Patent application for the industrial invention entitled: a composition based on hyaluronic acid and amino acids for use in the treatment of periodontal wounds

Abstract

The present invention describes a composition comprising high molecular weight hyaluronic acid (HA), L-proline, L-hydroxyproline, Glycine and L-lysine for use in the treatment of periodontal wounds. The composition of the present invention is surprisingly effective in controlling gum inflammation and bleeding, reduces the depth of gum pockets along with a significant reduction in epithelial and lymphocyte cell proliferation. Furthermore, it has a beneficial effect in the treatment of plaque-induced gingivitis.

Description

[0001] PATENT APPLICATION FOR THE INDUSTRIAL INVENTION ENTITLED:

[0002] A COMPOSITION BASED ON HYALURONIC ACID AND AMINO ACIDS FOR USE IN THE TREATMENT OF PERIODONTAL WOUNDS

[0003] FIELD OF THE INVENTION

[0004] The present invention relates to the field of compositions for use in treating periodontal wounds by promoting soft tissue healing and regeneration.

[0005] BACKGROUND ART

[0006] Tooth or molar extraction is one of the most common surgical procedures performed in dental clinics. The subsequent healing process of the socket has become an important topic of research and clinical practice for successful healing and restoration in the missing region with dental implants. The periodontium is a complex structure consisting of specialized tissues that support the teeth, and most periodontal surgeries are invasive procedures, including a resection of the gum or alveolar bone. The healing process of periodontal wounds is slightly different from the healing of skin wounds, and is similar to fetal healing, being almost free of scars. Wound healing undergoes four stages of tissue change sequences, namely, hemostasis, inflammation, proliferation, and remodeling. The healing of oral hard tissue and soft tissue wounds is largely dependent on the inflammatory response and vascular response.

[0007] Hyaluronic acid (HA), a non-sulfated glycosaminoglycan (GAG) has a considerable molecular weight but a simple structure and is the main component of the extracellular matrix (ECM) of the skin and has a valuable role in inflammatory, angiogenic and tissue regeneration processes. The characteristics of HA are strongly influenced by the molecular weight thereof: low molecular weight HA acts as a pro-inflammatory molecule, while high molecular weight HA acts as an antiinflammatory and immunosuppressive molecule. The structure of HA gives it a highly hydrophilic character, one of the main features for which it is used for dressing applications, being useful in the absorption of exudates and for cell adhesion. Furthermore, it is known that this GAG has excellent biocompatibility and adequate biodegradability. HA has been identified in all periodontal tissues, being particularly prominent in nonmineralized tissues such as the gums and periodontal ligament, and only in small amounts in mineralized tissues such as cementum and alveolar bone. The high molecular weight hyaluronate present in periodontal tissues is synthesized by hyaluronate synthase (HAS) enzymes (HAS1 , HAS2, and HAS3) in various cells of the periodontal tissues, including fibroblasts and keratinocytes in the gums and periodontal ligament, cementoblasts in the cementum, and alveolar bone osteoblasts.

[0008] Hyaluronic acid (HA) has a positive effect on the healing of soft tissues of periodontal wounds following non-surgical and surgical therapy.

[0009] Favia et al. (J. Biol. Regul. & Bioemost. Ag. 2008, 22(2), 109-116) describes an oral gel comprising Gly, Pro, Lys, HA and Leu which accelerates the healing of wounds of the oral mucosa and gums.

[0010] It is the aim of the present invention to provide a composition for use in the treatment of a periodontal wound to promote the healing and soft tissue regeneration thereof, in particular for use in the treatment of post-surgical wounds of the oral cavity.

[0011] SUMMARY OF THE INVENTION

[0012] The present invention relates to a composition comprising high molecular weight hyaluronic acid (HA), L-proline, L-hydroxyproline, Glycine and L-lysine for use in the treatment of periodontal wounds.

[0013] The composition of the present invention is surprisingly effective in controlling gum inflammation and bleeding, reduces the depth of gum pockets along with a significant reduction in epithelial and lymphocyte cell proliferation. Furthermore, it has a beneficial effect in the treatment of plaque-induced gingivitis.

[0014] The composition of the invention has the following advantageous features:

[0015] - Anti-inflammatory activity;

[0016] - Absorption capacity of exudate;

[0017] - Induces cellular adhesion;

[0018] - Stimulates gingival fibroblasts to proliferate and migrate;

[0019] - Stimulates the production of new type 1 / 3 collagen;

[0020] - Hemostatic capacity and control of proteolytic activity; - Induces rapid wound healing, totally biocompatible;

[0021] - Ability to activate bioactive amino acids during connection with the wound surface;

[0022] - Hypoallergenic, biocompatible, biodegradable;

[0023] - Provides a barrier to external contaminants;

[0024] - Stimulates growth factors and the production of granulation and re- epithelialization tissue;

[0025] - Minimizes trauma or maceration at wound edges;

[0026] - Accelerates bone regeneration through chemotaxis, proliferation and subsequent differentiation of mesenchymal cells. HA shares bone induction features with osteogenic substances such as bone morphogenetic protein 2 and osteopontin.

[0027] The composition of the present invention has a multifunctional role in the wound healing process, offering a much faster healing mechanism also with respect to also other HA-based products, potentially existing within the periodontal tissues.

[0028] DETAILED DESCRIPTION OF THE INVENTION

[0029] The composition of the invention is preferably indicated for use in the treatment of post-surgical wounds of the oral cavity.

[0030] Preferably, the composition of the invention is a sterile isotonic gel. The gel is preferably injectable, i.e. , the composition is administered by local injection into the wound site to be treated.

[0031] Preferably the composition of the invention comprises HA having a molecular weight of 2,000,000-3,000,000 Da.

[0032] Preferably the composition of the invention comprises HA in a concentration of 20- 30 mg / ml, more preferably 25 mg / ml.

[0033] Preferably the composition of the invention comprises L-proline in a concentration of 0.5-2.0 mg / ml, L-hydroxyproline in a concentration of 0.5-2.0 mg / ml, Glycine in a concentration of 0.5-2.0 mg / ml and L-lysine in a concentration of 0.5-2.0 mg / ml.

[0034] Preferably the composition of the invention further comprises a pharmaceutically acceptable carrier, more preferably water for injections (WFI), and pharmaceutically acceptable excipients. The excipients are preferably selected from the group consisting of monobasic sodium phosphate dihydrate and dibasic sodium phosphate dodecahydrate and mixtures thereof, more preferably present in the composition in the concentrations: monobasic sodium phosphate dihydrate 0.034-0.51 mg / ml; dibasic sodium phosphate dodecahydrate 0.180-2.70 mg / ml.

[0035] The present invention also relates to a method for treating post-surgical wounds of the oral cavity, said method comprising: injecting the composition into the surgical defect to be treated and between the flaps to be sutured. Preferably, injecting the composition at room temperature and under conditions of strict asepsis. The amount injected depends on the size of the wound.

[0036] The present invention can be better understood in the light of the following embodiments.

[0037] BRIEF DESCRIPTION OF THE FIGURES

[0038] Figure 1. depiction of the viability of cells treated with Proage®, Gumblast or Hyadent and compared with untreated cells (control) at 24, 48 and 72 hours.

[0039] Figure 2. Human fibroblast wound healing test comparing the effect of Proage®, Gumblast and Hyadent with control (no treatment). The images are representative of three replicas. (p<0.05)

[0040] Figure 3. Cytocompatibility of HAA and HAB, on SW982 synoviocyte cells. Cell viability was assessed after a 24-hour treatment by MTT test. The values are the mean ± SD of four separate experiments conducted in triplicate.

[0041] Figure 4. Effect of HAA and HAB (1 :500-1 :250 v / v) on IL-i p-induced ROS production in SW892 cells. Representative cytofluorimetry.

[0042] Figure 5. Effect of HAA and HAB (1 :500-1 :250 v / v) on IL-i p-induced ROS production in SW892 cells. Mean values ± SD of four separate experiments in triplicate. *p < 0.05, **p < 0.01 , ***p < 0.005, ****p < 0.001 (Unidirectional Anova associated with Tukey's post hoc test).

[0043] Figure 6. Effect of HAA and HAB (1 :500-1 :250 v / v) on IL-10-induced NO production in SW892 cells. Mean values ± SD of four separate experiments in triplicate. *p < 0.05, ***p < 0.005, ****p < 0.001 (Unidirectional Anova associated with Tukey's post hoc test). EXPERIMENTAL SECTION

[0044] EXAMPLE 1

[0045] Comparison of three different HA-based gels, Proage, Gumblast and Hyadent on wound proliferation and healing of human primary gingival fibroblasts.

[0046] Purpose of the study

[0047] This study aims to evaluate the effect of three different hyaluronic acid products, the composition of the present invention (Gumblast), Proage® and HyaDENT on the wound proliferation and healing capacity of human gingival fibroblasts.

[0048] Materials and methods

[0049] Materials

[0050] Proage®: Sodium hyaluronate 20 mg / ml, sodium chloride, monobasic sodium phosphate dihydrate, dibasic sodium phosphate dodecahydrate, water.

[0051] Gumblast: Sodium hyaluronate (3,000,000 Da) 25 mg / ml, L-proline 0.5 mg / ml, L- hydroxyproline 0.8 mg / ml, Glycine 1.8 mg / ml, L-lysine HCL 1.8 mg / ml, monobasic sodium phosphate dihydrate 0.03 mg / ml, dibasic sodium phosphate dodecahydrate 0.190 mg / ml, water.

[0052] Hyadent: Hyaluronic acid 2.0 mg, Cross-linked hyaluronic acid 16.0 mg, Sodium chloride 6.9 mg, Water for injections 1.0 ml.

[0053] Cell culture

[0054] The human fibroblast cell line was cultured in low glucose DMEM supplemented with fetal bovine serum (FBS) (10% v / v) (Gibco, Invitrogen) and penicillinstreptomycin (1 % v / v). The cells were seeded in a 12-well culture plate and maintained at 37°C in a humidified atmosphere with 5% v / v CO2 and the medium was changed every 48 hours.

[0055] Proliferation and vitality - MTT analysis

[0056] The MTT analysis (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide) is based on the conversion of MTT to formazan crystals by living cells, which determine mitochondrial activity. Since for most cell populations total mitochondrial activity is related to the number of viable cells, this test is widely used to measure cell proliferation and viability after treatment with active compounds.

[0057] Vitality=(cells treated with mean 0D) / (mean OD control)x100.

[0058] Wound healing assay The wound healing assay is one of the first methods developed to study directional cell migration in vitro. This method mimics cell migration during in vivo wound healing. Key steps involve creating a “wound” in a cell monolayer, imaging at the beginning and at regular intervals during cell migration to close the wound, and comparing the images to quantify the cell migration rate. It is particularly suitable for studies on the effects of cell-matrix and cell-cell interactions on cell migration. A wound healing test was performed to assess the ability of different products to activate human fibroblasts: the fibroblasts were seeded in a 24 multi-well in replicates. At about 80-90% confluence, a scratch was made in the center of the wells using a sterile 10 uL tip. The wound area was then recorded immediately after the creation of the "wound" and after 36 hours.

[0059] Results

[0060] Proliferation and vitality assessment

[0061] None of the HA-based gels tested were cytotoxic for the cells. However, Proage® stimulated fibroblast proliferation to a greater extent with respect to the control and to Gumblast and Hyadent at both 48 and 72 hours (p<0.05). (Fig. 1 ) Wound healing test

[0062] The wound healing test measures the ability of an active compound to stimulate or reduce the ability of fibroblasts to migrate and repair a wound. Polymers such as hyaluronic acid can affect both the cells and the extracellular matrix, typically accelerating the wound healing process. In this study we evaluated the effect of Proage®, Gumblast and Hyadent to stimulate wound closure. As clearly shown in Figure 2, all the products have an effect on wound closure with respect to the control; however, Gumblast has a significantly greater impact. In the wound healing test, Gumblast performs better than Proage® and Hyadent. This difference could be explained by the effect on amino acids during wound closure when the cells have high metabolic demands.

[0063] EXAMPLE 2

[0064] Comparative study on antioxidant and anti-inflammatory activity of amino acid-enriched hyaluronic acid formulations in a cellular model of rheumatoid arthritis This study reports the combined effect of HA with two different mixtures of amino acids on the inflammatory response of synovial fibroblasts induced by Interleukin- 1 P (IL-1 P), the main cytokine involved in rheumatoid diseases, to evaluate the potential use of these AAs as polysaccharide adjuvants in injectable preparations. Experimental protocols Reagents

[0065] Unless otherwise specified, all the reagents and chemicals were purchased from Merck (Milan, Italy) and are of the highest degree of purity available.

[0066] Composition of HA+AA solutions

[0067] Two HA+AA solutions, referred to as HAA and HAB, were prepared as follows: HAA containing sodium hyaluronate (HA, 2.5% w / v; MW 2.5 / 3 ,5-106Da) and a pool of amino acids (AA, 3.4% w / v) in sterile distilled water. The HAA AA pool according to the present invention consists of 1 % w / v L-hydroxyproline, 0.8% w / v L-lysine HCI, 0.8% w / v glycine and 0.8% w / v L-proline. HAB instead consisted of sodium hyaluronate (HA, 1.33% w / v; MW 1.64-106 Da) and a pool of amino acids (AA, 2% w / v) in sterile distilled water. The HAB AA pool, in accordance with Favia et al. (J. Biol. Regul. & Bioemost. Ag. 2008, 22(2), 109-116) consists of 1 % w / v glycine, 0.75% w / v proline, 0.15% w / v leucine and 0.1 % w / v lysine HCI. The prepared solutions were then diluted in series in DMEM.

[0068] Cell cultures

[0069] SW982 human synoviocytes were purchased from American Type Culture Collection (ATCC, Rockville, Maryland, USA) and were used between steps 4 and 10. The cells were grown in 75 cm2flasks 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% 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 / cm2in 24-well plates. After 24h, IL-1 [3 (MedChemExpress-MCE, New Jersey, USA) at 10 ng / mL was added and the treatment continued for 24 hours. Where indicated, cells were pre-incubated with HAA or HAB, at the indicated concentrations, for 1 h prior to cytokine addition.

[0070] Cytocompatibility The cytocompatibility of HAA and HAB 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. Briefly, the cells were seeded in 96-well plates (Corning 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 HAA and HAB. 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] Assessment of intracellular reactive oxygen species

[0072] Intracellular ROS levels were assessed cytofluorimetrically. In this regard, we analyzed the changes in fluorescence resulting from the intracellular oxidation of the fluorescent probe dichlorofluorescein (DCF, Merck, Milan, Italy), 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 10,000 cellular events per sample were taken into account for each analysis.

[0073] Evaluation of the release of nitrogen monoxide

[0074] The level of NO released into the culture medium was evaluated spectrophotometrically according to the reaction of the Griess assay (Thermo Fisher Scientific Inc., Waltham, MA, USA). Briefly, 100 pL of cell supernatant was 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.

[0075] Statistical analysis The results are expressed as mean ± standard deviation (SD) of n separate experiments conducted in triplicate. The statistical comparisons were performed by variance analysis (ANOVA-one way), followed by Tukey's correction for multiple comparisons, using Prism 9.5.0 (GraphPad Software Inc., San Diego, CA, USA). In all cases, the significance was accepted if the null hypothesis was rejected at the level p < 0.05.

[0076] Results

[0077] Cytocompatibility of HA A and HAB on SW982

[0078] The cytocompatibility of HAA and HAB against SW982 synoviocytes was evaluated by MTT assay, using a wide range of solution concentrations. Both compounds, HAA and HAB (in dilutions from 1 :250 to 1 :1000 v / v), did not show toxic effects on the cells (Figure 3). In consideration of the absence of toxicity even at the highest concentrations, all the subsequent experiments were conducted using the dilutions of 1 :250 and 1 :500 v / v.

[0079] Effect of HAA and HAB on IL-1 ^-induced ROS production in SW892 cells

[0080] The effect of HAA and HAB on ROS production in IL-1 [3-stimulated SW982 cells was assessed by cytofluorimetric analysis, using DCFDA as the appropriate fluorescent probe. After 24 hours of treatment, HAA, in the concentrations 1 :500- 1 :250 v / v, caused an inhibition of ROS production of 32.6% (p<0.001 ) and 46.1 % (p<0.001 ), respectively, while HAB, in the same concentrations, of 20.8% (p<0.01 ) and 31.9% (p<0.001 ). At both concentrations, HAA exerted a greater inhibitory effect on ROS production (p<0.05) (Figures 4 and 5).

[0081] Effect of HAA and HAB on IL-1 ^-induced NO release in SW892 cells

[0082] The effect of HAA and HAB on NO release, considered a reliable marker of inflammation, was evaluated by analyzing the amount of nitrite, the stable product thereof, in the culture medium. After 24 hours of treatment, HAA, in the concentrations 1 :500-1 :250 v / v, resulted in an inhibition of NO production of 46.6% (p<0.001 ) and 56.4% (p<0.001 ), respectively, while HAB, at the same concentrations, caused an inhibition of 22.1% (p<0.01 ) and 35.8% (p<0.001). Also for the inhibition of NO production, at both concentrations, the most marked effect was observed in the HAA treatments (p<0.001 ).

[0083] Effect of HAA and HAB on IL-1 ^-induced NO release in SW892 cells The effect of HAA and HAB on NO release, considered a reliable marker of inflammation, was evaluated by analyzing the amount of nitrite, the stable product thereof, in the culture medium. After 24 hours of treatment, HAA, in the concentrations 1 :500-1 :250 v / v, resulted in an inhibition of NO production of 46.6% (p<0.001 ) and 56.4% (p<0.001 ), respectively, while HAB, at the same concentrations, caused an inhibition of 22.1 % (p<0.01 ) and 35.8% (p<0.001 ). Also for the inhibition of NO production, at both concentrations, the most marked effect was observed in the HAA treatments (p<0.001 ).

[0084] Conclusions Overall, the data demonstrate that a pharmaceutical formulation containing the combination of HA (2.5% w / v; MW2.5 / 3 .5-106 Da) and a 3.4% w / v amino acid pool consisting of 1 % w / v L-hydroxyproline, 0.8% w / v L-lysine HCI, 0.8% w / v glycine and 0.8% w / v L-proline has a better antioxidant and anti-inflammatory effect with respect to a formulation containing HA (1 .33% w / v; MW 1 .64-106 Da) and a 2% amino acid pool containing 1 % w / v glycine, 0.75% w / v proline, 0.15% w / v leucine and 0.1 % w / v lysine HCI.

Claims

CLAIMS1 . A composition comprising high molecular weight hyaluronic acid (HA), L-proline, L-hydroxyproline, Glycine and L-lysine for use in the treatment of periodontal wounds.

2. The composition according to claim 1 wherein the wound is a post- surgical wound of the oral cavity.

3. The composition according to any one of claims 1 -2 in the form of a sterile isotonic gel.

4. The composition according to any one of claims 1 -3 wherein the composition is administered by local injection into the site of the wound to be treated.

5. The composition according to any one of claims 1 -4 wherein HA has a molecular weight of 2.000.000-3.000.000 Da.

6. The composition according to any one of claims 1 -5 wherein HA is in concentration 20-30 mg / ml.

7. The composition according to any one of claims 1 -6 wherein L-proline is in a concentration of 0.5-2.0 mg / ml, L-hydroxyproline is in a concentration of 0.5- 2.0 mg / ml, Glycine is in a concentration of 0.5-2.0 mg / ml and L-lysine is in a concentration of 0.5-2.0 mg / ml.

8. The composition according to any one of claims 1 -7 comprising a vehicle and pharmaceutically acceptable excipients.

9. The composition according to claim 8 wherein the vehicle is water for injections.

10. The composition according to any one of claims 1 -9 wherein the excipients are selected from the group consisting of monobasic sodium phosphate dihydrate and dibasic sodium phosphate dodecahydrate and mixtures thereof.

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