract high in maslinic acid according to any of claims 1 and 2.