Composition based on hyaluronic acid, epigallocatechin-3-o- gallate and / or xanthohumol

Abstract

The present invention describes a Hyaluronic acid-based homogenous composition with high molecular weight in combination with epigallocatechin-3-O-Gallate (EGCG) and / or xanthohumol (XAN) for use in treating rheumatic pathologies or for cosmetic use to prevent and treat skin aging.

Description

[0001] COMPOSITION BASED ON HYALURONIC ACID, EPIGALLOCATECHIN-3-O- GALLATE AND / OR XANTHOHUMOL

[0002] ***** ***** *****

[0003] FIELD OF THE INVENTION

[0004] The present invention relates to a composition comprising hyaluronic acid (HA), with high molecular weight, epigallocatechin-3-O-Gallate (EGCG) and / or xanthohumol (XAN) for use in treating rheumatic pathologies or for cosmetic use. The composition is suitable for intra-articular or subcutaneous or intradermal administration.

[0005] BACKGROUND ART

[0006] Rheumatic diseases are pathologies characterized by inflammation of joints, ligaments, tendons, bones or muscles and which, in some cases, can also include other organs. Some are classified as diseases of the connective tissue (connective tissue diseases), while others are considered as articular inflammatory diseases (arthritis).

[0007] Rheumatic pathologies exert substantial changes in the amount and molecular weight of hyaluronic acid in the synovial liquid, with important implications in seventy and progression of the disease.

[0008] Hyaluronic acid (HA) is a natural glycosaminoglycan consisting of repeated units of a disaccharide formed by glucuronic acid and N-acetyl glucosamine. The units thereof with high molecular weight (PM), which are long and devoid of ramifications, form a gel-like network, which provides hydration and lubrication for tissues, like the skin, cartilage and synovial liquid of the joints. Here, it acts as a buffer for mechanical knocks and cushions the friction between the bone ends. Additionally, HA performs an important anti-oxidant and immunomodulating action, which includes reducing the production of pro-inflammatory cytokines and inducing the release of anti-inflammatory cytokines in cells of the immune system and joints.

[0009] In inflammatory conditions, as in the case of rheumatic diseases, the cells of the cartilage lose the ability to regulate the process of synthesis and degeneration of HA, with progressive accumulation of fragments of polysaccharide in the articular joint, which, in turn, support the production of pro-inflammatory mediators and synovial hyperplasia. Furthermore, the properties of HA as an anti-oxidant and modulator of the inflammatory response of the joints, are lost, with an increase in the expression cells, dependent on oxidative stress, of inflammatory enzymes, such as cyclooxygenase 2 (COX-2) and metalloprotease of the matrix (MMP) and consequent exacerbation of the inflammatory process.

[0010] In this context, the intra-articular administration of HA is a commonly prescribed practice to support the tissues and modulate the inflammatory state thereof. This method of administration, which allows releasing the polysaccharide directly into the articular joint concerned, provides a localized therapeutic effect, with a reduction in pain and general improvement of joint function. Consolidated approaches for increasing the effect of HA provide the administration of the molecule in combination with corticosteroids or Non-Steroidal Anti-Inflammatory Drugs (NSAIDs), both conjugated and in free form, producing additional benefits for controlling the inflammatory state and progressive degeneration of the tissues.

[0011] However, the use of corticosteroids or NSAIDs has criticalities related, in particular, to the onset of relatively severe side effects, especially when used in the treatment of chronic pathologies.

[0012] Epigallocatechin-3-O-Gallate (EGCG), a catechin found in green tea, and xanthohumol (XAN), a prenylflavonoid contained in the inflorescence of hops, are polyphenols characterized by anti-oxidative and anti-inflammatory action.

[0013] EGCG has proven to be from 25 to 100 times more powerful than vitamin C and vitamin E in terms of anti-oxidant activity.

[0014] Xanthohumol (XAN) significantly inhibits inflammatory responses, weakens the expression of catabolic enzymes and improves ECM degeneration.

[0015] Therefore, epigallocatechin-3-O-Gallate (EGCG) and xanthohumol (XAN) carry out anti-oxidant and anti-inflammatory activities and thus play an important role in many inflammatory responses produced by nitric oxide (NO) and free radicals (ROS).

[0016] Free radicals (ROS) are also generated during skin aging. Skin aging can be divided into intrinsic aging (chronological aging) and extrinsic aging (photoaging).

[0017] Intrinsic aging is the natural process of aging induced by reduced activity of skin cells by ROS generated in the metabolism in skin cells. Extrinsic aging is caused by external environmental factors, such as ultraviolet irradiation and pollutants. The main causes of extrinsic aging are secondary reactions mediated by ROS, which occur when UV rays are absorbed by the skin. Excess ROS causes the formation of wrinkles through the scission and abnormal crossing of chains of fibrous proteins, like collagen, elastic fibers and glycosaminoglycans (GAG) in the skin’s extracellular matrix. The synthesis of collagen is inhibited by collagenase, including metalloproteinase-1 of the matrix (MMP-1 ). Finally, pro inflammatory cytokines increase inflammatory reactions and promote reactions linked to aging.

[0018] It is the aim of the present invention to provide an HA-based composition with improved anti-oxidant and anti-inflammatory activities for use in the treatment of rheumatic pathologies, which provides an alternative or more effective therapeutic strategy or with fewer side effects with respect to the known compositions of the state-of-the-art, or for preventing and treating skin aging.

[0019] SUMMARY OF THE INVENTION

[0020] The stated aim has been reached by means of an HA-based composition in combination with molecules of plant origin devoid of significant side effects.

[0021] Therefore, in a first aspect thereof, the present invention relates to a composition comprising hyaluronic acid, epigallocatechin-3-O-Gallate (EGCG) and / or xanthohumol (XAN).

[0022] The compositions comprising hyaluronic acid in combination with epigallocatechin-3- O-Gallate and / or xanthohumol are surprisingly effective in the treatment of rheumatic pathologies since the hyaluronic acid, theepigallocatechin-3-O-Gallate (EGCG) and / or xanthohumol (XAN) are shown to have a clear synergic effect on inhibiting the production of ROS and nitrite, the stable product of NO, both reliable markers of inflammation in osteoarthritis models.

[0023] The combination of hyaluronic acid and xanthohumol has also been shown to be particularly effective in reducing mechanical allodynia in rats with osteoarthritis in a statistically significant manner with respect to hyaluronic acid alone.

[0024] Therefore, in a second aspect, the present invention relates to a composition comprising hyaluronic acid, epigallocatechin-3-O-Gallate (EGCG) and / or xanthohumol (XAN) for use in the treatment of rheumatic pathologies, in particular, osteoarthritis.

[0025] In a third aspect, the present invention relates to a cosmetic composition comprising hyaluronic acid, epigallocatechin-3-O-Gallate (EGCG) and / or xanthohumol (XAN) for preventing and treating skin aging. Compositions of the invention surprisingly invert the degeneration of collagen in normal human dermal fibroblasts and reduce the expression and secretion of pro-inflammatory cytokines, including interleukin (IL)-1 (3 and IL-6, significantly improving both intrinsic and extrinsic skin aging.

[0026] DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 : Treatment plan for SW982 synoviocytes.

[0028] Figure 2: Cytotoxicity of HA, XAN and EGCG on SW982 synoviocytes. Cell viability was assessed after 24-hour treatment by MTT test. The values are the average ± DS of four separate experiments conducted in triplicate.

[0029] Figure 3: Effect of HA (0.05-2 pg / ml), EGCG (0.5-10 pg / ml), XAN (0.30-15 pg / ml) (A) and of HA combined with EGCG (20 / 0.4 pg; 40 / 0.8 pg; 100 / 2 pg; 200 / 4 pg) or with XAN (20 / 0.6 pg; 40 / 1.2 pg; 100 / 3 pg; 200 / 6 pg) (B) on the production of ROS induced by IL-1 p in SW892 cells and graph of the relevant combination index (Cl) with respect to the fraction of affected cells obtained by using the analysis program of the median effect (C).

[0030] Figure 4: Effect of HA (0.05-2 pg / ml), EGCG (0.5-10 pg / ml), XAN (0.30-15 pg / ml) (A) and of HA combined with EGCG (20 / 0.4 pg; 40 / 0.8 pg; 100 / 2 pg; 200 / 4 pg) or with XAN (20 / 0.6 pg; 40 / 1.2 pg; 100 / 3 pg; 200 / 6 pg) (B) on the release of NO induced by IL-1 p in SW892 cells and graph of the relevant combination index (Cl) with respect to the fraction of affected cells obtained by using the analysis program of the median effect (C).

[0031] Figure 5: Effect of XAN (A) and EGCG (B) in combination with HA on the production of ROS stimulated by IL-1 p in the synoviocytes. Representative images and average values ± SD of three separate experiments conducted in triplicate.

[0032] Figure 6: Treatment plan and measurement of mechanical allodynia.

[0033] Figure 7: Evaluation of mechanical allodynia in OA animals in the presence of vehicle, Hyaluronic acid, Xanthohumol, or Hyaluronic acid and Xanthohumol. The data is expressed as average ± S.E.M of retraction thresholds of the paw, measured in grams. Figure 8: Effect of HA, EGCG and XAN with equipotent concentrations (ICso), according to the Chou-Talalay theorem (A); graph of the values of the Combination Index (Cl) for the different fractions of inhibition (fa) on the production of ROS (B).

[0034] DETAILED DESCRIPTION OF THE INVENTION

[0035] In a first aspect, the present invention relates to a composition comprising hyaluronic acid, epigallocatechin-3-O-Gallate (EGCG) and / or xanthohumol (XAN).

[0036] Surprisingly, it was found that the simultaneous anti-oxidant activity of epigallocatechin-3-O-Gallate (EGCG) and / or xanthohumol (XAN), aided by the presence of HA, contributes to treating pathologies directly related to ROS, acting either directly with a reducing activity, or indirectly, inducing the cell’s defense mechanisms to overcome oxidative stress.

[0037] Advantageously, the combinations of hyaluronic acid with epigallocatechin-3-O- Gallate and / or xanthohumol, have shown to have a clear synergic effect in inhibiting the production of ROS and nitrite, the stable product of NO, both reliable markers of inflammation, in a cell model of rheumatoid arthritis.

[0038] The combination of hyaluronic acid and xanthohumol has also shown to be particularly effective in reducing mechanical allodynia in rats with osteoarthritis in a statistically significant manner with respect to hyaluronic acid alone.

[0039] Moreover, surprisingly, it was also found that compositions of the invention comprising hyaluronic acid (HA) in combination with epigallocatechin-3-O-Gallate (EGCG) and / or xanthohumol (XAN), also have a significant anti-aging effect. In fact, the compositions of the invention suppress the activity of collagenase MMP-1 inverting the degeneration of collagen induced by necrosis tumor-a (TNF-a) factor in normal human dermal fibroblasts; moreover, they weaken the expression and secretion of pro-inflammatory cytokines, including interleukin (IL)-1 (3 and IL-6, significantly improving both intrinsic and extrinsic skin aging.

[0040] According to a preferred embodiment of the invention, the composition comprising hyaluronic acid, epigallocatechin-3-O-Gallate (EGCG) and / or xanthohumol (XAN), is administered by intra-articular injection for rheumatic pathologies, or subcutaneously or intradermally for cosmetic use.

[0041] According to a preferred embodiment of the invention, the hyaluronic acid of the composition has a molecular weight ranging from 2,000,000 to 4,000.000 Da. More preferably, the hyaluronic acid has a molecular weight of 3,000.000 Da.

[0042] The composition of the invention preferably comprises hyaluronic acid (HA) having a concentration ranging from 15 to 30 mg / ml. More preferably, the concentration of hyaluronic acid (HA) is 25 mg / ml.

[0043] The composition of the invention preferably comprises epigallocatechin-3-O-Gallate (EGCG) having a concentration ranging from 3 to 16 mg / ml . More preferably, the concentration of epigallocatechin-3-O-Gallate (EGCG) ranges from 8 to 10 mg / ml.

[0044] The composition of the invention preferably comprises xanthohumol (XAN) having a concentration ranging from 5 to 12 mg / ml. More preferably, the concentration of xanthohumol (XAN) ranges from 6 to 8 mg / ml.

[0045] According to a preferred embodiment of the invention, the composition of the invention comprises hyaluronic acid (HA) having a concentration ranging from 20 to 30 mg / ml and epigallocatechin-3-O-Gallate (EGCG) having a concentration ranging from 8 to 10 mg / ml. The composition of the invention preferably comprises HA having a concentration of 25 mg / ml and epigallocatechin-3-O-Gallate (EGCG) having a concentration ranging from 8 to 10 mg / ml, more preferably, 8 mg / ml for intra-articular administration in rheumatic pathologies and 10 mg / ml for subcutaneous or intradermal administration for cosmetic use.

[0046] According to an alternative preferred embodiment of the invention, the composition of the invention comprises HA having a concentration ranging from 20 to 30 mg / ml and xanthohumol (XAN) having a concentration ranging from 6 to 8 mg / ml. The composition of the invention preferably comprises HA having a concentration of 25 mg / ml and xanthohumol (XAN) having a concentration ranging from 6 to 8 mg / ml, more preferably 8 mg / ml for intra-articular administration in rheumatic pathologies and 6 mg / ml for subcutaneous or intradermal administration for cosmetic use.

[0047] According to an alternative preferred embodiment of the invention, the composition of the invention comprises HA having a concentration ranging from 20 to 30mg / ml, epigallocatechin-3-O-Gallate (EGCG) having a concentration ranging from 8 to 10 mg / ml, and xanthohumol (XAN) having a concentration ranging from 6 to 8 mg / ml.

[0048] The composition of the invention preferably comprises HA having a concentration of 25 mg / ml, epigallocatechin-3-O-Gallate (EGCG) having a concentration of 8 mg / ml, and xanthohumol (XAN) having a concentration of 8 mg / ml for intra-articular administration in rheumatic pathologies.

[0049] The composition of the invention preferably comprises HA having a concentration of 25 mg / ml, epigallocatechin-3-O-Gallate (EGCG) having a concentration of 10 mg / ml, and xanthohumol (XAN) having a concentration of 6 mg / ml for subcutaneous or intradermal administration for cosmetic use.

[0050] According to a preferred embodiment of the invention, the composition comprises a vehicle and / or one or more pharmaceutically acceptable excipients.

[0051] The excipients are preferably selected from the group consisting of dibasic sodium phosphate dodecahydrate and dihydrate monobasic phosphate sodium.

[0052] In a second aspect thereof, the present invention relates to a composition comprising hyaluronic acid, epigallocatechin-3-O-Gallate (EGCG) and / or xanthohumol (XAN) for use in the treatment of rheumatic pathologies, in particular, osteoarthritis.

[0053] In a further aspect, the invention relates to a composition comprising hyaluronic acid, epigallocatechin-3-O-Gallate (EGCG) and / or xanthohumol (XAN) for use in the treatment of severe forms of acute pain in patients affected by arthrosis who do not respond adequately to pain relieving therapies with steroids and traditional antiinflammatories.

[0054] In another aspect, the present invention relates to a method for treating rheumatic pathologies, by administering, to a subject needing it, a composition comprising hyaluronic acid, epigallocatechin-3-O-Gallate (EGCG) and / or xanthohumol (XAN). The invention preferably relates to a method for treating osteoarthrosis, by administering, to a subject needing it, a composition comprising hyaluronic acid, epigallocatechin-3- O-Gallate (EGCG) and / or xanthohumol (XAN).

[0055] In another aspect, the present invention relates to a cosmetic composition comprising hyaluronic acid, epigallocatechin-3-O-Gallate (EGCG) and / or xanthohumol (XAN) for preventing and treating skin aging.

[0056] The invention is now shown by means of examples, which shall be understood for illustrative and non-limiting purposes thereof.

[0057] EXPERIMENTAL SECTION

[0058] Reagents

[0059] Unless otherwise specified, all the reagents and chemicals were purchased from Merck (Milan, Italy) and are of the highest degree of purity available.

[0060] SW982 human synoviocytes were purchased from American Type Culture Collection (ATCC, Rockville, Maryland, USA) and were used between steps 4 and 10.

[0061] Cell cultures and treatments

[0062] The cells were grown in 75 cm2 flasks in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1 % non-essential amino acids, 10 mM HEPES, 50 units / ml penicillin, 50 mg / ml streptomycin, and maintained at 37 °C at 5% of CO2”. The medium was changed every 48 hours. For the in vitro experiments, the cells were seeded in DMEM at the density of 6 x 104 cells / cm2 in 24-well plates. After 24 hours, IL-1 [3 (MedChemExpress-MCE, New Jersey, USA) at 10 ng / ml was added and the treatment continued for 24 hours. Where indicated, the cells were preincubated with HA, XAN and EGCG, alone or combined with the indicated concentrations, for 1 hour before adding the cytokine, as shown in the diagram in Figure 1 .

[0063] Examples

[0064] Example 1 : Composition for intra-articular use

[0065] The composition comprising hyaluronic acid, epigallocatechin-3-O-Gallate (EGCG) and xanthohumol (XAN) for intra-articular use is shown in Table 1 : Table 1

[0066] A similar composition can also be made in the absence of EGCG or XAN by modulating the amount of the excipients. Example 2 : Cosmetic composition

[0067] A cosmetic composition comprising hyaluronic acid (HA), epigallocatechin-3-O-Gallate

[0068] (EGCG) and / or xanthohumol (XAN) is shown in Table 2:

[0069] Table 2 A similar composition can also be made in the absence of EGCG or XAN, by modulating the amount of excipients.

[0070] Example 3: Cytotoxicity of HA, XAN or EGCG against SW982 cells The cytotoxicity of HA, XAN or EGCG against SW982 cells was determined by MTT colorimetric assay. This assay is based on the reduction of 3-(4,5-dimethyl-2 -thiazolyl) bromide-2.5-diphenyl-2-H tetrazolium to violet formazan by mitochondrial dehydrogenases of living cells. The cells were seeded in 96-well plates (Coming Costar, Milan, Italy) at a density of 7.5 x 104 cells / cm2, incubated overnight and then treated in the absence (control) or in the presence of HA, XAN and EGCG. After 24 hours of incubation, the medium was carefully removed and 20 pg of MTT was added per well. The supernatant was discarded after 2 hours of incubation at 37 °C and the formazan crystals formed inside the cells were dissolved in dimethyl sulfoxide (DMSO). The absorbance value at 575 nm characterizing formazan violet was measured with a microplate reader (LTek, INNO, Seongnam, Republic of Korea) and the value of the control cells was considered as 100% of the viability. Each experiment was repeated four times in triplicate.

[0071] As shown in Figure 2, HA (from 0.05 to 5.0 mg / ml) and EGCG (from 0.5 to 100 pg / ml) do not exert any toxic effect on the cells, XAN in a concentration > 20 pg / ml produces a reduction in dose-dependent cell viability.

[0072] Example 4: Evaluation of the effect of HA in combination with EGCG and / or XAN on the production of ROS

[0073] The effect of HA, XAN, EGCG and HA in combination with XA and / or EGCG on the production of ROS in SW892 cells stimulated by IL-1 (3, was studied by cytofluorometer analysis. The changes in fluorescence resulting from intracellular oxidation of the fluorescent probe dichlorofluorescein (DCF, Merck, Milan, Italy) were analyzed, added in the dark and 30 minutes before the end of the treatment, at a final concentration of 0.5 pM. After trypsinization, the cells were isolated by centrifugation (2000 x g, 4 °C, 5 min), washed and resuspended in 400 pl of PBS ready for cytofluorometer analysis (CytoFLEX, Beckmann Coulter, California, USA). At least 10000 cell events per sample were considered for each analysis.

[0074] After 24 hours of treatment with the individual compounds, the concentration values of HA, XAN and EGCG, which caused 50% inhibition in the production of ROS were 200±0.01 pg / ml, 6.17±0.51 pg / ml and 4.60±0.39(n=3) respectively, as shown in Figure 3A.

[0075] Thus, the inhibitory effect of the co-treatment with HA / XAN, HA / EGCG and HA / XAN / EGCG was studied using various dilutions of an equipotent mixture of the molecules, according to the Chou-Talalay theorem (Chou, T.C. Drug combination studies and their synergy quantification using the Chou-Talalay method. Cancer Res. 2010, 70, 440-446).

[0076] The values of the Combination Index (Cl) for the different fractions of inhibition (fa) on the production of ROS are shown in Figure 3B and in Figure 8A.

[0077] The combination index (Cl) was calculated using CompuSyn software (ComboSyn, Paramus, NJ, USA) to define the type of effect. Cl was calculated for each fraction value of the effect (fa), where fa is defined as the inhibition percentage / 100. Cl > 1 , Cl < 1 and Cl =1 indicate antagonism, synergism or additive effect, respectively.

[0078] A complex inhibitory effect with both combinations is apparent, with a clear synergic effect between HA / XAN and HA / EGCG at fa values of>0.3. This result may be interesting in the light that for anti-cancer or antiviral agents, synergy with high effect levels (e.g. at fa >0.8) is more relevant for therapy than at low effect levels (fa <0.2). Furthermore, the results show that when HA is in the simultaneous presence of EGCG and XAN, an additive effect is obtained on the inhibition of the production of ROS, at least up to fa values of<0.75.

[0079] Table 3 shows the quantitative data of the dose-response relationship of HA, XAN, EGCG individually or combined on the production of ROS induced by IL-1 [3 in synoviocytes in culture. The values of the Dose Reduction Index (DRI) indicate that the effective concentration of HA, in synergic combination with XAN or with EGCG, is reduced by about 2.5 to 3.5 times.

[0080] Table 3

[0081] DRI indicates the dose reduction index; r indicates the correlation coefficient.

[0082] A greater DRI value indicates a greater reduction in the dose for a given therapeutic effect. Example 5: Evaluation of the effect of HA in combination with EGCG or XAN on the release of nitric oxide

[0083] The effect of the combination of HA with XAN or EGCG on the release of NO into the culture medium was spectrophotometrically evaluated according to the reaction of the Griess assay (Thermo Fisher Scientific Inc., Waltham, MA, USA). 100 pl of cell supernatant were incubated with an equal volume of Griess reagent (1 % sulfanilamide in 5% phosphoric acid and 0.1 % N-(l -naphthyl)-ethylenediamine). After incubating the reaction mixture for 10 minutes, the absorbance was read by plate reader (LTek, HYMN) set at 540 nm.

[0084] A clear synergic effect with Cl< 1 , calculated as in Example 2, of the inhibition of the production of nitrite, the stable product of NO, is apparent also for this parameter, for fa values>0.3, as shown in Figure 4 and in Table 4.

[0085] Table 4

[0086] DRI indicates the dose reduction index; r indicates the correlation coefficient.

[0087] Example 6: Evaluation of the strengthening effect of anti-oxidant activity of HA in combination with XAN or EGCG

[0088] In order to evaluate the strengthening effect of the anti-oxidant action of HA from XAN or EGCG, SW982 cells were treated with IL-1 [3 (10 ng / ml) in the absence or in the presence of 200 pg / ml HA in combination with non-effective concentrations of XAN (0.175 and 0.35 pg / ml) or EGCG (0.05 and 0.25 pg / ml). After 24 hours of treatment, the cells were collected and subjected to cytofluorometer analysis to determine the ROS as described previously.

[0089] While the phytochemicals XAN and EGCG, at 0.35 pg / ml and 0.25 pg / ml, respectively, are not individually able to alter the production of ROS in SW892 cells stimulated by IL-1 [3, when added in the presence of HA 0.2 mg / ml, they significantly strengthen the inhibitory effect of the polysaccharide as shown in Figure 5. Example 7: Evaluation of the pain-relieving effect of the combination of hyaluronic acid (HA) and Xanthohumol (XAN) in an osteoarthrosis model (OA) of a rat.

[0090] The aim of the study was to evaluate the pain-relieving effect of the combination of hyaluronic acid (HA) and Xanthohumol (XAN) in an osteoarthrosis model (OA) of a rat. The study was carried out on male Wistar rats to avoid bias caused by the alteration of the pain threshold in females during the different phases of the menstrual cycle.

[0091] Young male Wistar rats were subjected to an injection (1 mg / 50ul) of monosodium iodoacetate (MIA) or vehicle (saline) in the right joint. Pain was monitored as the measurement of mechanical allodynia by means of Von frey test at days 0 (pretreatment) and days 7, 14, 15, 21 , 26, 33, 37, 44 and 51 after induction. The pharmacological treatment by intra-articular route with hyaluronic acid 2% / 50ul and Xanthohumol100ug / 50ul was carried out on day 14 and 26 after the measurement (figure 1 ). The doses of the tested compounds were selected according to preceding studies published in literature (Chen et al., 2021 ; Kroin et al., 2016).

[0092] Treatment groups (N= 5)*

[0093] 1 . Negative control (Vehicle / vehicle)

[0094] 2. Positive control (MIA+vehicle)

[0095] 3. Xanthohumol (MIA+Xanthohumol)

[0096] 4. Hyaluronic acid (MIA+Hyaluronic acid)

[0097] 5. Combination (MIA+Hyaluronic acid+Xanthohumol)

[0098] The data is expressed as average ± S.E.M of the retraction thresholds of the paw, measured in grams. A comparison between the groups was made by means of the ANOVA variance test for repeated measurements, followed by Tukey’s correction (post hoc) test for multiple comparisons. P values of less than 0.05 were considered statistically significant.

[0099] OA was induced by means of intra-articular injection of monosodium iodoacetate (MIA), an inhibitor of glyceraldehyde-3-phosphate dehydrogenase activity. It induces death of the chondrocytes and determines cartilaginous lesions with loss of matrix and proteoglycans and functional impairment of the joint, reproducing conditions observed in human OA. Said model represents the most suitable model for evaluating the pain component.

[0100] Tactile allodynia was evaluated as painful symptomology. This is the symptom that principally limits the quality of life for patients affected by different forms of chronic pain. In fact, it represents a painful integration to a stimulus, which is harmless in itself, such as the bristle of a toothbrush or fabric (e.g. an item of clothing) rubbed on the skin. This is due to phenomena of peripheral sensitization of the nociceptive fibers and tactile fibers.

[0101] Figure 6 shows the plan for treating and measuring mechanical allodynia.

[0102] Measurement of tactile allodynia was carried out by means of the Von Frey test, which provides the application of filaments of nylon onto the plantar surface of the animal’s paw. The filaments have different thicknesses and exert a specific force depending on the size thereof (0.008, 0.02, 0.04, 0.07, 0.16, 0.4, 0.6, 1.0, 1 .4 and 2.0 g cut-off). The animals were stabilized in plastic compartments with a metal net bottom, inside which they are free to move. After a period of adaptation of about 1 hour, beneath the plantar surface of the paw of each rat, ipsi lateral ly to the articular lesion, the Von Frey filaments were applied with a linear and increasing force for 3-4 seconds. The response to the filament applied as the mechanical nociceptive threshold of the paw (PWT) was recorded and expressed in grams (g) for the reflected response of avoiding or retracting the stimulated paw.

[0103] The condition of OA was induced by means of injection of MIA (1 mg / 50ul) into the right joint. OA animals subjected to treatment with vehicle (saline solution) (MIAA / ehicle) showed a reduction in the mechanical threshold with respect to the control animals (VehicleA / ehicle). Said effect was apparent from day 7 after induction and was maintained for the entire duration of the period of observation [7g: 42 ± 11 .02 vs 85.2 ± 14.8 gr, P=0.046],

[0104] In animals with OA, treatment with Xanthohumol (100pg / 50pl) determined a partial increase in the mechanical threshold even if it was not statistically significant with respect to the group treated with vehicle.

[0105] With respect to the vehicle, treatment with hyaluronic acid did not alter the mechanical threshold in animals with OA. On the contrary, the Hyaluronic acid and Xanthohumol combination increased the mechanical threshold in a significantly statistical manner, thus determining a reduction in painful behavior. A single administration carried out on days 14 and 26 induced an analgesic effect lasting from 10 to 18 days. The effects induced by the Hyaluronic acid and Xanthohumol combination were statistically different with respect to the treatment with hyaluronic acid alone [15g: 15.6 ± 4.2 vs 68± 8, P=0.0079; 33g:16.8±10.8 vs 75±16.88, P=0.0022], as shown in Figure 7.

[0106] The data obtained indicates that the combination hyaluronic acid and xanthohumol reduces mechanical allodynia in rats with osteoarthritis in a statistically significant manner, with respect to hyaluronic acid alone. Furthermore, the duration of the anti- allodynic effect lasts over time.

Claims

CLAIMS1. A composition comprising hyaluronic acid (HA), epigallocatechin-3-O-Gallate (EGCG) and / or xanthohumol (XAN).

2. The composition of claim 1 , wherein the administration is by intra-articular or subcutaneous or intradermal route.

3. The composition of claims 1 or 2, in which hyaluronic acid has a molecular weight ranging from 2,000,000 to 4,000,000 Da.

4. The composition of anyone of claims 1 -3, wherein the concentration of hyaluronic acid (HA) ranges from 15 to 30 mg / ml.

5. The composition of anyone of claims 1 -4, wherein the concentration of epigallocatechin-3-O-Gallate (EGCG) ranges from 3 to 16 mg / ml.

6. The composition of anyone of claims 1 -5, wherein the concentration of xanthohumol (XAN) ranges from 5 to 12 mg / ml.

7. The composition of anyone of claims 1 -6, wherein the concentration of hyaluronic acid (HA) is 25 mg / ml, the concentration of epigallocatechin-3-O-Gallate (EGCG) ranges from 8 to 10 mg / ml and / or the xanthohumol (XAN) concentration ranges from 6 to 8 mg / ml.

8. The composition of anyone of claims 1 -7, wherein the concentration of hyaluronic acid (HA) is 25 mg / ml, the concentration of epigallocatechin-3-O-Gallate (EGCG) is 8 mg / ml and the xanthohumol (XAN) concentration is 8 mg / ml, or epigallocatechin-3-O- Gallate (EGCG) concentration is 10 mg / ml and xanthohumol (XAN) concentration is 6 mg / ml.

9. The composition of anyone of claims 1 -8 comprising a vehicle and / or one or more pharmaceutically acceptable excipients.

10. A composition of anyone of claims 1 -9 for use as a medicinal product.

11. A composition of anyone of claims 1 -9 for use in the treatment of rheumatic pathologies, preferably osteoarthritis.

12. A composition of anyone of claims 1 -9 for use in the treatment of severe forms of acute pain in patients suffering from osteoarthritis who do not respond adequately to pain-relieving therapies with steroids and traditional anti-inflammatories.

13. Use of the composition of anyone of claims 1 -9 as a cosmetic, preferably to prevent and treat skin aging.

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