Compositions and kits for use in wound healing, tissue regeneration, and / or bone regeneration

Abstract

The present invention relates to a composition and a kit for use in the prevention or treatment of wounds and / or pathological conditions requiring wound healing, tissue regeneration and / or bone regeneration, comprising hyaluronic acid or a pharma- ceutically acceptable salt thereof and one or more inorganic peroxides (CaO2, ZnO2 and / or MgO2).The present invention is particularly suitable for the prevention or treatment of gingival diseases.

Description

[Technical field]

[0001] The present invention relates to compositions and kits for use in the prevention or treatment of wounds and / or pathological conditions requiring wound healing, tissue regeneration, and / or bone regeneration, comprising hyaluronic acid or a pharma- ceutically acceptable salt thereof and an inorganic peroxide selected from calcium peroxide (CaO2), magnesium peroxide (MgO2), and / or zinc peroxide (ZnO2).

[0002] More specifically, the present invention relates to compositions and kits comprising hyaluronic acid or a pharma- ceutically acceptable salt thereof and a peroxide selected from calcium peroxide (CaO2), magnesium peroxide (MgO2), and / or zinc peroxide (ZnO2) for use in the prevention or treatment of gum disease. [Background technology]

[0003] Oxygen is involved in various biological processes, such as cellular metabolism and signal transduction. Particularly during the wound healing process, the temporary oxidative stress caused by oxygen helps increase cellular activity, secrete wound growth factors, and promote neovascularization. At sites where a chronic inflammatory response can be detected, oxygen consumption increases and blood flow is stimulated. This change in local tissue pO2 is due in part to increased oxygen consumption, such as oxygen consumption by resident and infiltrating defense cells, in part to endothelial injury and vasoconstrictive microcirculation, as well as reduced oxygen availability by facultative anaerobic bacteria. These metabolic processes are found in all tissues. During the periodontal process, a hypoxic metabolic shift is almost always observed due to a disruption in the balance between oxygen supply and consumption in the tissues (Gordon, HA; Rovin, S.; Bruckner, G. Blood flow, collagen components of oral tissue and salivary kallikrein in young to senescent, germfree and conventional rats. A study on the etiologic factors of periodontal disease. Gerontology 1978, 24, 1-11).

[0004] "Burst release" in the physiological context of cell defense, also referred to as "respiratory burst", refers to a sudden increase in oxygen that generates oxygen radicals, for example for defense against microorganisms (Goncalves, RV; Costa, AMA; Grzeskowiak, L. Oxidative Stress and Tissue Repair: Mechanism, Biomarkers, and Therapeutics. Oxidative Medicine and Cellular Longevity 2021, 2021, 6204096). However, for tissue regeneration, burst release is undesirable. Indeed, a constant sustained release of oxygen is advantageous, providing sufficient oxygen to the local microenvironment without the generation of unhealthy radicals that lead to tissue damage.

[0005] In the field of tissue engineering, oxygen-releasing materials have been developed to address the problem of insufficient oxygen supply due to the lack of vascularization of large artificial tissue fragments (Wang, J.; Zhu, Y.; Bawa, HK; Ng, G.; Wu, Y.; Libera, M.; van der Mei, HC; Busscher, HJ; Yu, X. Oxygen-Generating Nanofiber Cell Scaffolds with Antimicrobial Properties. ACS Applied Materials & Interfaces 2011, 3, 67-73; Lv, X.; Li, Z.; Chen, S.; Xie, M.; Huang, J.; Peng, X.; Yang, R.; Wang, H.; Xu, Y.; Feng, C. Structural and functional evaluation of oxygenating keratin / silk fibroin scaffolds and initial assessment of their potential for urethral tissue engineering. Biomaterials 2016,84,99-110;Park,S.;Park,KMHyperbaric oxygen-generating hydrogels.Biomaterials 2018,182,234-244).

[0006] Calcium (Ca), zinc (Zn), and magnesium (Mg) are natural elements in many tissues and are involved in many physiological processes such as bone metabolism or wound healing (Chen, Z.; Zhang, W.; Wang, M.; Backman, LJ; Chen, J. Effects of Zinc, Magnesium, and Iron Ions on Bone Tissue Engineering. ACS Biomater Sci Eng 2022, 8, 2321-2335). It is known that zinc is released when zinc peroxide is decomposed and accelerates the wound healing process (Lin, PH; Sermersheim, M.; Li, H.; Lee, PHU; Steinberg, SM; Ma, J. Zinc in Wound Healing Modulation. Nutrients 2017, 10). Calcium and magnesium have been shown to increase bone regeneration (Zhou, H.; Liang, B.; Jiang, H.; Deng, Z.; Yu, K. Magnesium-based biomaterials as emerging agents for bone repair and regeneration: from mechanism to application. Journal of Magnesium and Alloys 2021, 9, 779-804; Aquino-Martinez, R.; Angelo, AP; Pujol, FV Calcium-containing scaffolds induce bone regeneration by regulating mesenchymal stem cell differentiation and migration. Stem Cell Research & Therapy 2017, 8, 265). Thus, these elements have great potential for tissue engineering and regeneration. However, the effect is dose-dependent, and higher doses may negatively affect tissue regeneration or even induce apoptosis.It is known that very large amounts of insoluble Ca salts are deposited in elastic tissues such as skin as dermal calcification, causing tissue damage (Pugashetti, R.; Shinkai, K.; Ruben, BS; Grossman, ME; Maldonado, J.; Fox, LPCalcium may preferentially deposit in areas of elastic tissue damage. J Am Acad Dermatol 2011,64,296-301). Zn has shown cytotoxic effects in vitro, so overdosing should also be avoided when using Zn for optimal tissue engineering effects. The cytotoxic effects of zinc are due to the fact that it is able to transport Ca into cells using channels that are also used for calcium. 2+ By regulating the ingress of Ca 2+ (Borovansky, J.; Riley, PAC Cytotoxicity of zinc in vitro. Chem Biol Interact 1989, 69, 279-291). 2+ and Zn 2+ Similarly, Mg 2+ are known to cause cytotoxic effects both in vivo and in vitro, and these effects are dose-dependent. (Sanders, T.; Liu, YM; Tchounwou, PBCytotoxic, genotoxic, and neurotoxic effects of Mg, Pb, and Fe on pheochromocytoma (PC-12) cells. Environ Toxicol 2015, 30, 1445-1458).

[0007] Gingivitis, periodontitis, and peri-implantitis are the most common biofilm-associated inflammatory gum diseases in humans or mammals. If untreated, these periodontal diseases result in the loss of teeth or implants. Periodontitis and peri-implantitis develop as a result of dysbiosis of bacterial populations in susceptible individuals and are associated with dysregulation of immune inflammatory responses. Thus, dysbiosis is involved in the development of microbial-associated pathologies and host-mediated destruction of connective tissue and alveolar bone (Darveau, RP Periodontitis: A polymicrobial disruption of host homeostasis. Nat. Rev. Microbiol. 2010, 8, 481-490.). Periodontitis and peri-implantitis also involve the destruction of connective tissue, including collagen, proteoglycans, and other extracellular matrix components. Gingivitis is an inflammatory response of gingival tissue to bacterial metabolic products and pathogenic toxins found in oral plaque.

[0008] The transition from periodontal health to disease is associated with a dramatic shift from a commensal microbial community composed primarily of facultative bacterial genera such as Actinomycetes and Streptococcus to a dysbiotic microbial makeup composed primarily of anaerobic genera from the phyla Firmicutes, Proteobacteria, Spirochaetes, Bacteroidetes, and Synergistes (Hajishengallis G. Periodontitis: from microbial immune subversion to systemic inflammation. Nat Rev Immunol. 2015;15(1):30-44. doi:10.1038 / nri3785).

[0009] The gram-negative anaerobic flora induces a series of immune responses that promote the destruction of tissues. This results in the degradation of soft tissues and alveolar bone, allowing the gram-negative anaerobic bacteria to grow under optimal conditions in the periodontal pocket. However, the pathogenesis of periodontitis and peri-implantitis diseases is based on a dysbiotic microbial structure, and although some well-characterized pathogens exhibit destructive virulence factors, the pathogenesis of periodontitis and peri-implantitis is essentially mediated by the host response (Van Dyke, TE; Serhan, CN Resolution of inflammation: A new paradigm for the pathogenesis of periodontal diseases. J. Dent. Res. 2003, 82, 82-90.0).

[0010] There are various approaches to the treatment of gingivitis, periodontitis or peri-implantitis, most of which involve the application of antibacterial compositions (Lasserre, JF; Toma, S.; Bourgeois, T.; El Khatmaoui, H.; Marichal, E.; Brecx, MC Influence of low direct electric currents and chlorhexidine upon human dental biofilms. Clin. Exp. Dent. Res. 2016, 2, 146-154.).

[0011] As one common approach, biodegradable chlorhexidine (CHX) chips are used as an adjunct to scaling and root planing (SRP) in the treatment of patients with moderate to severe periodontitis (Medaiah S, Srinivas M, Melath A, Girish S, Polepalle T, Dasari AB. Chlorhexidine chip in the treatment of chronic periodontitis - a clinical study. J Clin Diagn Res. 2014; 8(6): ZC22-ZC25. doi: 10.7860 / JCDR / 2014 / 8808.4477). However, studies have shown that using CHX as an adjunct after SRP does not have a significant positive effect on microbiota and tissue attachment (Grisi, DC, et al., 2002; Carvalho, J., et al., 2007), possibly because CHX is toxic to fibroblasts and osteoblasts and inhibits tissue regeneration.

[0012] Some other treatment products contain sodium perborate gel, which can be placed in the pocket after SRP to release oxygen. However, sodium perborate is known to be a corrosive and irritating substance, and, despite its antibacterial effect, is cytotoxic and therefore inhibits tissue regeneration. Another approach involves compositions containing hyaluronic acid, which have been described in relation to the treatment of gingival disease.

[0013] Treatment of gingivitis or periodontitis using a liquid composition comprising a non-biodegradable thermosensitive pharma- ceutical acceptable polyalkylene oxide block copolymer, hyaluronic acid or a salt thereof having a molecular weight of up to about 50,000 Daltons, and optionally a therapeutic agent is described in WO 2018 / 158764(A1). Hyaluronic acid and its potential role in periodontal healing, particularly in the wound healing process associated with periodontal disease, have been reported (MOSLEY R ET AL: "Hyaluronan and its potential role in periodontal healing", Dental update, update publ., London, GB, vol. 29, no. 3, 1 April 2002, pages 144-148). Hyaluronic acid also has benefits in periodontal therapy (DAHIYA PARVEEN ET AL: "Hyaluronic Acid: A Boon in periodontal Therapy", NORTH AMERICAN JOURNAL OF MEDICALK SCIENCES, vol. 5, no. 5, 1 May 2013, pages 309-315).

[0014] German Utility Model No. 20201602375 discloses hyaluronic acid in combination with the disinfectant octenidine dihydrochloride, which is suitable for the treatment and prevention of several diseases, such as stomatological complications, gum damage after dental scaling, oral mucosal or tongue wounds, or other types of wounds and damage in the oral cavity.

[0015] EP 1 638 582 discloses the use of hyaluronic acid in the preparation of a composition for the treatment of recurrent oral aphthae.

[0016] EP 3056195 A1 discloses compositions of hyaluronic acid and beta-glucan and their use for the treatment of, inter alia, periodontitis and gingivitis.

[0017] WO 2018 / 158764 discloses certain compositions of polyalkylene oxide block copolymers and hyaluronic acid and their use for the treatment of, inter alia, periodontitis, gingivitis, and peri-implantitis.

[0018] Furthermore, a root filler that uses CaO2 as an antimicrobial agent is known (Malyk Yuriy (2005), "In vitro studies on the use of calcium peroxide (CaO2)-containing sealer materials in endodontic therapy", doctoral thesis, Poliklinik fur Zahnerhaltung und Parodontologie der Ludwig-Maximilians-Universitat Munchen).

[0019] WO 2018 / 026204 discloses an in-situ crosslinked polymer hydrogel containing calcium peroxide that releases high concentrations of oxygen in a sustained release form. It also describes an implant material for tissue regeneration and filling that contains such an in-situ crosslinked sustained release oxygen releasing hydrogel.

[0020] CN112826761 and CN112870109 disclose methods for preparing dentifrices with antibacterial effects containing calcium peroxide.Canadian Patent Invention No. 1334282 describes periodontal compositions and methods containing peroxide and bicarbonate.

[0021] US Patent No. 5,616,313 describes a method for minimizing damage to gums and periodontal tissues. The method includes delivering a first composition containing a zinc salt to a container, delivering a second composition containing a bicarbonate salt to the container, and transferring the combination to the gums and periodontal tissues in the mouth within 5 minutes after delivery to the container. However, the released oxygen is only available once (burst release) at very high concentrations for a very short time. High concentrations of peroxide are required to achieve antibacterial effects.

[0022] Various compositions are known for the treatment of various gingival diseases in humans or animals, but they do not combine a sustained antibacterial effect while at the same time supporting the regeneration of gingival tissue, which reduces the efficiency of the treatment of wounds or inflammatory lesions, in particular gingivitis, periodontitis and peri-implantitis. [Prior art documents] [Patent documents]

[0023] [Patent Document 1] German Utility Model No. 20201602375 [Patent Document 2] European Patent No. 1638582 [Patent Document 3] European Patent Application Publication No. 3056195 [Patent Document 4] International Publication No. 2018 / 158764 [Patent Document 5] International Publication No. 2018 / 026204 [Patent Document 6] China Patent Application Publication No. 112826761 [Patent Document 7] China Patent Application Publication No. 112870109 [Patent Document 8] Canadian Patent No. 1334282 [Patent Document 9] U.S. Pat. No. 5,616,313 [Non-patent literature]

[0024] [Non-Patent Document 1] Gordon,HA;Rovin,S.;Bruckner,G.Blood flow,collagen components of oral tissue and salivary kallikrein in young to senescent,germfree and conventional rats.A study on the etiologic factors of periodontal disease.Gerontology 1978,24,1-11 [Non-Patent Document 2] Goncalves,RV;Costa,AMA;Grzeskowiak,L.Oxidative Stress and Tissue Repair:Mechanism,Biomarkers,and Therapeutics.Oxidative Medicine and Cellular Longevity 2021,2021,6204096 [Non-Patent Document 3] Wang, J.; Zhu, Y.; Bawa, H. K.; Ng, G.; Wu, Y.; Libera, M.; van der Mei, H. C.; Busscher, H. J.; Yu, X. Oxygen-Generating Nanofiber Cell Scaffolds with Antimicrobial Properties. ACS Applied Materials & Interfaces 2011, 3, 67 - 73; Lv, X.; Li, Z.; Chen, S.; Xie, M.; Huang, J.; Peng, X.; Yang, R.; Wang, H.; Xu, Y.; Feng, C. Structural and functional evaluation of oxygenating keratin / silk fibroin scaffold and initial assessment of their potential for urethral tissue engineering. Biomaterials 2016, 84, 99 - 110; Park, S.; Park, K. M. H. Hyperbaric oxygen-generating hydrogels. Biomaterials 2018, 182, 234 - 244 Non-Patent Document 4 Chen, Z.; Zhang, W.; Wang, M.; Backman, L. J.; Chen, J. Effects of Zinc, Magnesium, and Iron Ions on Bone Tissue Engineering. ACS Biomater Sci Eng 2022, 8, 2321 - 2335 Non-Patent Document 5 Lin, P. H.; Sermersheim, M.; Li, H.; Lee, P. H. U.; Steinberg, S. M.; Ma, J. Zinc in Wound Healing Modulation. Nutrients 2017, 10 Non-Patent Document 6 Zhou,H.;Liang,B.;Jiang,H.;Deng,Z.;Yu,K.Magnesium-based biomaterials as emerging agents for bone repair and regeneration:from mechanism to application.Journal of Magnesium and Alloys 2021,9,779-804;Aquino-Martinez,R.;Angelo,AP;Pujol,FVCalcium-containing scaffolds induce bone regeneration by regulating mesenchymal stem cell differentiation and migration.Stem Cell Research&Therapy 2017,8,265

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[0025] Against this background, it is an object of the present invention to provide a medicamentously active composition for the prevention or treatment of wounds and inflammatory diseases, which increases the amount of available oxygen and releases oxygen over a long period of time.

[0026] This object is solved by the subject matter of claim 1, in particular by a composition comprising hyaluronic acid or a pharma- ceutically acceptable salt thereof and one or more inorganic peroxides. [Means for solving the problem]

[0027] The present invention is based on the surprising discovery that when at least one inorganic peroxide is combined with hyaluronic acid in a composition, the amount of available oxygen is increased and the oxygen is released in a delayed manner.More specifically, it has been found that by combining at least one inorganic or metal peroxide with hyaluronic acid, the amount of available oxygen is increased by up to 5 times depending on the condition.In a preferred embodiment, the composition of the present invention comprises at least one inorganic peroxide selected from the group consisting of calcium oxide (CaO2), zinc peroxide (ZnO2) and / or magnesium peroxide (MgO2), and hyaluronic acid or its pharma-ceutically acceptable salt.

[0028] It has also been found that by adding MgO2 and / or ZnO2 the amount of available oxygen can be further increased or altered and therefore tailored to the individual application and needs of the tissue. It is particularly surprising and important that oxygen is released over an extended period of time.

[0029] The continuous and steady release of antibacterial oxygen over an extended period of time makes the treatment of wounds or inflammatory conditions more efficient and less dangerous to the subject by avoiding high concentrations of peroxides which would cause localized oxygen burst releases and thus generate large amounts of antibacterial oxygen radicals which damage local tissue.

[0030] As shown herein, the composition of the present invention is suitable for tissue regeneration or bone regeneration, because it has been surprisingly found that low concentrations of metal peroxides or inorganic peroxides, when combined with hyaluronic acid or its pharma- ceutically acceptable salts, show unexpected properties.The delayed release of oxygen from metal peroxides such as CaO2, ZnO2 and / or MgO2 is due to the matrix formed by the hyaluronic acid composition.In addition, the delayed release of oxygen from inorganic peroxides, especially calcium peroxide, bound to hyaluronic acid can avoid burst release (a single high concentration of oxygen) and harmful concentrations of oxygen to surrounding wound tissue.

[0031] In a preferred embodiment, the composition of the present invention comprises hyaluronic acid or a pharma- ceutically acceptable salt thereof, and one or more inorganic peroxides for use in the treatment of wounds or inflammatory conditions. Examples of inflammatory conditions to be treated include, but are not limited to, conditions or pathologies requiring wound healing, tissue regeneration, and / or bone regeneration. Preferably, the inflammatory condition is a gum disease. More preferably, the gum disease is selected from the group consisting of gingivitis, periodontitis, or peri-implantitis. In a preferred embodiment, the composition of the present invention comprises hyaluronic acid or a pharma- ceutically acceptable salt thereof, and one or more inorganic peroxides selected from CaO2, ZnO2, and / or MgO2 for use in the prevention or treatment of gingivitis, periodontitis, or peri-implantitis. In a further preferred embodiment, the composition of the present invention comprises at least calcium peroxide (CaO2) and hyaluronic acid as active compounds, and is suitable for use in wound healing, tissue regeneration, and / or bone regeneration. In a preferred embodiment, such a composition further comprises ZnO2 and MgO2 to increase the amount of available oxygen.

[0032] In an alternative embodiment, the composition comprises two or more different inorganic peroxides, and in yet another embodiment, the composition comprises three different inorganic peroxides. Thus, in addition to oxygen release, the ionic end product of the inorganic peroxides can also be optimized for tissue and specifically tailored for the indication.

[0033] In a first variant, the composition comprises CaO2 and hyaluronic acid or a pharma- ceutically acceptable salt thereof. In a second variant, the composition comprises ZnO2 and hyaluronic acid or a pharma- ceutically acceptable salt thereof. In a third variant, the composition comprises MgO2 and hyaluronic acid or a pharma- ceutically acceptable salt thereof. In a fourth variant, the composition comprises CaO2 and hyaluronic acid or a pharma- ceutically acceptable salt thereof, and at least one further peroxide, such as, but not limited to, ZnO2 or MgO2. In a fifth variant, the composition consists of CaO2 and hyaluronic acid or a pharma- ceutically acceptable salt thereof, and ZnO2. In a sixth variant, the composition consists of CaO2 and hyaluronic acid or a pharma- ceutically acceptable salt thereof, and MgO2. In a seventh variant, the composition comprises MgO2 and hyaluronic acid or a pharma- ceutically acceptable salt thereof, and at least one further peroxide, such as, but not limited to, CaO2 or ZnO2. In an eighth variant, the composition consists of MgO2 and hyaluronic acid or a pharma- ceutically acceptable salt thereof, and CaO2. In a ninth variant, the composition consists of MgO2 and hyaluronic acid or a pharma- ceutically acceptable salt thereof, and ZnO2. In a further variant, the composition comprises ZnO2 and hyaluronic acid or a pharma- ceutically acceptable salt thereof. In a further variant, the composition comprises ZnO2 and hyaluronic acid or a pharma- ceutically acceptable salt thereof, and MgO2 and / or CaO2. In a preferred embodiment, the composition consists of a combination of two inorganic peroxides selected from the group consisting of CaO2, ZnO2, and MgO2, and hyaluronic acid or a pharma- ceutically acceptable salt thereof. Preferably, such a formulation comprises two inorganic peroxides, CaO2 and ZnO2, or alternatively CaO2 and MgO2, and hyaluronic acid or a pharma- ceutically acceptable salt thereof. A preferred alternative formulation includes three inorganic peroxides, CaO2, ZnO2 and MgO2, and hyaluronic acid or a pharma- ceutically acceptable salt thereof. The combination of peroxides can be adjusted depending on the condition being treated. Combining different peroxides (CaO2, ZnO2 and / or MgO2) increases the amount of available oxygen and increases the concentration of the final product (CaO2, ZnO2 and / or MgO2). 2+ , Zn2+ or Mg 2+ ) becomes available to the tissue being treated. Most importantly, burst release of toxic amounts of peroxides and oxygen is avoided.

[0034] In a further variation, the composition is a pharmaceutical composition comprising at least a diluent, solvent or carrier, at least one inorganic peroxide and hyaluronic acid or a pharma- ceutically acceptable salt thereof.

[0035] In one aspect, the compositions of the present invention allow for the treatment of various types of wounds, including venous leg ulcers, diabetic foot ulcers, pressure sores, arterial ulcers, burns, and chronic wounds such as non-healing surgical wounds. Additionally, the compositions of the present invention are useful for treating abrasions, lacerations, minor cuts, scalding wounds, burns, and other partial thickness wounds.

[0036] It is desirable to treat wounds contaminated with harmful pathogenic microorganisms. For example, the periodontal pocket is an ideal environment for anaerobic pathogens due to its anaerobic condition. As a result, inflamed tissues produce increased gingival crevicular fluid, which is the main source of nutrition for subgingival microorganisms.

[0037] One of the advantages of the composition of the present invention is that the effective period of oxygen to destroy biofilms and bacteria in diseased tissues, such as periodontal pockets, is significantly extended, resulting in more effective treatment of inflammatory conditions, such as gum diseases, such as gingivitis, periodontitis, or peri-implantitis.As a result, tissue destruction can be avoided, or at least delayed or stopped. At the same time, tissue regeneration is stimulated by hyaluronic acid and is not adversely affected by the action of inorganic peroxides.The composition of the present invention is used for the treatment of diseases characterized by tissue destruction or bone destruction, depending on the condition.The composition of the present invention is therefore suitable for use in tissue regeneration, tissue engineering, or bone regeneration.

[0038] Combining calcium peroxide with hyaluronic acid would not be expected to slow down oxygen release. In fact, hyaluronic acid only forms a loose mesh matrix, which increases the likelihood of oxygen release in the same way as in water. It is therefore surprising that combining hyaluronic acid with an inorganic peroxide, such as calcium peroxide, results in a slowdown in oxygen release without adversely affecting the surrounding tissue, while at the same time maintaining the antimicrobial properties of the metal peroxide. As exemplified herein, the growth or biofilm formation of periodontal pathogenic bacteria and the immune inflammatory response can be effectively inhibited. Most importantly, no toxic effects were observed on fibroblasts or osteoblasts.

[0039] The inclusion of different peroxide combinations in hyaluronic acid allows the oxygen delivery to be tailored to the needs of different targets (e.g. bone, skin, cartilage). By using different peroxides, overdosing of one ion can be avoided and the biomaterial can be tailored to the needs of different targets for different ions.

[0040] The present invention can address the underlying causes of periodontitis or peri-implantitis by (a) increasing antimicrobial efficacy against obligate anaerobes, thereby restoring the commensal flora, and (b) accelerating tissue regeneration and preventing bacterial recolonization in the periodontal pocket.

[0041] The composition of the present invention is preferably formulated as a pharmaceutical composition.Preferably, the pharmaceutical composition comprises one or more pharma- ceutically acceptable carriers, solvents, diluents or excipients, and is preferably prepared as a formulation for topical administration in the oral cavity.

[0042] Preferably, the composition of the present invention is formulated in the form of a solution, suspension, emulsion, cream, or gel. Such formulations include a therapeutically effective concentration of hyaluronic acid or its pharma- ceutically acceptable salt, and a therapeutically effective concentration of one or more inorganic peroxides selected from CaO2, ZnO2, or MgO2. In a preferred embodiment, the composition of the present invention is provided as a gel, ready to be injected into the space between the teeth and gums by a delivery means such as a syringe. The combination of the present invention allows the gel to have a bactericidal effect against periodontal pathogens, prevent new colonization after mechanical cleaning, and accelerate tissue regeneration (e.g., bone, gums). In addition, the gel temporarily seals the gap between the teeth and gums where pathogens may grow.

[0043] The compositions of the present invention preferably include sterile aqueous solutions or dispersions, and sterile powders for preparing sterile injectable solutions or dispersions. Injectable solutions must be sterile and fluid to the extent that they can be injected by any suitable delivery means for oral cavity, such as a syringe. Injectable solutions preferably include a carrier, which can be any suitable solvent or dispersion medium, including, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, or vegetable oils. The effective dosage of the active ingredient used can vary depending on the specific compound used, the mode of administration, and the severity of the condition being treated. However, generally, satisfactory results can be obtained when the compounds of the present invention are administered in a single dose of up to 3mL per dose.

[0044] A preferred dosage form suitable for administration to the oral cavity comprises about 15 mg / mL to 35 mg / mL hyaluronic acid and about 100 mg / L to 500 mg / L calcium peroxide mixed with a pharma- ceutically acceptable carrier. The dosage regimen may be adjusted to provide an optimal therapeutic response. The combination of the present invention may be administered to the oral cavity of a human or animal suffering from gum disease. Typically, veterinary dosage corresponds to human dosage, with dosage being proportional to the animal's body weight compared to adult humans.

[0045] For example, when administered to the oral cavity, the pharmaceutical composition of the present invention may contain a pharma- ceutically acceptable carrier, such as a solvent or diluent, that does not adversely affect the activity of hyaluronic acid and calcium peroxide.Such carriers include, but are not limited to, agents, stabilizers, absorption retarders, disintegrants, emulsifiers, antioxidants, binders, lubricants, moisture absorbents, etc.For example, the solvent may be selected from the group consisting of water and sucrose solution, the diluent may be selected from the group consisting of lactose, starch, and microcrystalline cellulose, the absorption retarder may be selected from the group consisting of chitosan and glycosyl glycan, and the lubricant may be selected from the group consisting of magnesium stearate, talc, sodium stearyl fumarate, or a combination thereof.

[0046] In preferred embodiments of the present invention, the compositions of the present invention preferably further comprise a carrier, a thickening agent, and one or more additional antimicrobial agents. According to various aspects of these embodiments, the compositions may include moisturizers, migration enhancing agents, oils, and the like.

[0047] Preferably, the pharmaceutically acceptable salt of hyaluronic acid is a hyaluronate, including but not limited to an alkali metal salt or alkaline earth metal salt of hyaluronic acid, such as sodium hyaluronate, potassium hyaluronate, and calcium hyaluronate.In one embodiment, the pharmaceutically acceptable salt of hyaluronic acid is sodium hyaluronate.In an alternative embodiment, the pharmaceutically acceptable salt of hyaluronic acid is potassium hyaluronate or calcium hyaluronate.The composition of the present invention may include a combination of sodium hyaluronate, potassium hyaluronate, and / or calcium hyaluronate.

[0048] Preferably, in the composition of the present invention, hyaluronic acid or its pharma- ceutically active salt is provided in the form of a matrix capable of binding calcium peroxide.The hyaluronic acid matrix according to the present invention promotes tissue regeneration and fibroblast survival.Furthermore, the hyaluronic acid matrix prevents calcium peroxide from dissolving, reducing the risk of further attachment of new potentially pathogenic bacteria.When calcium peroxide is bound to the hyaluronic acid matrix, oxygen is uniformly released from the calcium peroxide component in the composition, inhibiting the growth of periodontal pathogens over a long period of time. Examples of such periodontal pathogens include obligate anaerobic bacteria, particularly periodontal pathogens, including, but not limited to, Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans or gram-positive aerobic bacteria comprised of Streptococcus spp., e.g., Streptococcus oralis and Actinomyces spp.

[0049] The hyaluronic acid or its salt used in the composition of the present invention may be either high molecular weight hyaluronic acid or low molecular weight hyaluronic acid or their salts. In the context of the present invention, high molecular weight hyaluronic acid is preferred. The high molecular weight hyaluronic acid or its salt preferably has an average molecular weight of about 300,000 to about 6 million Daltons (Da). Most preferably, it has an average molecular weight of about 500,000 to about 3 million Daltons. The low molecular weight hyaluronic acid or its salt contained in the composition of the present invention has a molecular weight of up to about 50,000 Da and is optionally hydrolyzed or partially hydrolyzed. In certain embodiments, the low molecular weight hyaluronic acid has a molecular weight of about 3,000 to about 50,000 Da, about 4,000 to about 20,000 Da, or about 6,000 to about 10,000 Da.

[0050] In a preferred embodiment, the hyaluronic acid or its pharma- ceutically acceptable salt contained in the composition of the present invention is present in an amount of about 1 mg / mL to about 100 mg / mL, preferably about 5 mg / mL to about 80 mg / mL, about 5 mg / mL to about 50 mg / mL, or about 10 mg / mL to about 40 mg / mL, more preferably about 10 mg / mL to about 30 mg / mL, most preferably 20 mg / mL to about 25 mg / mL, for example, 21 mg / mL, 22 mg / mL, 23 mg / mL, 24 mg / mL, or 25 mg / mL.Preferably, the hyaluronic acid is present in the composition at the concentration shown as sodium hyaluronate.

[0051] The inorganic peroxide component of the composition of the present invention is included in an amount sufficient to uniformly release oxygen when the composition is applied to the oral cavity. Typically, the peroxide component can be used in the composition of the present invention in an amount such that at least about 1 mg / L of the composition contains an inorganic peroxide selected from CaO2, ZnO2, or MgO2.

[0052] In a preferred embodiment, calcium peroxide is present in the composition at a concentration of about 1 mg / L to 5000 mg / L (1.4×10 -5 In a more preferred embodiment, calcium peroxide is present in the composition of the present invention in an amount of about 10 mg / L to 1000 mg / L (1.4×10 -4 ~0.014 mol / L), preferably about 10 mg / L to about 800 mg / L (respectively 1.4 × 10 -4 ~0.011 mol / L), about 100 mg / L to about 800 mg / L (1.4 × 10 -3 ~0.011 mol / L), or about 100 mg / L to about 500 mg / L (1.4 × 10 -3 and more preferably from about 200 mg / L to about 500 mg / L (respectively 0.0028 to 0.0069 mol / L), for example, 200 mg / L, 250 mg / L, 300 mg / L, 400 mg / L or 500 mg / L.

[0053] The concentration of zinc peroxide is preferably 1 mg / L to 10,000 mg / L (1.02 × 10 -5 ~0.103 mol / L).

[0054] The concentration of magnesium peroxide is preferably 1 mg / L to 15,000 mg / L (1.77 × 10 -5 ~0.266 mol / L).

[0055] Further aspects of the present invention relate to additional components preferably present in the composition to increase the efficiency or therapeutic effect of the composition. In the first aspect of the present invention, the composition comprises one or more additives including, but not limited to, plant extracts, calcium hydroxyapatite, and / or collagen. For example, the composition of the present invention preferably further comprises one or more plant extract preparations, preferably from plant species selected from the group consisting of eucalyptus, rhubarb root, clove oil, aloe vera, and / or cumin. The presence of the plant extract in the composition of the present invention inhibits bacterial growth, making it a viable alternative to antibiotics. Thus, the plant extract, when present in the composition of the present invention, improves the antibacterial effect of CaO2, ZnO2 or MgO2.

[0056] In a further preferred embodiment of the present invention, the additive in the composition of the present invention is calcium hydroxyapatite, which has been found to induce the formation and storage of natural collagen. Additionally, calcium hydroxyapatite promotes the remodeling of teeth and bones.

[0057] In a further preferred embodiment of the invention, the additive in the composition of the invention is collagen, which, when present in the composition of the invention, supports the attachment of gums to teeth.

[0058] When the composition includes one or more additives selected from the group consisting of a plant extract, calcium hydroxyapatite, or collagen, the plant extract is present in the composition of the present invention in an amount of about 0.1% to about 10% by weight, more preferably in an amount of about 0.3% to about 3% by weight, and most preferably at a concentration of 0.5% by weight.

[0059] In a further preferred embodiment, calcium hydroxyapatite is present in the compositions of the invention in an amount of about 1% to about 30% by weight, more preferably in an amount of about 5% to about 20% by weight, and most preferably at a concentration of 10% by weight, with a calcium / phosphate ratio of 0.8 to 20.

[0060] In a further preferred embodiment of the present invention, collagen is present in the composition of the present invention in an amount of about 0.9% to about 25% by weight, more preferably in an amount of about 5% to about 20% by weight, and most preferably at a concentration of 15% by weight.

[0061] The present invention also includes modified or variant derivatives of the compounds described herein, in particular chemically or biologically modified forms of hyaluronic acid or its salts, calcium peroxide, plant extracts, calcium hydroxyapatite or collagen.

[0062] The pH value of the composition may vary depending on the particular application. According to various embodiments of the present invention, the pH value is about 4 to about 9, preferably, the pH is about 6 to 8.5, and most preferably, the pH is 6.5 to 8.

[0063] The composition of the present invention exerts a bactericidal effect on periodontal pathogens, not only temporarily prevents their recolonization, but also actively supports tissue regeneration.In particular, the hyaluronic acid matrix can promote oxygen release for a long period of time, and at the same time has a positive effect on tissue regeneration.Therefore, the composition of the present invention results in an increase in the success rate of treatment of gingival disease and a decrease in the possibility of retreatment.It is clear to those skilled in the art that the composition of the present invention, due to its general properties, can be suitable for the treatment of any gingival disease, but is particularly useful for the treatment or prevention of gingivitis, periodontitis, or peri-implantitis.

[0064] In a preferred embodiment of the invention, the amount of available oxygen can be increased by applying a special process in the preparation of the formulation. The process to obtain the combination of the invention is: 1. Dissolving a preservative in a liquid matrix; 2. Dissolving any soluble ingredients; 3. Suspending the insoluble components; and 4. Incorporating hyaluronic acid while stirring vigorously.

[0065] One example of a preservative that may be used in the context of the present invention is methyl 4-hydroxybenzoate (MHB).

[0066] The preferred liquid matrix is ​​PBS buffer or other suitable solution. The soluble components constitute additives such as plant extracts. Preferred insoluble components include, but are not limited to, peroxide, calcium hydroxyapatite, or other types of plant extracts. By applying this procedure, the amount of available oxygen can be significantly increased compared to when the compounds are simply mixed and then dissolved and suspended.

[0067] The present invention further comprises a kit for targeted delivery of the composition of the present invention. In a preferred embodiment, the present invention relates to a kit for wound healing, tissue regeneration and / or bone regeneration comprising a composition according to the present invention and a delivery means for applying the composition to a wound, tissue or bone.

[0068] In a preferred embodiment, the kit of the present invention is configured to deliver a composition to a gingival pocket, a periodontal pocket, or a pocket caused by peri-implantitis, the kit comprising a composition according to the present invention and a delivery means for locally applying the composition to the gingival pocket, the periodontal pocket, or a pocket caused by peri-implantitis.

[0069] In an alternative preferred embodiment, the kit of the invention is configured to apply the composition onto or into the oral mucosa, the kit comprising a composition according to the invention and a delivery means for topically applying the composition onto or into the oral mucosa.

[0070] In a preferred embodiment, the composition is provided in the kit of the present invention in the form of a gel that is preferably applied in the oral cavity with a syringe. Preferably, the gel is provided in the gingival pocket, the periodontal pocket, or the pocket formed as a result of peri-implantitis. In a preferred embodiment, the delivery means of the composition in the kit is a syringe. In an alternative embodiment, the composition of the present invention is applied on or in the oral mucosa. The composition in the kit of the present invention allows for a sustained release dosage form that can be administered in the oral cavity of animals or humans.

[0071] Taking a combination of peroxides together can optimally provide oxygen and ions to various tissues without cytotoxicity. As shown herein, the compositions and kits of the present invention allow for the delayed release of oxygen from antimicrobial calcium peroxide, thereby successfully treating infectious or inflammatory diseases such as gum diseases, including but not limited to gingivitis, periodontitis, and peri-implantitis, without adversely affecting surrounding tissues such as teeth or implant tissues.

[0072] The present invention is described in further detail in the following examples, which are intended to be of an illustrative nature only. The examples and embodiments described herein do not in any way limit the scope of the present invention. The present invention also includes any variations, modifications, and / or combinations of the components described herein. [Brief description of the drawings]

[0073] [Figure 1] Shows the cytotoxicity of hyaluronic acid, calcium peroxide, and their combination in human primary fibroblasts in vitro. [Diagram 2] In vitro cytotoxicity of hyaluronic acid, calcium peroxide, and their combination in human primary osteoblasts. [Diagram 3] In vitro cytotoxicity of hyaluronic acid, calcium peroxide, and their combination in primary human umbilical vein endothelial cells (HUVEC). [Figure 4] The biocompatibility of hyaluronic acid, calcium peroxide, and their combination in the chorioallantoic membrane assay is shown (Figure 4A: vascularized surface of the sponge; Figure 4B: degradation rate of the sponge). [Diagram 5] The effect of calcium peroxide on planktonic bacterial colonies of Streptococcus oralis (S. oralis) and Porphyromonas gingivalis (P. gingivalis) is shown. [Figure 6]1 shows the effect of hyaluronic acid and calcium peroxide on mature P. gingivalis biofilms. [Figure 7] 1 shows the effect of hyaluronic acid and calcium peroxide on mature S. oralis biofilms. [Figure 8] 1 shows sustained release of oxygen from a combination of hyaluronic acid and calcium peroxide compared to calcium peroxide alone and hyaluronic acid alone. [Figure 9] Figure 1 shows the oxygen content of formulations with and without hyaluronic acid. The formulations contain a combination of hyaluronic acid with calcium peroxide, zinc peroxide, and magnesium peroxide. [Figure 10] 1 shows the effect of the manufacturing process for a formulation containing hyaluronic acid and calcium peroxide when the ingredients are simply mixed but not processed correctly. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0074] Formulations suitable for application in the buccal cavity: The following non-limiting examples describe formulations suitable for treating multiple gum diseases, including but not limited to gingivitis, periodontitis, peri-implantitis, or other gum disease-related inflammatory diseases. The formulations may further include any suitable carrier or excipient known to those skilled in the art. The dosage regimen may be adjusted to provide optimal therapeutic response. All formulations exhibit the desired therapeutic effect.

[0075] Example 1: The formulation contains 256 mg / L calcium peroxide suspended in phosphate-buffered saline (PBS) to stabilize the pH in the range of 7 to 7.5. Then, 23 mg / mL hyaluronic acid or its salts are added with vigorous stirring until a homogenous gel is produced.

[0076] Example 2: The formulation contains 256 mg / L calcium peroxide suspended in phosphate buffered saline (PBS) to stabilize the pH in the range of 7 to 7.5. Then, 23 mg / mL hyaluronic acid or its salts and 4 mg / L rhubarb root extract are added with vigorous stirring until a homogenous gel is produced.

[0077] Example 3: The formulation contains 256 mg / L calcium peroxide suspended in phosphate-buffered saline (PBS) to stabilize the pH in the range of 7 to 7.5. Then, 23 mg / mL hyaluronic acid or its salts and 100 mg / L calcium hydroxyapatite are added with vigorous stirring until a homogenous gel is produced.

[0078] Example 4: The formulation contains 500 mg / L calcium peroxide suspended in phosphate-buffered saline (PBS) to stabilize the pH in the range of 7 to 7.5. Then, 30 mg / mL hyaluronic acid or its salts and 100 mg / L calcium hydroxyapatite are added with vigorous stirring until a homogenous gel is produced.

[0079] Example 5: The formulation contains 256 mg / L calcium peroxide suspended in phosphate-buffered saline (PBS) to stabilize the pH in the range of 7 to 7.5. Then, 23 mg / mL hyaluronic acid or its salts and 10 mg / L collagen are added with vigorous stirring until a homogenous gel is produced.

[0080] Example 6: The formulation contains 115 mg of sodium hyaluronate and 50 mg of CaO2 diluted in 5 mL of H2O.

[0081] Example 7: The formulation contains 250 mg of sodium hyaluronate, 50 mg of CaO2, 250 mg of MgO2, and 200 mg of ZnO2 diluted in 5 mL of PBS buffer.

[0082] Tissue compatibility testing of substances used in the present invention To investigate the cytotoxicity of calcium peroxide, hyaluronic acid, and their combination on cells, these substances were administered in vitro to human primary fibroblasts, osteoblasts, and human umbilical vein endothelial cells (HUVECs) and compared with untreated cells and control dead cells. For this purpose, cytotoxicity tests were performed by quantitative and calorimetric measurement of cell viability according to ISO10993-5. In addition, biocompatibility tests were performed with the chorioallantoic membrane assay (CAM assay) in fertilized chicken eggs. All tests were repeated at least three times independently. In all tests, 30 mg / mL of hyaluronic acid and 0.256 mg / mL of CaO2 were used. According to ISO10993-5, cytotoxicity is defined by a reduction in the number of viable cells by more than 30% due to the substance. These results are shown in Figures 1-4.

[0083] Figure 1 shows the cell viability of human primary fibroblasts obtained from human oral mucosa. Fibroblasts were treated with hyaluronic acid or a combination of calcium peroxide and hyaluronic acid. Both treatments showed a decrease in cell viability compared to untreated fibroblasts (=100% cell viability). This indicates that hyaluronic acid alone or in combination with calcium peroxide is not cytotoxic. In contrast, fibroblasts treated with 0.256 mg / mL calcium peroxide showed a decrease in cell viability of more than 30%, indicating cytotoxicity of calcium peroxide alone.

[0084] Figure 2 shows the cell viability of human primary osteoblasts. Osteoblasts were treated with hyaluronic acid, calcium peroxide, or a combination thereof. Osteoblasts showed little loss of viability after treatment compared to untreated osteoblasts (=100% cell viability). This indicates that both of these substances, alone or in combination, have no cytotoxic effect on osteoblasts.

[0085] Figure 3 shows the cell viability of human primary umbilical vein endothelial cells (HUVEC). Both of these substances, even in combination, had no cytotoxic effect on HUVEC. Compared to treating with hyaluronic acid as a single substance, the combination of both substances slightly increased the viability of HUVEC.

[0086] To investigate the biocompatibility of hyaluronic acid, calcium peroxide, and their combination, a CAM assay was performed (Figure 4). The data in Figures 4A and 4B show that none of the test substances had a cytotoxic effect on the CAM. The degradation of sponges with the different substances was not different from that of the empty sponge, and no adverse tissue reaction to the substances was observed on the CAM. Angiogenesis, which can be measured by the difference in the area of ​​the sponge covered with blood vessels, was not significantly different in CAM tissues treated with hyaluronic acid, calcium peroxide, or their combination, compared to untreated CAM tissue.

[0087] In summary, the combination according to the invention was shown to be non-cytotoxic in all cell types investigated and was proven to be biocompatible in the CAM assay.

[0088] Inhibitory efficacy of calcium peroxide against planktonic bacteria. Selected concentrations of calcium peroxide were used to demonstrate the inhibitory efficacy of calcium peroxide against planktonic bacteria. According to Figure 5, different concentrations of calcium peroxide were applied to mature biofilms in combination with hyaluronic acid. Calcium peroxide was added to the planktonic bacterial strains S. oralis and P. gingivalis at concentrations from 0 mg / L (control) to 500 mg / L, and hyaluronic acid at a concentration of 11.550 mg / L. In each case, the optical density at 600 nm, the parameters of growth of the bacterial culture, and the relative metabolic activity are shown. At concentrations above 125 mg / L, the optical density decreased, indicating a significant decrease in the growth of P. gingivalis, a bacterium that causes periodontitis.

[0089] The upper left part of Figure 5 shows that the optical density of S. oralis, a planktonic bacterium of the oral flora, was significantly decreased at calcium peroxide concentrations of 250 mg / L and 500 mg / L compared to the control group. The lower left part shows that the relative metabolic activity of S. oralis was unchanged compared to the control. The upper right part of Figure 5 shows that the optical density of P. gingivalis, a bacterium that causes periodontitis, was significantly decreased at calcium peroxide concentrations of 125 mg / L, 250 mg / L, and 500 mg / L compared to the control group. The lower right part shows that the relative metabolic activity of P. gingivalis was significantly decreased at calcium peroxide concentrations of 125 mg / L, 250 mg / L, and 500 mg / L compared to the control group.

[0090] These results show that a calcium peroxide concentration of 125 mg / L has a statistically significant inhibitory effect on the periodontitis-causing bacterium P. gingivalis, but not on S. oralis, a gram-positive facultative anaerobic bacterium of the family Streptococcus found in physiological oral flora. At concentrations of about 500 mg / L, increasing concentrations of calcium peroxide induce a stress response in S. oralis, whereas a stress response is already induced in the periodontal pathogen P. gingivalis at significantly lower concentrations. However, at concentrations above 125 mg / L, oxygen showed a toxic effect on P. gingivalis. On the other hand, the growth of bacteria of the commensal flora, represented by S. oralis, is only slightly inhibited by a concentration of 125 mg / L. The data show that while harmful bacteria are selectively inhibited, the physiological flora remains largely unaffected.

[0091] Inhibitory Efficacy of Calcium Peroxide against Biofilms To demonstrate the effect of the combination of calcium peroxide and hyaluronic acid on biofilms, different concentrations were used to measure the biofilm volume, percentage of live and dead cells, and relative metabolic activity of P. gingivalis, a periodontitis-causing bacterium, and S. oralis, a physiological bacterium of the oral flora, and the results are shown in Figures 6 and 7.

[0092] Figure 6 shows the effect of hyaluronic acid and calcium peroxide on mature P. gingivalis biofilms. On the left side of Figure 6, the biofilm volume [μm 3 ] was significantly decreased. The upper right part of Figure 6 shows that the percentage of dead bacteria was significantly increased at calcium peroxide concentrations of 3.9 mg / L, 125 mg / L, and 500 mg / L compared to the control group. Furthermore, the percentage of dead bacteria was significantly increased at calcium peroxide concentration of 500 mg / L compared to 3.9 mg / L and 125 mg / L. The lower right figure shows that there was no significant difference in metabolic activity.

[0093] Figure 7 shows the effect of hyaluronic acid and calcium peroxide on mature S. oralis biofilms. On the left side of Figure 7, the biofilm volume [μm 3 ] has been shown to increase significantly.

[0094] The upper right part of Figure 7 shows that the proportion of dead bacteria increased significantly at calcium peroxide concentrations of 3.9 mg / L, 125 mg / L, and 500 mg / L compared to the control group. The lower right part of Figure 7 shows that the metabolic activity increased significantly at calcium peroxide concentration of 500 mg / L compared to the control group.

[0095] These results show that calcium peroxide concentrations of 3.9, 125 and 500 mg / L in hyaluronic acid, when applied to biofilms according to the invention, lead to a significant inhibition of the growth of the anaerobic periodontitis-causing bacterium P. gingivalis. In contrast, aerobic bacteria of the physiological oral flora, such as S. oralis, are only affected by the oxygen released from the matrix.

[0096] Figure 8 shows the sustained release effect of oxygen with the combination of hyaluronic acid and calcium peroxide compared to calcium peroxide or hyaluronic acid alone. It has been shown that the release of oxygen is significantly increased when calcium peroxide is encapsulated in a hyaluronic acid matrix. Furthermore, the combination of hyaluronic acid and calcium peroxide increased the amount of available oxygen as shown by the enlarged area under the curve (AUC). Experiments were performed at different pH levels (pH 2 for Figure 8A, pH 6 for Figure 8B, and pH 8 for Figure 8C) and 50 mg of calcium peroxide dissolved in 5 mL of water (HO) or 5 mL of hyaluronic acid matrix were compared to baseline over a period of 850 minutes.

[0097] FIG. 9 shows the oxygen content of different biomaterial combinations with and without hyaluronic acid. Baseline: Normalized dissolved oxygen content (norm.DO) as a function of time in 0.9 w% NaCl solution with 200 mL 0.1 M citric acid at 34.1° C. Blank curve Composition I: Addition of 5 mL H2O and 250 mg CaO. Composition I: 5 mL H2O containing 115 mg sodium hyaluronate and 250 mg CaO. Blank curve Composition II: Addition of 5 mL PBS buffer containing 10 mg MHB, 50 mg CaO2, 250 mg MgO2 and 200 mg ZnO2. Composition II: Addition of 5 mL PBS buffer containing 10 mg MHB and 250 mg sodium hyaluronate, 50 mg CaO2, 250 mg MgO2, 200 mg ZnO2. MHB: Preservative (methyl 4-hydroxybenzoate).

[0098] The data show that the amount of available oxygen is increased by up to 5-fold when inorganic peroxide is combined with hyaluronic acid or its derivatives. The amount of available oxygen (corresponding to the area under the curve in Figure 9) can be further increased or modified by using MgO2 and / or ZnO2 and can be tailored to the needs of individual applications and tissues. In particular, it was a surprising discovery that oxygen is released over a longer period of time when inorganic peroxide is combined with hyaluronic acid or its derivatives.

[0099] Figure 10 shows that special treatments during the manufacturing process can improve the efficiency of the formulation and increase oxygen concentration over a long period of time. If the calcium peroxide and hyaluronic acid components of the composition are simply mixed and dissolved / suspended, the amount of available oxygen is significantly reduced. By first dissolving the preservative in a liquid matrix (e.g., PBS buffer), then dissolving the soluble components (e.g., plant extracts), then suspending the insoluble components (e.g., peroxide, hydroxyapatite, plant extracts, etc.), and finally inserting the hyaluronic acid with vigorous stirring, oxygen concentration and oxygen release can be increased over a long period of time.

[0100] In summary, the results show that the combination of one or more inorganic peroxides and hyaluronic acid effectively minimizes the growth of periodontal anaerobic bacteria without affecting physiological bacterial flora.Oxygen is released slowly because inorganic peroxides (exemplified by CaO2) are trapped in the hyaluronic acid matrix, so that only harmless concentrations of oxygen reach cells through the hyaluronic acid matrix.At the same time, tissue regeneration is promoted and not adversely affected.

[0101] Most importantly, the beneficial effects of the combination of CaO2 and hyaluronic acid can be enhanced when additional inorganic peroxides, i.e., MgO2 and / or ZnO2, are present in the composition. In other words, not only can the amount of available oxygen be further increased or modified (Figure 9), but also the amount of utilized inorganic peroxide end products available to the tissue (CaO2+Hyaluronic acid) can be further increased or modified (Figure 9).2+ , Zn 2+ or Mg 2+ ) may also be adapted according to the requirements of the organization.

[0102] Materials and Methods Preparation of inorganic peroxide and hyaluronic acid Calcium peroxide (Sigma Aldrich, St. Louis, MO, US), zinc peroxide and / or magnesium peroxide were suspended in water or an aqueous buffer solution. The desired amount of hyaluronic acid sodium salt (HERRLAN-PSM eK) was added with vigorous stirring.

[0103] Preparation of inorganic peroxide and hyaluronic acid for bacterial assays Autoclaved calcium peroxide (CaO2) (Sigma Aldrich, St. Louis, MO, US), ZnO2 and / or MgO2 were sterile filtered, deionized, and dissolved in autoclaved water. Solutions were adjusted to concentrations of 3.9–500 mg / l CaO2 for bacterial experiments by serial dilution with water (for plankton experiments) or BHI / VitK (for biofilm experiments). All solutions were freshly prepared for each experiment.

[0104] Cell isolation Primary human cells were obtained from patients undergoing surgery at the University Medical Center Mainz (Germany). The use of residual material was approved by the ethical committee of the Landesarztekammer Rheinland-Pfalz in accordance with the principles set out in the Declaration of Helsinki and the ICH-GCP (International Harmonized Guidelines for good clinical practice). All patients provided written informed consent.

[0105] Fibroblasts were obtained from human oral mucosa. Tissue samples were cut into small pieces of approximately 2x2 mm with a sterile disposable scalpel. Before cell isolation, tissue pieces were stepwise sterilized with 70% ethanol, sterillium® classicpure (Bode Chemie GmbH, Hamburg, Germany), and again with 70% ethanol. They were then transferred to 5–10 mL (depending on the amount of tissue) of phosphate-buffered saline (PBS; Sigma-Aldrich, St. Louis, MO, US) containing 0.5% protease solution (P6141, Sigma-Aldrich, St. Louis, MO, US) and incubated overnight at 4 °C. The next day, the protease solution was incubated with shaking at 37 °C for an additional 15 min. The samples were then passed through a cell sieve (EASYstrainer® 70 μm sterile, Greiner bio-one, Kremsmunster, Austria) using a cell scraper (Falcon®, Corning, NY, US). Cells were pelleted by centrifugation (1,500 rpm, 5 min) and transferred to cell culture medium and cultured on a plate with a growth area of ​​25 cm. 2 Cells were seeded into small cell culture flasks at 37 °C. Cells were morphologically characterized and used for up to 10 passages to confirm their original identity. Cells were maintained at 37 °C and 5% CO2 in DMEM / Ham's F12 (Gibco, ThermoFisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum and antibiotics (10,000 U / mL penicillin and 10 mg / mL streptomycin, Sigma-Aldrich, St. Louis, MO, USA).

[0106] Primary human osteoblasts were isolated according to the following protocol. Human bone fragments were obtained during hip or knee replacement surgery. The use of residual material was approved by the ethical committee of the Landesarztekammer Rheinland-Pfalz, in accordance with the principles set out in the Declaration of Helsinki and the ICH-GCP (International Harmonized Guidelines for good clinical practice). All patients provided written informed consent.

[0107] A bone rongeur was used to remove the cancellous bone fragments from the bone fragments. The isolated fragments were washed several times with PBS (Sigma-Aldrich, St. Louis, MO, USA) until a clear supernatant was obtained. The supernatant was discarded and 15 mL of collagenase type I solution (1 mg / mL in medium 199) was added. Collagenase digestion was performed in a 37°C water bath under mechanical agitation. After 45 min, the fragments were washed again several times with PBS (Sigma-Aldrich). The washed bone fragments were transferred with a sterile rongeur into a 6-well tissue culture plate and DMEM / F-12 medium supplemented with 20% fetal calf serum (FCS) and 1% penicillin / streptomycin (PS) was added.

[0108] After the first passage, human osteoblasts were cultured in DMEM / Ham's F12 (Gibco, ThermoFisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum and antibiotics (10,000 U / mL penicillin and 10 mg / mL streptomycin, Sigma-Aldrich, St. Louis, MO, USA). The medium was changed twice a week. For osteoblast differentiation, the medium was supplemented with 10 nM dexamethasone, 3.5 mM b-glycerophosphate, and 50 μg / mL ascorbic acid.

[0109] Primary human umbilical vein endothelial cells (HUVECs) were isolated from umbilical veins. The use of residual material was approved by the ethical committee of the Landesarztekammer Rheinland-Pfalz in accordance with the principles set out in the Declaration of Helsinki and the International Harmonized Guidelines for good clinical practice (ICH-GCP). All patients provided written informed consent.

[0110] The umbilical cord was flushed with PBS (Sigma-Aldrich) until the buffer solution was clear, and blood clots in the vein were removed. Collagenase solution was then injected into the umbilical cord, placed in warmed PBS, and incubated at 37°C for 12 min. After incubation, the collagenase solution containing the endothelial cells was flushed from the umbilical cord while running PBS (Sigma-Aldrich) through the vein. The effluent was collected and centrifuged at room temperature for 10 min at a maximum of 420 g. During the first cell passage, the cells were stored at 37°C in 3 mL of 0.1% gelatin-coated culture dishes. The cells were incubated overnight at 37°C and washed the next day with PBS (Sigma-Aldrich). Endopan 3 (PAN Biotech, Aidenbach, Germany) containing 3% fetal serum was used as the culture cell medium.

[0111] Cell viability assay Cells were seeded in 24-well plates and allowed to adhere overnight. The number of cells per well was 50.000 for fibroblasts and HUVECs, and 40.000 for osteoblasts, in 1.5 mL medium / well, respectively. After 24 hours, the cell culture medium was replaced with 400 μl of fresh medium. Cells were treated with 0.256 mg / mL CaO2 (Sigma Aldrich, St. Louis, MO, USA), or 30 mg / mL hyaluronic acid (Herrlan PSM eK), or a combination of both. Untreated cells served as control. These substances were applied to inserts with a 10 μm thick semi-transparent polycarbonate membrane with 0.4 μm pores (Corning Inc., New York, USA). These inserts were inserted into 24-well plates and incubated overnight in direct contact with the cell culture medium of the cells. The inserts were removed after 24 h, the cell culture medium was replaced with 1 mL of medium containing 10% AlamarBlue® Cell Viability Reagent (ThermoFisher Scientific, Waltham, MA, USA), and the cells were incubated for 4 h at 37°C. After 4 h, the liquid (200 μl / well) was transferred from the 24-well plate to a black 96-well plate (Greiner bio-one GmbH, Frickenhausen) for measurement. The AlamarBlue assay is based on the blue color change of a non-fluorescent indicator dye (resazurin) after receiving an electron, which goes from an oxidized to a reduced state to a fluorescent pink compound. Fluorescence was measured with a fluorescence microplate reader (Fluoroskan Ascent Microplate reader, ThermoFisher Scientific, Waltham, MA, USA). Results are presented as relative fluorescence using a 538 nm excitation filter and a 600 nm emission filter and were normalized to untreated controls.

[0112] Chorioallantoic membrane assay (CAM assay) Fertilized White Leghorn chicken eggs (LSL Rhein-Main GmbH, Dieburg, Germany) were incubated at 38°C in an incubator (Type 3000 digital fully automatic, Siepmann GmbH, Herdecke, Germany) at constant humidity of 55 rH. During the first 3 days, eggs were placed horizontally on one side to ensure that the CAM was detached from the eggshell facing up. On day 3 of embryonic development (EDD), eggs were prepared by removing 5-6 mL of egg white to widen the space between the eggshell and the CAM. A small window measuring 3 x 2 cm was cut in the upward-facing part of the eggshell. This window was covered with Parafilm® (Sigma-Aldrich, St. Louis, MO, USA) to prevent evaporation. For EDD-8, Gelaspon Strips (Bausch & Lomb Inc.; New York, USA) were cut into slices measuring 1 x 0.5 x 2 mm. 20 μl of each substance (CaO2 512 mg / L, sodium hyaluronate 10 mg / mL, and their combination) was added to the sponge. 20 μl of water was used as a control. Further evaluation of potential tissue adverse events was performed in a blinded manner. After 3 hours, at EDD-10 and EDD-12, pictures were taken at 100x magnification with a digital vital fluorescence microscope (Olympus BXFM, Olympus Deutschland GmbH, Hamburg, Germany) using the cellSens Dimension software package.

[0113] Microorganisms and culture conditions Streptococcus oralis ATCC 9811 was obtained from the American Type Culture Collection (ATCC), and Porphyromonas gingivalis DSM 20709 was obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ). Strains were grown in anaerobic conditions (80% N2, 10% H2, 10% CO2) in 50 mL screw-cap tubes (Sarstedt, Numbrecht, Germany) containing brain heart infusion medium (BHI; Oxoid, Wesel, Germany) containing 10 μg / mL vitamin K (Roth, Karlsruhe, Germany; BHI / VitK) at 37 °C for 24 h, and 24-h-old precultures were obtained for further processing.

[0114] Investigation of the growth and metabolic activity of planktonic bacteria S. oralis and P. gingivalis under the influence of CaO2 To determine the effect of CaO2 on planktonic S. oralis and P. gingivalis, 24-h-old precultures were pelleted by centrifugation at 4,000g for 15 min at 4°C, the supernatant was discarded, and the bacterial pellet was resuspended in fresh 2x concentrated BHI / VitK medium (2xBH / VitK). Optical density was measured at 600 nm (OD 600 The OD of the freshly prepared CaO2 solution was measured with a BioPhotometer, Eppendorf, Hamburg, Germany, adjusted to 0.2, and diluted 1:10 in 2xBHI / VitK to 0.02. 600 = 0.02) and the final OD 600 = 0.01 and the final concentrations of CaO2 were as follows:

[0115] [Table 1]

[0116] As a positive control, bacterial cultures (OD 600= 0.02) was mixed 1:2 with sterile filtered, deionized, autoclaved water (growth control). As negative controls, 2xBHI / VitK was mixed with equal volumes of sterile filtered, deionized, autoclaved water (medium control) and CaO2 solution was mixed 1:2 with 2xBHI / VitK (CaO2 solution control). Finally, 150 μL of each suspension was transferred to each well of a 96-well plate and incubated for 24 h at 37 °C with rotation (180 rpm) under anaerobic conditions (Anaerobic Jar and AnaeroGen; Oxoid, Wesel, Germany). All experiments were performed in three biological replicates and two technical replicates.

[0117] To evaluate the effect of CaO on the growth of planktonic S. oralis and P. gingivalis cultures, 24-h-old bacterial cultures were mixed and the optical density (OD 600 ) was measured in a plate reader (Tecan, Mennedorf, Switzerland). Metabolic activity was measured using the BacTiter-Glo® Microbial Viability Assay (Promega, Mannheim, Germany). 50 μL of BacTiter-Glo® reagent and 50 μL of well-mixed bacterial culture were added to an opaque 96-well plate. After 5 min of incubation at room temperature in the dark, the samples were mixed again and the amount of adenosine triphosphate (ATP) was measured by measuring luminescence using a plate reader. All results were normalized to medium or CaO2 solution controls.

[0118] Investigating the effect of CaO2 on S. oralis and P. gingivalis biofilms To evaluate the effect of CaO2 on biofilms of S. oralis and P. gingivalis, OD 600 The 24-h-old precultures were treated as described for the planktonic experiments, except that the OD was adjusted to 0.1 and diluted to 0.01. 600= 0.01) volume of 2 mL was added to each well of a 6-well plate and incubated for 24 h at 37 °C under anaerobic conditions (Anaerobic Jar and AnaeroGen; Oxoid, Wesel, Germany). All experiments were performed in three biological replicates and two technical replicates.

[0119] After 24 h of growth, the pH values ​​of all samples and controls were measured using indicator sticks (PH-FIX indicator sticks pH 0-14; Macherey-Nagel, Duren, Germany). The supernatant was removed and 2 mL of fresh BHI / VitK was added to the controls and 2 mL of CaO2 solution was added to the biofilms that needed to be treated with the following concentrations:

[0120] [Table 2]

[0121] After 2 h of incubation at 37 °C under anaerobic conditions (Anaerobic Jar and AnaeroGen; Oxoid, Wesel, Germany), the pH values ​​of all samples and controls were measured again. Metabolic activity was measured using the BacTiter-Glo Microbial Viability Assay (Promega, Mannheim, Germany). Biofilms were rinsed twice with phosphate-buffered saline (PBS; Biochrom GmbH, Berlin, Germany) and 1 mL BacTiter-Glo reagent was added to each biofilm. Biofilms were rinsed with the reagent by pipetting up and down several times. After rotation (180 rpm) and incubation for 5 min in the dark, 100 μL of the solution was added to an opaque 96-well plate. Samples were mixed again and luminescence was measured in a plate reader (Tecan, Mennedorf, Switzerland). To analyze the effect of CaO2 on the amount and live / dead distribution of biofilms, biofilms were stained with SYTO 9 and propidium iodide using the LIVE / DEAD BacLight Bacterial Viability Kit (Life Technologies, Carlsbad, California, USA) according to the manufacturer's recommendations. Biofilms were then rinsed twice with PBS and fixed for 30 min with PBS containing 2.5% glutaraldehyde (Roth, Karlsruhe, Germany). After fixation, biofilms were washed twice and covered with 3 mL of PBS. They were analyzed microscopically using a confocal laser scanning microscope (CLSM; Leica TCS SP8, Leica Microsystems, Mannheim, Germany) at excitation and emission wavelengths of SYTO9 (488 nm and 500–550 nm) and propidium iodide (552 nm and 675–750 nm). Three images were taken from each biofilm using a z-step of 2 μm. 3D image processing and analysis of biofilm volume and percentage of dead and live bacteria were performed using Imarisx64 software (version 8.4.1, Bitplane AG, Zurich, Switzerland).

[0122] Oxygen release measurement 50 mg of CaO2 (75% 200 mesh, SigmaAldrich) was suspended in 5 mL of deionized water (Millipore grade). 115 mg of hyaluronic acid sodium salt (1,5-2,5 M Da: Herrlan-PSM, Alpen, Germany) was added under vigorous stirring. The mixture was stirred for at least 4 h and filled into a syringe to avoid air bubbles.

[0123] The concentration profile of dissolved oxygen was measured by HI 5421 (Hanna Instruments, Vohringen, Germany). The sensor was placed in a temperature-controlled beaker directly above a magnetic stirring bar. The beaker was filled with 200 mL of 0.9 wt% NaCl aqueous solution containing 0.1 M citric acid (≧99.5%: CarlRoth, Karlsruhe, Germany). The pH was adjusted by 1 M NaOH (Carl Roth). A cellulose filter bag was placed in the solution to allow the dissolved material to move freely, but not the hyaluronic acid matrix or undissolved CaO2 particles.

[0124] The detector started to detect dissolved oxygen concentration (DO mode) and a baseline was detected for approximately 50 minutes. Then, within 30 seconds, 5 mL of test solution was added to the cellulose filter bag. Dissolved oxygen concentration profiles were detected over several hours in 5 mL of water containing 50 mg of CaO2, both with and without hyaluronic acid sodium salt, as well as baseline. Dissolved oxygen concentrations were normalized to the dissolved oxygen concentration at the time the sample was added.

[0125] statistical analysis Graphical processing and statistical analysis were performed using GraphPad Prism software 8.4 (GraphPad Software Inc., La Jolla, USA). The Kolmogorov-Smirnov normality test was applied to determine whether the data were normally distributed. If the data were normally distributed, a regular one-way analysis of variance with Dunnett's correction for multiple comparisons was used to determine statistically significant differences between treated and control samples. If the data did not meet the assumption of normal distribution, the Kruskal-Wallis test with Dunn's correction for multiple comparisons was used. For all comparisons, the significance level was set at p ≤ 0.05.

Claims

1. A composition comprising hyaluronic acid or a pharma- ceutically acceptable salt thereof and one or more inorganic peroxides for use in the prophylaxis or treatment of a wound or a pathological condition requiring wound healing, tissue regeneration, or bone regeneration.

2. The peroxide is calcium peroxide (CaO ２ ), magnesium peroxide (MgO ２ ), and / or zinc peroxide (ZnO ２ 2. The composition of claim 1, wherein the compound is selected from the group consisting of phenyl, ...

3. The inorganic peroxide is CaO ２ , MgO ２ and ZnO ２ The composition of claim 1 which is a combination of

4. a) plant extracts; b) calcium hydroxyapatite; c) one or more additives selected from the group consisting of collagen; The composition of claim 1.

5. 5. The composition of claim 4, wherein the plant extract is obtained from eucalyptus, rhubarb root, clove oil, aloe vera and / or cumin.

6. The composition of claim 1 , wherein the pathological condition is gum disease.

7. The composition of claim 6, wherein the gum disease is selected from gingivitis, periodontitis, or peri-implantitis.

8. The CaO ２ The composition of any one of claims 2 to 7, wherein is present in the composition in an amount of about 1 mg / L to 5000 mg / L.

9. The CaO ２ The composition of any one of claims 2 to 7, wherein is present in the composition in an amount of about 20 mg / L to 1000 mg / L.

10. The composition of any one of claims 2 to 9, wherein the hyaluronic acid or a pharma- ceutically acceptable salt thereof is present in the composition in an amount of about 1 mg / mL to about 100 mg / mL.

11. The composition of any one of claims 4 to 10, wherein the plant extract is present in the composition in an amount of about 0.1% to about 10% by weight.

12. 12. The composition of any one of claims 4 to 11, wherein the calcium hydroxyapatite is present in the composition in an amount of about 1% to about 30% by weight.

13. 13. A composition according to any one of claims 4 to 12, wherein the calcium hydroxyapatite is present in the composition in a calcium / phosphate ratio of from 0.8 to 20.

14. The composition of any one of claims 4 to 13, wherein the collagen is present in the composition in an amount of about 0.9% to about 25% by weight.

15. The ZnO ２ The composition of any one of claims 2 to 14, wherein is present in the composition in an amount of about 1 mg / L to 10,000 mg / L.

16. The MgO ２ The composition of any one of claims 2 to 15, wherein is present in the composition in an amount of about 1 mg / L to 15,000 mg / L.

17. The composition according to any one of claims 1 to 16, wherein the pH of the composition is in the range of 6 to 8.

5.

18. The composition of claim 1 , wherein the composition is formulated as a pharmaceutical composition for topical administration.

19. The composition of any one of claims 1 to 18, wherein the composition is formulated as a solution, suspension, emulsion, cream, or gel.

20. The composition according to any one of claims 1 to 19, which is subjected to the following process: (1) dissolving a preservative in a liquid matrix; (2) dissolving the soluble components; (3) suspending the insoluble components; and (4) incorporating hyaluronic acid while stirring vigorously.

21. 21. A kit for wound healing, tissue regeneration and / or bone regeneration comprising a composition according to any one of claims 1 to 20 and a delivery means for applying said composition to a wound, tissue or bone.

22. The kit according to claim 21, wherein the kit is configured to deliver a composition to a gingival pocket, a periodontal pocket, or a pocket caused by peri-implantitis, the kit comprising a composition according to any one of claims 1 to 20 and a delivery means for locally applying the composition to the gingival pocket, the periodontal pocket, or the pocket caused by peri-implantitis.

23. 22. The kit of claim 21, wherein the kit is configured to apply the composition onto or into the oral mucosa, the kit comprising a composition according to any one of claims 1 to 20 and a delivery means for topically applying the composition onto or into the oral mucosa.

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