Common peptides in HYA

Abstract

Pharmaceutical and / or cosmetic formulations containing artificial peptides and their medical uses are described, the artificial peptides being characterized by proline sequences that are ubiquitous in key stromal cell proteins involved in wound healing, bone and cartilage formation, and the maintenance of connective tissue in all vertebrates. This invention relates to a pharmaceutical formulation in gel form, comprising a. a substance comprising Pro-X-X-Pro-Y-Y-Y-Pro-X-X-Pro-Y-Y-Pro-X-X-Pro-Y-Y-Y-Y-Pro-Y-Y-Y-Pro-Y-Y-Y-Y-Pro-X-X-Pro-X-Pro-Y-Y-Y-Y-Pro-X-X-Pro-Y-Y-Y-Pro-Y-Y-Pro-X-X-Pro-Y-Y-Pro-Y-Pro-X-X-Pro-Y-Pro-Y-Pro-X-X-Pro-Y-Pro-Y-Pro-X-X-Pro-Y-Y-Pro-X-X-Pro-Y-X ... Artificial peptides comprising the amino acid sequence of NO:1) and / or artificial peptides comprising or composed of the amino acid sequence Pro-X-X-Pro-Y-Y-Pro-X-X-Pro-Y-Y-Pro-X-X-Pro-Y-Y-Pro-X-Pro-X-Pro-Pro (SEQ ID NO:2), wherein i) Pro is proline; ii) X is an amino acid selected from Ala, Ile, Leu, Met, Phe, Trp and Val; iii) Y is an amino acid selected from Asn, Cys, Gln, Ser, Thr and Tyr; b. cross-linked hyaluronic acid fibers (HA-XL) and c. linear hyaluronic acid fibers (HA).

Description

Technical Field

[0001] This invention relates to the field of soft tissue healing and / or improvement.

[0002] Pharmaceutical and / or cosmetic formulations containing artificial peptides are described. These artificial peptides are characterized by proline sequences ubiquitous in key stromal cell proteins involved in wound healing, bone and cartilage formation, and the maintenance of connective tissue in all vertebrates. The medical uses of these pharmaceutical and / or cosmetic formulations are also described. This article describes the formulation of artificial peptides in hydrogels containing a combination of hyaluronic acid and cross-linked hyaluronic acid for minimally invasive administration and / or controlled release, for the treatment of soft tissue symptoms, diseases, and / or disorders, and for cosmetic uses such as improving skin elasticity and / or hydration. Background Technology

[0003] Soft tissue refers to all tissues in the human body that have not undergone ossification or calcification (such as bones and teeth) and have not hardened. Soft tissue connects, surrounds, or supports internal organs and bones, and includes muscles, tendons, ligaments, fat, fibrous tissue, lymphatic and blood vessel systems, fascia, and synovium. The term "soft tissue" is commonly used to describe muscles, tendons, ligaments, and / or fascia, but several other tissue types and body systems also include soft tissue, including fat, skin, nerves, and blood vessels.

[0004] Soft tissue disorders, diseases, and injuries are medical symptoms that affect soft tissues. They include trauma, wounds, and soft tissue damage to connective tissue and / or epithelial tissue.

[0005] In the craniofacial complex, soft tissue disorders include periodontitis, peri-implantitis, peri-mucosal inflammation, gingivitis, aphthous stomatitis, and other oral infections and / or inflammations.

[0006] Skin and soft tissue infections remain among the most common infections following surgical procedures, ranging in severity from mild cellulitis to severe necrotizing infections, with high morbidity and mortality rates. Most commonly, these infections arise from skin lesions in susceptible hosts, but sometimes they can also develop from previously unknown lesions via hematogenous transmission.

[0007] Biomaterials scientists have designed organic bone substitutes specifically based on the biochemical characteristics of simulated tissues to achieve near-native functions. Several non-collagenous proteins in bone are called intrinsically disordered proteins (IDPs) because they lack detectable ordered domains and fixed 3D structures under physiological conditions.

[0008] EP2118136 discloses artificial IDP peptides with improved properties in inducing and / or stimulating mineralization in vivo and in vitro. These peptides are readily synthesized, and their methods of use can be employed to induce and / or stimulate mineral precipitation and / or biomineralization.

[0009] The amino acid signature of the artificial peptide disclosed in EP2118136 is a proline-rich sequence ubiquitous in key stromal cell proteins involved in wound healing, bone and cartilage formation, and connective tissue maintenance in all vertebrates. This core proline sequence is highly conserved in vertebrates, possesses inherent disorder, and does not elicit any immunogenic response in humans.

[0010] In contrast to the successful application of peptides in hard tissue healing, the need for equally effective solutions in soft tissue healing and regeneration remains largely unmet. Furthermore, given the susceptibility of soft tissues to complications and disorders following surgery and / or treatment, finding minimally invasive methods to deliver this advanced peptide technology is preferable.

[0011] Hydrogels are a class of extensively studied biomaterials, with an increasing number of products being applied clinically. Hydrogels are a group of biomaterials composed of polymer or colloidal networks that swell upon contact with water. As viscoelastic materials, hydrogels can structurally mimic the extracellular matrix (ECM), creating a favorable environment for cell proliferation and tissue regeneration, thus attracting significant attention in regenerative medicine. The viscoelastic properties of hydrogels make them suitable for use as stem cell carriers or drug-controlled release scaffolds. Different tissues require different loading patterns and levels, and the clinical requirements for materials vary, necessitating a wide range of diverse applications. Therefore, a "one-size-fits-all" hydrogel strategy is not feasible.

[0012] This invention addresses both the need for advanced peptide technologies to promote soft tissue healing, growth, and differentiation, and the need for novel methods to apply such active substances to and / or to affected soft tissues, particularly craniofacial complex soft tissues, in a minimally invasive manner. Summary of the Invention

[0013] The inventors have demonstrated that the artificial IDP peptides disclosed in EP2118136 can unexpectedly not only effectively stimulate the regeneration of mineralized tissues, but also promote the growth and / or differentiation of connective tissue cells, and through them produce secondary promoting effects on the growth and spread of epithelial cells, such as promoting local angiogenesis, regulating acute and chronic inflammation, revitalizing senescent cells (such as cells senescent due to radiation damage), and reducing pain and swelling when applied topically.

[0014] To address the issue of complications, diseases, and impairments that easily occur in soft tissues after surgery and / or treatment, the inventors have also developed a novel artificial IDP peptide hydrogel formulation that can be minimally invasively applied to soft tissues.

[0015] Therefore, the present invention relates to a pharmaceutical and / or cosmetic formulation in the form of a gel (such as a hydrogel) comprising a. an artificial peptide characterized by a proline sequence ubiquitous in key stromal cell proteins involved in wound healing, bone and cartilage formation, and connective tissue maintenance in most vertebrates; b. cross-linked hyaluronic acid filaments (HA-XL); and c. linear hyaluronic acid filaments (HA).

[0016] This invention relates to a pharmaceutical and / or cosmetic formulation in the form of a gel (such as a hydrogel) comprising or consisting of a. an artificial peptide, b. cross-linked hyaluronic acid fibers (HA-XL), and c. linear hyaluronic acid fibers (HA), wherein the artificial peptide comprises: the amino acid sequence Pro-XX-Pro-YYY-Pro-XX-Pro-YY-Pro-XX-Pro-X-Pro-YYYYYY-Pro-YYYYY-Pro-XX-Pro-X-Pro-YYY-Pro-Y-Pro-Y-Pro-XX-Pro-Y-Pro-Y-Pro-XX-Pro-Y-Pro-XX-Pro-Y-Pro-XX-Pro-Y-Pro-XX-Pro-Y-Pro-XX-Pro-Y-Pro-X-Pro-Pro-XXXXXXXX-Pro-XX-Pro-XXXX (SEQ ID NO: 1), wherein i) Pro is proline; ii) X is an amino acid selected from Ala, Ile, Leu, Met, Phe, Trp and Val; iii) Y is an amino acid selected from Asn, Cys, Gln, Ser, Thr and Tyr, and the amount of cross-linked hyaluronic acid cellulose (HA-XL) is 1-40 mg / mL, such as 1.0, 2.5, 4, 10, 20, 25 or 40 mg / mL, or 1-30 mg / mL, and the amount of linear hyaluronic acid cellulose (HA) is 1-40 mg / mL, such as 1.0, 2.5, 4, 10, 20, 25 or 40 mg / mL, or such as 1-30 mg / mL.

[0017] Typically, the pharmaceutical and / or cosmetic formulations of the present invention comprise a. 0.1-250 µg / mL of an artificial peptide, such as 0.1, 1.0, 5.0, 10, 50, 100, 200, or 250 µg / mL; b. 1-40 mg / mL of cross-linked hyaluronic acid fibers (HA-XL), such as 1.0, 2.5, 4, 10, 20, 25, or 40 mg / mL; and c. 1-40 mg / mL of linear hyaluronic acid fibers (HA), such as 1.0, 2.5, 4, 10, 20, 25, or 40 mg / mL, or composed thereof, wherein the artificial peptide comprises the amino acid sequence Pro-XX-Pro-YYY-P. ro-XX-Pro-YY-Pro-XX-Pro-X-Pro-YYYYYY-Pro-YYYYYY-Pro-XX-Pro-X-Pro-YYY-Pro-YY-Pro-Y-Pro-XX-Pro-Y -Pro-YY-Pro-XX-Pro-YY-Pro-XX-Pro-YY-Pro-XX-Pro-Y-Pro-Pro-X-Pro-Pro-XXXXXXXX-Pro-XX-Pro-XXXX(SEQ IDNO: 1).

[0018] Alternatively, or additionally, the pharmaceutical and / or cosmetic formulations of the present invention comprise, or are composed of, an artificial peptide, said artificial peptide comprising the amino acid sequence Pro-XX-Pro-YY-Pro-XX-Pro-YY-Pro-XX-Pro-YY-Pro-Y-Pro-X-Pro-Pro (SEQ ID NO: 2).

[0019] Typically, pharmaceutical and / or cosmetic formulations according to the present invention comprise a. 0.1-250 µg / mL of an artificial peptide, such as 0.1, 1.0, 5.0, 10, 50, 100, 200, or 250 µg / mL; b. 1-40 mg / mL of cross-linked hyaluronic acid fibers (HA-XL), such as 1.0, 2.5, 4, 10, 20, 25, or 40 mg / mL; and c. 1-40 mg / mL of linear hyaluronic acid fibers (HA), such as 1.0, 2.5, 4, 10, 20, 25, or 40 mg / mL, wherein the artificial peptide comprises the amino acid sequence Pro-XX-Pro-YY-Pro-XX-Pro-YY-Pro-XX-Pro-YY-Pro-Y-Pro-X-Pro-Pro (SEQ ID NO: 2).

[0020] In embodiments, the pharmaceutical and / or cosmetic formulations of the present invention comprise one or more artificial peptides selected from artificial peptides comprising the amino acid sequences of SEQ ID NO: 4 (P2) (peptide 2) and SEQ ID NO: 5 (P6), or wherein the artificial peptide has at least 90% identity with artificial peptides comprising the amino acid sequences of SEQ ID NO: 4 and SEQ ID NO: 5.

[0021] The pharmaceutical and / or cosmetic formulations according to the present invention may comprise, or be composed of, cross-linked hyaluronic acid fibers (HA-XL), which comprise hyaluronic acid fibers cross-linked with 1,4-butanediol diglycidyl ether (HA-XL(BDDE)) and / or hyaluronic acid fibers cross-linked with diglycidyl ether (HA-XL(BDDE) or HA-XL(PEGDE)).

[0022] In the presently preferred embodiments, the pharmaceutical and / or cosmetic formulations of the present invention comprise a. 50 µg / mL of an artificial peptide having the amino acid sequences SEQ ID NO: 4 (P2) and / or SEQ ID NO: 5 (P6), b. 20 mg / mL of hyaluronic acid fibers crosslinked with 1,4-butanediol diglycidyl ether (HA-XL(BDDE)) and / or hyaluronic acid fibers crosslinked with diglycidyl ether (HA-XL(BDDE) or HA-XL(PEGDE)), and c. 2.5 mg / mL of linear hyaluronic acid fibers (HA).

[0023] In pharmaceutical and / or cosmetic formulations according to the present invention, the hyaluronic acid fibers are typically 0.7-4.0 MDa, such as 1.0-2.0, 1.5-1.8, or 3.0-3.3 MDa. In one embodiment, the hyaluronic acid fibers are 3.0-3.3 MDa, such as 1.5 MDa.

[0024] Typical pharmaceutical and / or cosmetic formulations according to the present invention are hydrogels.

[0025] The pharmaceutical and / or cosmetic formulations according to the present invention may further comprise: d. one or more buffers; e. a fluoride source; f. one or more salts; and g. water.

[0026] In addition, the pharmaceutical and / or cosmetic formulations of the present invention may also contain one or more components selected from the following: d. water; e. sodium fluoride (NaF); f. citric acid; g. sodium hydroxide; h. sodium chloride; i. disodium hydrogen phosphate; and j. sodium phosphate.

[0027] The pharmaceutical and / or cosmetic formulations according to the present invention may also contain one or more components selected from the following: sorbitol, xylitol, NaOH, HCl, phosphate-buffered saline (PBS), acetic acid, citric acid, and maleic acid.

[0028] The pharmaceutical formulation according to the invention may also contain mesenchymal stem cells (MSCs), such as 100,000-10,000,000 MSCs / ml, preferably about 1,000,000 MSCs / ml. Furthermore, the pharmaceutical formulation according to the invention can promote MSC homing.

[0029] The pharmaceutical and / or cosmetic formulations according to the present invention are stable in RT for at least 1-2 years and / or stable in vivo in the form of a gel for at least 30 days.

[0030] In one aspect, pharmaceutical and / or cosmetic formulations according to the invention are prepared at a controlled rate (such as 1... (g / hour) releases the artificial peptides contained therein.

[0031] In embodiments, the pharmaceutical and / or cosmetic formulations according to the present invention may also contain another active ingredient, such as, but not limited to, active ingredients selected from growth factors, plasma-rich fibrin / plasma, and enamel matrix derivatives.

[0032] One aspect relates to novel pharmaceutical formulations according to the invention, which are used in medicine, particularly for soft tissue healing, such as, but not limited to, for wound healing, inducing angiogenesis, inducing re-epithelialization, stimulating collagen production, and / or promoting targeted collagen formation.

[0033] The pharmaceutical formulations according to the present invention contain anti-inflammatory properties. These anti-inflammatory properties can be measured by a reduction in pro-inflammatory cytokines. In embodiments, the pro-inflammatory cytokines include one or more cytokines selected from IL-23, IL-1α, IL-1β, TNF-α, MCP-1, IL-12P70, IFN-γ, IFN-β, IL-6, IL-10, IL-27, IL-17A, and GM-CSF.

[0034] The novel pharmaceutical formulations according to the present invention can be used for anti-inflammatory and / or antimicrobial therapy for the treatment of periodontitis, mucositis, peri-implantitis, and / or cartilage regeneration. In the treatment of craniomaxillofacial complex disorders, diseases, and / or impairments, the novel pharmaceutical formulations according to the present invention can be used to treat periodontitis, peri-implantitis, peri-mucositis, gingivitis, aphthous stomatitis, and other oral infections and / or inflammations.

[0035] One aspect of the present invention relates to a method for preparing pharmaceutical and / or cosmetic formulations according to the invention, the method comprising: a. providing an artificial peptide as defined herein; b. providing hyaluronic acid with a molecular weight of 0.7-4 MDa (such as 1.0-2.0, 1.5-1.8, or 3.0-3.3 MDa, such as 1.5 MDa), wherein said hyaluronic acid comprises or is composed of a mixture of linear hyaluronic acid and hyaluronic acid crosslinked with BDDE and / or PEGDE; c. The artificial peptide is mixed at a concentration of 0.1-250 µg / mL and the hyaluronic acid mixture at a concentration of 1-40 mg / mL, and optionally, a fluoride source is added to the mixture, wherein the osmotic pressure of the pharmaceutical and / or cosmetic formulation is 50-400 mOsm / L, such as 100-310 mOsm / L, or such as 125-175 mOsm / L, such as about 150 mOsm / L, or such as 275-325 mOsm / L, such as about 300 mOsm / L.

[0036] In an additional embodiment, the method includes a. providing an artificial peptide at a concentration of up to 100 mg / mL, such as 0.01 μg / mL to 100 mg / mL; b. adding hyaluronic acid fibers dissolved in 0.3 M NaOH at a concentration of 10% by weight; c. mixing a crosslinking agent, such as BDDE and / or PEGDE; d. heating the reaction vessel to 20-100°C and incubating for 1-2 hours to induce gelation; e. cooling and neutralizing the solution; f. homogenizing the gel; g. dialyzing the gel in sterile PBS for at least 18 hours; h. adding linear hyaluronic acid in an amount of about 10% of the crosslinked hyaluronic acid; i. homogenizing the formulation to ensure uniform distribution of the peptides; and j. optionally adding phosphate buffered solution (PBS).

[0037] In an embodiment of the method for preparing a pharmaceutical and / or cosmetic formulation according to the invention, the artificial peptide in step a. is dissolved in 1% acetic acid at a concentration of 10 mg / mL, then b. hyaluronic acid cellulose (3.0-3.3 MDa) dissolved in an alkaline solution (preferably NaOH) at a concentration of 10% by weight is added, c. 1,4-butanediol diglycidyl ether (BDDE) and / or poly(ethylene glycol) diglycidyl ether (PEGDE) is mixed, d. the reaction vessel is heated to about 40°C and incubated for about 4 hours, e. the solution is cooled and neutralized with an acidic solution (preferably HCl), f. the gel is homogenized into particles of 100-400 µm. In an embodiment, the method further includes g. dialyzing the gel in a biocompatible solution (such as PBS or physiological saline), h. adding linear hyaluronic acid in an amount of about 10% by weight of cross-linked hyaluronic acid, i. homogenizing the formulation to ensure uniform distribution of the peptides, and j. optionally adding phosphate buffer solution (PBS).

[0038] In another aspect, a method for preparing a pharmaceutical and / or cosmetic formulation according to any one of the preceding claims is disclosed, comprising: a. providing an artificial peptide at a concentration of up to 100 mg / mL; b. adding hyaluronic acid fibers dissolved in 0.3 M NaOH at a concentration of 10% by weight; c. mixing a crosslinking agent; d. heating a reaction vessel to 20-100°C and incubating for 1-2 hours; e. stopping heating and neutralizing with a solution such as HCl; f. homogenizing the gel; g. dialyzing the gel in a solution such as sterile water, sterile PBS, or sterile saline; h. adding linear hyaluronic acid, such as about 10% of the amount of crosslinked hyaluronic acid; i. homogenizing the formulation to ensure uniform distribution of the peptide; and j. optionally adding sterile water, sterile PBS, or sterile saline.

[0039] In another aspect, a method for preparing pharmaceutical and / or cosmetic formulations according to this disclosure is disclosed, said method comprising, a. To provide the artificial peptide as defined in any one of claims 1-3, b. Provide linear hyaluronic acid with a molecular weight of 0.7-4 MDa (such as 1.0-2.0, 1.5-1.8, or 3.0-3.3 MDa, such as 1.5 MDa). c. Use a cross-linking agent (such as BDDE or PEGDE) to cross-link linear hyaluronic acid to obtain cross-linked hyaluronic acid (HA-XL). d. Optionally, the cross-linked hyaluronic acid is dialyzed. e. Mix 0.1-250 µg / mL of the artificial peptide and 1-40 mg / mL of the cross-linked hyaluronic acid, and add 1-40 mg / mL of linear hyaluronic acid to obtain a mixture comprising: - Cross-linked hyaluronic acid, - Linear hyaluronic acid, and - Artificial peptides.

[0040] In another aspect, a method for preparing pharmaceutical and / or cosmetic formulations according to this disclosure is disclosed, said method comprising, a. To provide the artificial peptide as defined in any one of claims 1-3, b. Provide linear hyaluronic acid with a molecular weight of 0.7-4 MDa (such as 1.0-2.0, 1.5-1.8, or 3.0-3.3 MDa, such as 1.5 MDa). c. Mix 0.1-250 µg / mL of the artificial peptide and 1-40 mg / mL of the hyaluronic acid. d. Crosslink the mixture using a crosslinking agent (such as BDDE or PEGDE) to obtain a mixture of internally and / or inter-crosslinked peptides with hyaluronic acid. e. Add linear hyaluronic acid (HA) to obtain a mixture of internally cross-linked and / or inter-cross-linked artificial peptides and hyaluronic acid, as well as linear hyaluronic acid.

[0041] In the implementation method, the method further includes: f. Mixing 1-40 mg / mL of internally cross-linked and / or inter-cross-linked artificial peptides and hyaluronic acid with 0.1-250 µg / mL of additional artificial peptides to obtain a mixture comprising: - Artificial peptides and hyaluronic acid with internal and / or inter-crosslinking. - Linear hyaluronic acid, and - Artificial peptides.

[0042] Therefore, the present invention also relates to pharmaceutical preparations obtained according to the method of the present invention and their use in medicine, as well as cosmetic preparations and their use.

[0043] Definitions and abbreviations

[0044] In this document, "artificial peptide" refers to a non-natural peptide, that is, a peptide that does not normally exist in nature but is produced by assembling and selecting amino acids in a specific order, number, and manner, and such peptide is applicable to this invention. Even if an artificial peptide may include some or all of the peptides that happen to exist in nature, "artificial peptide" is still the peptide included in this invention. "Artificial" may be used interchangeably with terms such as "synthetic" or "non-natural".

[0045] In this article, "Pro" refers to the amino acid proline.

[0046] In this article, "X" represents a hydrophobic amino acid. Hydrophobic amino acids are defined as amino acids selected from Ala, Ile, Leu, Met, Phe, Trp, and Val.

[0047] In this article, "Y" represents a polar amino acid. Polar (hydrophilic) amino acids are defined as amino acids selected from Asn, Cys, Gln, Ser, Thr, and Tyr.

[0048] In this article, common terms are used to represent amino acids. Thus, for example, A is Ala (hydrophobic), C is Cys (polar), F is Phe (hydrophobic), H is His, I is Ile (hydrophobic), L is Leu (hydrophobic), M is Met (hydrophobic), N is Asn (polar), Q is Gln (polar), S is Ser (polar), T is Thr (polar), V is Val (hydrophobic), W is Trp (hydrophobic), and Y is Tyr (polar).

[0049] In this document, "surface" means any surface that may be associated with the artificial peptides of this invention, such as metallic surfaces, such as those of titanium, zirconium, tantalum, aluminum, gold, surgical steel, or nickel, or alloys thereof, or their metal oxides, or their metal hydroxides or hydrides, or hydroxyapatite, aragonite, bioglass, glass, or polyurethane. Another example of a surface according to the invention is a biological surface, such as a transplant surface, a wound surface, etc. Additional examples of surfaces include mucosal surfaces, skin surfaces, and other equivalent surfaces.

[0050] In this article, "subjects" refers to any vertebrate, such as birds, reptiles, mammals, primates, and humans.

[0051] Hyaluronic acid; Hyaluronic acid (usually abbreviated as HA) is a natural linear carbohydrate polymer belonging to the non-sulfated glycosaminoglycan class. As used herein, linear hyaluronic acid refers to non-crosslinked (linear) hyaluronic acid, and is referred to as HA. It consists of repeating disaccharide units of β-1,3-N-acetylglucosamine and β-1,4-glucuronic acid, with a molecular weight (MW) up to 6 MDa. Hyaluronic acid is found in hyaline cartilage, synovial fluid, and skin tissues, including the dermis and epidermis.

[0052] BDDE: 1,4-Butanediol diglycidyl ether is a type of crosslinking agent whose molecular weight can be designed according to specific applications, such as a molecular weight of approximately 200 Da (M W ).

[0053] PEGDE: Polyethylene glycol diglycidyl ether is a type of crosslinking agent with repeating polyethylene glycol motifs. Its molecular weight can be designed for specific applications, such as a molecular weight of approximately 500 Da (M... W ).

[0054] HA-XL(BDDE) is used in this document to indicate that hyaluronic acid is cross-linked using BDDE as defined above.

[0055] HA-XL(PEGDE) is used in this document to indicate that hyaluronic acid is cross-linked using PEGDE as defined above.

[0056] As used in this article, the term "biocompatibility" means that it will not cause clinically relevant tissue irritation, damage, toxicity or immune response in living tissue.

[0057] The term "cell" as used in this article refers to the structural and functional unit of a living organism, which is the smallest unit of an organism classified as living.

[0058] As used herein, the term "compatible" means that the components of a composition can be combined with each other in such a way that no interaction occurs under normal use conditions that would substantially reduce the effectiveness of the composition.

[0059] As used in this article, the term "component" refers to a part, element, or ingredient.

[0060] As used herein, the term “symptom” refers to a variety of health conditions and is intended to include disorders or diseases caused by any underlying mechanism or disturbance, injury, or factor contributing to the health of tissues and organs.

[0061] As used in this article, the term “differentiation” refers to the developmental process in which cells or tissues become more organized or complex, while simultaneously becoming more specialized in function.

[0062] As used in this article, the terms “disease” and “disorder” refer to symptoms of impaired health or dysfunction.

[0063] As used herein, the term "mucosa" refers to the mucous tissue lining the various tubular structures composed of epithelium, lamina propria, and the smooth muscle layer in the digestive tract. As used herein, the term "mucosal graft" refers to a graft of mucous membrane.

[0064] As used in this article, the term "patient" refers to any mammal. Examples of mammals include humans, farm animals, and livestock.

[0065] The term “peptide” as used in this article refers to two or more amino acids linked by peptide bonds.

[0066] It must be noted that, as used herein and in the appended claims, the singular forms “a (a, an)” and “the” include plural references, unless the context clearly specifies otherwise.

[0067] As used herein, the term "comprising" means the presence of a feature, integer, step, or component as described in the claims, but does not exclude the presence or addition of one or more other features, integers, steps, components, or combinations thereof. The term "comprising" is intended to include embodiments covered by the terms "substantially constitutes" and "consisting of". Similarly, the term "substantially constitutes" is also intended to include embodiments covered by the term "consisting of".

[0068] As used herein, the term "about" when used to modify the quantity of an ingredient or reactant means that the value may vary, for example, due to typical measurement and liquid handling procedures in practice when preparing concentrates or using solutions; or due to unintentional errors in these procedures; or due to differences in the manufacture, source, or purity of the ingredients used in preparing the composition or carrying out the method. The term "about" also covers differences in quantity due to different equilibrium conditions of composition resulting from a particular initial mixture. Whether or not the term "about" is used, the claims include the content equivalent to the quantity.

[0069] In this article, the abbreviation "u" as used in "um", "ug", or "uL" can be used interchangeably with "micro", "mcg", or "micro". "Use them interchangeably. For example, microgram is a unit of mass, equal to one millionth of a gram (1 × 10⁻⁶). -6 ).

[0070] When providing numerical ranges, it should be understood that the values ​​between the upper and lower limits of the range, as well as any other values ​​or intermediate values ​​within the range, are included in this invention; the intermediate values ​​may be accurate to one-tenth of the lower limit unit, unless the context clearly specifies otherwise. The upper and lower limits of these smaller ranges may be independently included within the smaller range and are also covered by this invention; however, this is contingent upon the absence of explicitly excluded limits within the range. When the range includes one or two limits, this invention also includes ranges excluding both limits.

[0071] It should be understood that the terminology used herein is for describing particular implementations only and is not intended to limit the scope of the invention, which is limited only by the appended claims.

[0072] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of this invention, exemplary methods and materials are described herein. All publications mentioned herein are incorporated to disclose and describe methods and / or materials relevant to the cited publications. Attached Figure Description

[0073] Figure 1 : Schematic diagram of the preparation steps for manufacturing (XL) hyaluronic acid gel crosslinked with BDDE or PEGDE.

[0074] Figure 2 A. Rheological shear and frequency sweeps of HA-XL (BDDE) and HA-XL (PEGDE) gels. B. Rheological shear and frequency sweeps of the two gels. C. Real-time crosslinking monitoring at 40℃ for 5 hours. D. FTIR analysis of the two gels.

[0075] Figure 3: A. HA-XL (BDDE) and B. HA-XL (PEGDE) before dialysis in distilled water (top of A and B) and The leaching medium after 18 hours of dialysis (see the lower diagram in A and B) 1 H NMR.

[0076] Figure 4 Comparison of P6 biotin release in cross-linked HA-XL (BDDE) and linear hyaluronic acid gels.

[0077] Figure 5 (a) sham surgery, (b) Emdogain (Straumann, Basel, CH), (c) P2-containing cross-linked HA+ Comparison of wound healing performance of HA-XL(BDDE).

[0078] Figure 6 HA+HA-XL(BDDE) gel containing P2, P6, or P2+P6, compared with sham surgery and "empty" HA+HA-XL(BDDE) gel. Wound healing performance compared to glue. a) Wound cavity area, b) Re-epithelialization score, c) Granulation formation score, d) Infiltration score.

[0079] Figure 7 : HA+HA-XL(BDDE) gel containing P2 or P6 and EMD and HA+HA-XL(BDDE) gel containing EMD Comparison of wound healing performance. a) Wound cavity area, b) Re-epithelialization score, c) Granulation formation score, d) Infiltration score.

[0080] Figure 8 Example images of histological wound staining. (Top) Re-epithelialization: 3 - Completely intact; Granulation formation: 3 - None. Infiltration: 2 - Some infiltration is present around the wound. (Medium) Re-epithelialization: 3 - Completely intact, granulation tissue formation: 1 - Some infiltration is present around the wound. Degree, Infiltration: 0 - Subeschar infection / necrotic tissue + peri-wound infiltration. (Below) Re-epithelialization: 3 - Complete healing, granulation formation: 0 - Extensive, infiltrative; 1 - Peri-wound infiltration. This score shows that HA+HA- XL containing P2 is significantly different from HA+HA- XL alone (BDDE). The regeneration potential of XL(BDDE).

[0081] Figure 9 Based on specific biomarkers, flow cytometry analysis confirmed that inflammation decreased three weeks after tissue healing. light.

[0082] Figure 10 Based on specific biomarkers, flow cytometry analysis confirmed that inflammation decreased three weeks after tissue healing. light.

[0083] Figure 11 Based on specific biomarkers, flow cytometry analysis confirmed that inflammation decreased three weeks after tissue healing. light.

[0084] Figure 12 Microcomputed tomography reconstruction of multiple traumatic femoral fractures in mice 3 weeks after healing. Then, HA+HA-XL(BDDE) gel (Hyd), HA+HA-XL(BDDE)+P2 (Hyd+P2), and HA+HA-XL(BDDE)+P2+MSC were used. Treatment was administered using (Hyd+P2+MSC).

[0085] Figure 13 The parameters are output after morphological analysis following reconstruction by micro-computed tomography.

[0086] Figure 14 : Using HHA+HA-XL(BDDE)(A), HA+HA-XL(BDDE)+P2(C), HA+HA-XL(BDDE)+P2+ von Kossa / van Gieson staining for fracture healing after MSC(E) treatment, and HA+HA-XL(BDDE) staining 3 weeks after fracture. Movat staining of (B), HA+HA-XL(BDDE)+P2 (D), HA+HA-XL(BDDE)+P2+MSC(F).

[0087] Figure 15 Morphometric analysis of histological data.

[0088] Figure 16 Small-angle X-ray scattering / X-ray diffraction (SAXS / X-ray diffraction) of osteogenic and biomineralization processes after healing XRD analysis. The full width at half maximum (fwhm) HAp reflects the degree of orientation, and the T-parameters of the defect points are shown in the figure above.

[0089] Figure 17 Block diagram of small-angle X-ray scattering / X-ray diffraction (SAXS / XRD) analysis.

[0090] Figure 18 Cross-linked HA-XL (BDDE) [HA-XL], linear hyaluronic acid [HA], and cross-linked hyaluronic acid and linear... A comparison of the change in %P2-peptide release over time in a hyaluronic acid combination gel [HA+HA-XL(BDDE)], including the variation in bubble size. It indicates the percentage change in relative weight.

[0091] Figure 19 Representative Masson Goldner trichrome stained section, with the active inflammatory area circled by a dashed line (prosthetic hand). Technique: Necrotic / encapsulated tissue; HA+HA-XL (BDDE): Inflammatory infiltration; Emdogain®: Active infection), and dashed lines indicate water. Tumor (ae). All histological images, except for Emdogain® (pig 6), are from pig 2. Scale bar: 1 mm. Images show the groups. Mean inflammation score (f) [1 = damaged, 3 = normal]. The inflammation score for each sample is based on epithelial physiology, edema, and inflammatory infiltration. The average value of the three indicators of infiltration / active infection, sham surgery, HA+HA-XL(BDDE)+P2, and HA+HA-XL(BDDE)+P6 was 6. And HA+HA-XL(BDDE) and Emdogain® are 3.

[0092] Figure 20 Cell viability was measured using the CCK8 assay. n=8 p≤0.5, p≤0.01, p≤0.001。 Detailed Implementation

[0093] This invention relates to a pharmaceutical and / or cosmetic formulation in the form of a gel (such as a hydrogel) comprising a. an artificial peptide characterized by a proline sequence ubiquitous in key stromal cell proteins involved in wound healing, bone and cartilage formation, and connective tissue maintenance in most vertebrates; b. cross-linked hyaluronic acid filaments (HA-XL); and c. linear hyaluronic acid filaments (HA).

[0094] In one aspect, the formulations of the present invention comprise one or more artificial peptides loaded into a hydrogel carrier. The one or more artificial peptides comprised in the formulations of the present invention are characterized by comprising or consisting of the amino acid sequence Pro-XX-Pro-YYY-Pro-XX-Pro-YY-Pro-XX-Pro-X-Pro-YYYYYY-Pro-YYYYY-Pro-XX-Pro-X-Pro-YYY-Pro-YY-Pro-Y-Pro-XX-Pro-Y-Pro-XX-Pro-YY-Pro-XX-Pro-YY-Pro-XX-Pro-Y-Pro-XX-Pro-X-Pro-Pro-XXXXXXXX-Pro-XX-Pro-XXXX (SEQ ID NO: 1) or thereof, wherein i) Pro is proline; ii) X is an amino acid selected from Ala, Ile, Leu, Met, Phe, Trp, and Val; and iii) Y is an amino acid selected from Asn, Cys, Gln, Ser, Thr, and Tyr.

[0095] Alternatively, or additionally, one or more artificial peptides contained in the formulations of the present invention are characterized by comprising or consisting of the amino acid sequence Pro-XX-Pro-YY-Pro-XX-Pro-YY-Pro-XX-Pro-YY-Pro-Y-Pro-Y-Pro-X-Pro-Pro (SEQ ID NO: 2), wherein i) Pro is proline; ii) X is an amino acid selected from Ala, Ile, Leu, Met, Phe, Trp and Val; and iii) Y is an amino acid selected from Asn, Cys, Gln, Ser, Thr and Tyr.

[0096] In one embodiment, the formulation is delivered minimally invasively via a syringe. In vitro and in vivo studies have shown that the peptide-containing gel formulation described herein stimulates periodontal tissue formation with comparable or superior active ingredients to those of leading market products. Furthermore, the formulations of this invention are fully synthetic, thus offering scalability and price competitiveness. Moreover, the formulations disclosed herein do not require the use of animal tissue. The hydrogel carrier of this invention uses covalently cross-linked hyaluronic acid, thereby overcoming limitations such as, but not limited to, rapid post-application clearance and gingival flap collapse.

[0097] Artificial peptides

[0098] In one aspect, the present invention relates to an unexpected insight that the artificial peptides disclosed in EP2118136 can be used to promote connective tissue cells and, through them, produce secondary promoting effects on the growth and spread of epithelial cells, such as promoting local angiogenesis and regulating acute and chronic inflammation, revitalizing senescent cells (e.g., cells damaged by radiation), and reducing pain and swelling when applied topically.

[0099] In Examples 11 and 12, the role of peptides in inflammation is also highlighted, showing that the disclosed formulations can reduce the levels of several inflammatory cytokines, thereby alleviating the inflammatory response after injury. Furthermore, the formulations provided can accelerate the first stage of healing by upregulating genes such as collagen biosynthesis and modification enzymes, and improve the healing process of cartilage and bone through processes such as heparan sulfate degradation, glycosaminoglycan degradation, assembly of collagen fibrils and other polymeric structures, collagen biosynthesis and modification enzymes, ECM-receptor interactions, extracellular matrix organization, and mineral resorption.

[0100] The artificial peptides according to the present invention are biomimetic peptides inspired by motifs found in amelogenin. Due to their inherent disorder, they are flexible peptides capable of dynamically adapting to their local environment. Therefore, they can interact with other structural polymers such as collagen, thereby improving appearance. For example, peptides can fold into temporary extracellular matrix, enabling them to bind to surfaces such as mucous membranes. This can protect the soft tissue of the mucous membrane, providing a protective effect. Studies have also demonstrated that peptides can nucleate calcium phosphate and directionally grow crystals into thin, lamellar plates, thus enabling the formation of a protective mineral layer on tooth enamel.

[0101] The artificial peptides according to the present invention are biomimetic intrinsic disordered proteins (IDPs).

[0102] Under physiological conditions, i.e., the external or internal environmental conditions that organisms (such as humans) may encounter in nature, peptides are typically inherently disordered. The release of peptides from the gel matrix is ​​generally related to the structure of the peptide / protein, where inherently disordered proteins / peptides, due to their high conformational flexibility, are generally more easily released from the gel than structured proteins and / or peptides. This characteristic makes the artificial peptides of this disclosure particularly useful as components released from gels (such as cross-linked HA-XL gels and / or combined HA-HA-XL gels).

[0103] One advantage of using synthetic peptides is that using natural peptides and / or proteins for medical and / or cosmetic purposes is not always practical. For example, natural proteins are often long, meaning they are difficult to synthesize using chemical methods and cell expression systems. Generally, artificial peptides are easier to synthesize than large, intact proteins because peptides typically lack complex higher-order structures, while large proteins are difficult to synthesize. Furthermore, natural proteins contain only natural amino acids and may therefore be prone to rapid degradation. Additionally, if purified from the natural environment (such as developing teeth), there is always a risk of contamination from other products, which could potentially cause allergic reactions. Moreover, long, natural proteins often have multiple functions in vivo and may not be optimized for the intended use.

[0104] Furthermore, the active motifs of the synthetic peptides in the formulations of the present invention can be designed to specifically stimulate the healing and / or growth of soft tissues and skin.

[0105] The formulations of this invention contain artificial peptides characterized by proline sequences that are ubiquitous in key stromal cell proteins involved in wound healing, bone and cartilage formation, and the maintenance of connective tissue in most vertebrates.

[0106] The artificial peptide contained in the formulation of the present invention is characterized by comprising or consisting of the amino acid sequence Pro-XX-Pro-YYY-Pro-XX-Pro-YY-Pro-XX-Pro-X-Pro-YYYYYY-Pro-YYYYY-Pro-XX-Pro-X-Pro-YYY-Pro-YY-Pro-Y-Pro-XX-Pro-Y-Pro-Y-Pro-XX-Pro-Y-Pro-XX-Pro-Y-Pro-XX-Pro-Y-Pro-XX-Pro-X-Pro-Pro-XXXXXXXX-Pro-XX-Pro-XXXX (SEQ IDNO: 1) or thereof, wherein i) Pro is proline; ii) X is an amino acid selected from Ala, Ile, Leu, Met, Phe, Trp and Val; iii) Y is an amino acid selected from Asn, Cys, Gln, Ser, Thr and Tyr.

[0107] Alternatively, or additionally, the formulations of the present invention comprise one or more artificial peptides characterized by comprising or consisting of the amino acid sequence Pro-XX-Pro-YY-Pro-XX-Pro-YY-Pro-XX-Pro-YY-Pro-Y-Pro-X-Pro-Pro (SEQ ID NO: 2), wherein i) Pro is proline; ii) X is an amino acid selected from Ala, Ile, Leu, Met, Phe, Trp and Val; and iii) Y is an amino acid selected from Asn, Cys, Gln, Ser, Thr and Tyr.

[0108] Furthermore, the peptides according to the invention may contain 20 to 120 amino acids, such as, but not limited to, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-60, 60-70, 70-80, 80-90, 90-100, or 100-120 amino acids, such as, but not limited to, 21, 22, 23, 24, 26, 27, 28, 29, 30, 32, 33, 37, 42, 47, 49, 51, 53, 57, 59, 63, 65, 75, 85, 87, 88, 92, 95, 105, or 115 amino acids. In a preferred embodiment, the peptides according to the invention contain 20-50 amino acids.

[0109] The amino acids in the artificial peptides of this invention can be further modified chemically, isometrically, or otherwise, provided the peptide sequence is intact. Modification of the amino acids in the artificial peptides of this invention can improve the peptide's activity, stability, biocompatibility, or clinical performance, or reduce its toxicity and adverse reactions. Examples of chemical modifications include, but are not limited to, glycosylation and methylation. The amino acids can also be in all different types of stereoisomers, such as the D or L form of the amino acid, or the S or R isomer. The amino acids in the artificial peptides of this invention can also be replaced with their synthetic analogs. For example, using synthetic analogs can make the peptide more stable and less prone to degradation. Examples of non-natural amino acids include α-amino acids. and α-disubstituted amino acids, N-alkyl amino acids , lactic acid Halogenated derivatives of natural amino acids, such as trifluorotyrosine p-Cl-phenylalanine p-Br-phenylalanine p-I-phenylalanine L-Allylglycine β-alanine La-aminobutyric acid Lg-aminobutyric acid La-aminoisobutyric acid Le-aminohexanoic acid#, 7-aminoheptanoic acid L-methionine sulfone# L-leucine L-valine p-Nitro-L-phenylalanine L-Hydroxyproline#, L-Thioproline Methyl derivatives of phenylalanine (Phe), such as 4-methyl-Phe Pentamethyl-Phe L-Phe (4-amino)#, L-Tyr (methyl) L-Phe(4-isopropyl) L-Tic (1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) L-Diaminopropionic acid and L-Phe(4-benzyl) This article uses symbols. The symbol # indicates the hydrophobicity of the derivative, while # indicates its hydrophilicity. It indicates gender characteristics.

[0110] The peptides identified above are artificial (synthetic) peptides containing a polyproline concordance sequence, as well as hydrophobic amino acids ("X") and polar amino acids ("Y"). They can induce and / or stimulate soft tissue healing in biological systems and can also be used clinically, industrially, chemically, or otherwise to stimulate soft tissue formation. Protein sequences used to construct artificial peptides include those of collagen 1 and 2 (human, mouse, and rat), amelogenin (human, mouse, rat, rabbit, pig, and bovine), ameloblastin (human, rat), osteosialyl protein (human, mouse), and amelogenin (human, mouse).

[0111] The artificial peptides in the formulations of this invention are particularly suitable for inducing and / or stimulating soft tissue healing because their amino acid sequences have been optimized for this purpose. The use of the artificial peptides of this invention is advantageous because their shorter length compared to natural peptides facilitates their synthesis and allows for the use of amino acid analogs as explained herein. Furthermore, the use of artificial peptides allows for modification of the amino acid sequence to enable binding to, for example, metal surfaces or facilitate purification, such as by selecting the amino acid sequence of the peptide itself or by using N-terminal and / or C-terminal tags.

[0112] Therefore, in one aspect, the present invention relates to formulations comprising an artificial peptide containing the amino acid sequence of SEQ ID NO: 1 and / or SEQ ID NO: 2, which is capable of inducing and / or stimulating soft tissue growth and / or differentiation. Preferably, such artificial peptide consists of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0113] One embodiment of the present invention relates to a formulation comprising a short common peptide sequence comprising, or consisting of, the amino acid sequence Pro-XX-Pro-YY-Pro-XX-Pro-YY-Pro-XX-Pro-YY-Pro-Y-Pro-X-Pro-Pro (SEQ ID NO: 2), wherein: i) Pro is proline; ii) X is an amino acid selected from Ala, Ile, Leu, Met, Phe, Trp and Val, preferably Ile, Leu, Val and Met; iii) Y is an amino acid selected from Asn, Cys, Gln, Ser, Thr and Tyr, with Ser and Gln being preferred.

[0114] SEQ ID NO: 2 is assembled from amino acids at positions 47-50, 53-66, and 70-76 of SEQ ID NO: 1 (i.e., the underlined amino acids in SEQ ID NO: 1 above). A preferred embodiment of the invention relates to a formulation comprising an artificial peptide containing the amino acid sequence shown in SEQ ID NO: 2, more preferably consisting of the amino acid sequence shown in SEQ ID NO: 2. Another preferred embodiment relates to a formulation comprising an artificial peptide consisting of the amino acid sequence shown in SEQ ID NO: 2, which is capable of inducing and / or stimulating soft tissue growth and / or differentiation. Due to the short length of SEQ ID NO: 2, it is advantageous for synthetic preparation.

[0115] The present invention also relates to a formulation according to the invention comprising other artificial peptides containing a specified amino acid sequence included in the conserved sequence of the invention. In a first aspect, such artificial peptide is PLV PSY PLV PSY PLVPSY PYP PLPP (SEQ ID NO: 3). Another preferred amino acid sequence is PLV PSQ PLV PSQ PLV PSQ PQPPLPP (SEQ ID NO: 4). These two sequences are the two most conserved sequences in the present invention.

[0116] In embodiments, the artificial peptides included in the formulations according to the present invention comprise the amino acid sequence shown in SEQ ID NO: 4 (peptide 2 or P2) or SEQ ID NO: 5 (peptide 6 or P6). As illustrated by example, in embodiments, the artificial peptides included in the formulations according to the present invention consist of the amino acid sequence shown in SEQ ID NO: 4 (peptide 2 or P2) or SEQ ID NO: 5 (peptide 6 or P6).

[0117] Table 1. Example peptide sequences

[0118] In one aspect, the present invention relates to a formulation according to the invention comprising an artificial peptide having the amino acid sequence of SEQ ID NO: 4, which is capable of inducing and / or stimulating soft tissue growth and / or differentiation. Preferably, such artificial peptide consists of the amino acid sequence shown in SEQ ID NO: 4.

[0119] In one aspect, the present invention relates to a formulation according to the invention comprising an artificial peptide having the amino acid sequence of SEQ ID NO: 5, which is capable of inducing and / or stimulating soft tissue growth and / or differentiation. Preferably, such artificial peptide consists of the amino acid sequence shown in SEQ ID NO: 5.

[0120] Other aspects of the invention relate to formulations according to the invention comprising one or more artificial peptides having 80-100% identity with any sequence of SEQ ID NO: 1-5, such as peptides having 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, or 99% identity with the sequences of SEQ ID NO: 1-5.

[0121] In a preferred embodiment, the formulation according to the invention comprises one or more artificial peptides disclosed herein, each consisting of any one of the sequences in SEQ ID NO: 1-5.

[0122] In the formulations according to the invention, the peptide may further comprise an N-terminal and / or C-terminal tag containing the amino acids His and / or Met. Met contains sulfur, which, as previously mentioned, facilitates binding to metal surfaces. His has a strong affinity for, for example, Ni and other metals. Therefore, the advantage of using these tags is that the peptide can attach to metal surfaces such as titanium, zirconium, aluminum, tantalum, gold, surgical steel, and nickel, or metal oxide hydroxide and / or hydride surfaces, etc. C-terminal and / or N-terminal tags are also useful in the purification of the prepared peptide, as is well known to those skilled in the art. Using N-terminal and / or C-terminal tags also allows the peptide to be fully exposed, i.e., the tag is used to bind the peptide to the surface, while the rest of the peptide is free to interact with atoms, molecules, cells, and tissues, etc. Using a tag at each end of the peptide can be very useful in the preparation of the peptide, allowing one end of the peptide to be attached to a chromatographic column while the other end is available for binding to the target surface when purifying the target peptide from an incomplete peptide product.

[0123] Therefore, a preferred embodiment of the present invention relates to a formulation according to the invention comprising an artificial peptide as defined herein, further comprising an N-terminal and / or C-terminal histidine tag. As previously described, such a tag may comprise methionine and / or histidine residues that are linked to the artificial peptide according to the invention. In a preferred embodiment, the tag comprises three or more residues, such as three to five or five to ten residues. The tag may comprise any number of residues linked to the artificial peptide according to the invention, which can still provide a stable composition with the artificial peptide according to the invention without negatively affecting the secondary structure of the artificial peptide. Preferably, the histidine tag consists of five histidine residues. In another preferred embodiment, the artificial peptide comprises an N-terminal and / or C-terminal methionine tag, preferably consisting of five methionine residues. In another preferred embodiment, the peptide of the present invention comprises a methionine tag at its C-terminus or N-terminus and a histidine tag at the other end.

[0124] SEQ IDS

[0125] In embodiments, the pharmaceutical and / or cosmetic formulations according to the present invention comprise one or more artificial peptides comprising, or consisting of, one or more amino acid sequences selected from, or composed of, the sequences shown in Table 2 below: Table 2 Peptide Sequences

[0126] In this document, Pro represents proline (Pro); L represents leucine (Leu); V represents valine (Val); S represents serine (Ser); Q represents glutamine (Gln); H represents histidine (His); M represents methionine (Met); C represents cysteine ​​(Cys); X represents an amino acid selected from A (alanine, Ala), I (isoleucine, Ile), L (leucine, Leu), M (methionine, Met), F (phenylalanine, Phe), W (tryptophan, Trp), and V (valine, Val), preferably Ile, Leu, Val, and Met; Y represents an amino acid selected from N (asparagine, Asn), C (cysteine, Cys, CysH), Q (glutamine, Gln), S (serine, Ser), T (threonine, Thr), and Y (tyrosine, Tyr), preferably Ser and Gln.

[0127] In embodiments, the pharmaceutical and / or cosmetic formulations according to the present invention comprise one or more artificial peptides selected from artificial peptides comprising the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7.

[0128] In embodiments, the pharmaceutical and / or cosmetic formulations according to the present invention comprise one or more artificial peptides selected from the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7.

[0129] In embodiments, the pharmaceutical and / or cosmetic formulations according to the present invention comprise one or more artificial peptides selected from artificial peptides having at least 90% identity with the amino acid sequences of SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6 and SEQ ID NO 7, for example having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequences of SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6 or SEQ ID NO 7.

[0130] Typically, pharmaceutical and / or cosmetic formulations according to the present invention contain 0.1-250 µg / mL, such as 0.1, 1.0, 5.0, 10, 50, 100, 200 or 250 µg / mL of the artificial peptide according to the present invention.

[0131] Typically, the artificial peptides contained in the formulations of this invention are designed to have a length not exceeding 30 amino acids, such as not exceeding 29 amino acids, not exceeding 28 amino acids, not exceeding 27 amino acids, not exceeding 26 amino acids, not exceeding 25 amino acids, not exceeding 24 amino acids, not exceeding 23 amino acids, not exceeding 22 amino acids, not exceeding 21 amino acids, not exceeding 20 amino acids, not exceeding 19 amino acids, not exceeding 18 amino acids, not exceeding 17 amino acids, not exceeding 16 amino acids, not exceeding 15 amino acids, not exceeding 14 amino acids, not exceeding 13 amino acids, or not exceeding 12 amino acids. Furthermore, the artificial peptides contained in the formulations of this invention are designed to retain the proline site and to systematically modify other constituent amino acids, as defined in SEQ ID NO 1 and SEQ ID NO 2.

[0132] The designed peptides (P2, P4, P5, and P6) differ in their amino acid composition and, apart from the conserved proline positions in their sequences, exhibit distinct physical, chemical, and structural properties. A recently developed network-based peptide design approach was employed to explore potential correlations between the structure of specific peptides and biological responses.

[0133] Hyaluronic acid

[0134] This invention relates to a pharmaceutical and / or cosmetic formulation in gel form comprising a. an artificial peptide characterized by a proline sequence ubiquitous in key stromal cell proteins involved in wound healing, bone and cartilage formation, and connective tissue maintenance in most vertebrates; and b. hyaluronic acid fibers, wherein the hyaluronic acid fibers comprise cross-linked hyaluronic acid fibers (HA-XL) and non-cross-linked (linear) hyaluronic acid (HA). The gel, referred to as HA+HA-XL, is a gel comprising a mixture of linear hyaluronic acid (HA) and cross-linked hyaluronic acid (HA-XL). Example 2 illustrates a method for preparing such a mixture, wherein 10% HA and 90% HA-XL are used. It should be understood that the amounts of HA and HA-XL can be adjusted according to the specific intended use.

[0135] Hyaluronic acid is a natural polymer that naturally exists in the extracellular matrix of human cells. It contributes to a high-water-content environment, enabling cells to attach and proliferate. Furthermore, it has been shown to possess inherent antibacterial and anti-inflammatory properties, which are beneficial for preventative functions. Hyaluronic acid has been widely used in pharmaceutical formulations, medical devices, and cosmetics. Therefore, pharmaceutical-grade raw materials derived through bacterial fermentation are readily available.

[0136] Due to its unique physical and biological properties (including biocompatibility and biodegradability), hyaluronic acid is widely used in various fields, both current and developing.

[0137] WO 03 / 061626 discloses an injectable, insertable, or implantable drug delivery system that forms a hydrogel upon implantation, injection, or insertion, comprising hyaluronic acid and pharmaceutically effective compounds.

[0138] Hyaluronic acid is a long-chain polysaccharide composed of repeating units of glucuronic acid and N-acetylglucosamine. It is a natural polymer with wide clinical applications in medical fields such as ophthalmology, aesthetics (dermal fillers), and orthopedics (viscosity supplements). Hyaluronic acid is a naturally occurring component of the extracellular matrix in the human body, which has inspired its use in medical devices, and it does indeed exhibit high bioactivity. Initially, hyaluronic acid was derived from animal sources, including roosters, which resulted in high prices and difficulties in overcoming regulatory hurdles. However, in the past few decades, bacterial fermentation has become the gold standard for producing hyaluronic acid, offering high reproducibility, low cost, and regulatory facilitation. A significant limitation is that hyaluronic acid is rapidly eliminated from the body by hyaluronidase, typically with a half-life of several hours to several days. Covalent cross-linking has become a popular strategy to overcome this problem and improve the therapeutic effects of hyaluronic acid gels. A popular strategy is to crosslink hyaluronic acid with materials such as 1,4-butanediol diglycidyl ether (BDDE) or polyethylene glycol diglycidyl ether (PEGDE), thereby forming covalent bonds through ring-opening esterification via the nucleophilic attachment of the active hydroxyl groups found in hyaluronic acid.

[0139] Crosslinked hyaluronic acid gels can also be achieved via a base-catalyzed reaction in an organic solvent, where the carboxyl group is activated and subsequently undergoes nucleophilic acyl substitution with the hydroxyl group. Similarly, carbodiimides (such as 1-ethyl-3-(3-dimethylaminopropyl) (EDC)) can be used as catalysts to induce crosslinking between hyaluronic acid polymers in a two-step process, where the carboxyl group of hyaluronic acid is first activated by EDC in an acidic environment, followed by the formation of an ester bond between the carboxyl group of hyaluronic acid and the adjacent hydroxyl group, and the recovery of EDC. Crosslinking can also be induced by photopolymerization, for example, by functionalizing the carboxyl group with methacrylate terminal groups.

[0140] Although there are various alternative strategies for cross-linking hyaluronic acid groups to functionalize carboxyl and hydroxyl terminal groups, cross-linked hyaluronic acid BDDE (referred to in this article as HA-XL(BDDE)) remains the most popular in clinical practice. It is primarily used for dermal fillers.

[0141] Hyaluronic acid has diverse biomedical applications, and hyaluronic acid-based products achieve their therapeutic effects through various pathways. Dermal fillers require materials that can fill volume and maintain volume stability over a long period. In ophthalmology, hyaluronic acid can be used for both lubrication and moisture retention in patients prone to dry eye. In dentistry, hyaluronic acid cross-linked with BDDE has been used to fill periodontal pockets and as a scaffold for gingival regeneration.

[0142] While the applications and mechanisms of action of hyaluronic acid are diverse, a common requirement is high biocompatibility. Hyaluronic acid is a native component of the human extracellular matrix and exhibits high affinity for cells through binding to the CD44 receptor. This gives hyaluronic acid its inherent anti-inflammatory properties by reducing NF-κB release. When hyaluronic acid is cross-linked, it does occupy some carboxylic acid and hydroxyl groups, which reduces its ability to bind to the CD44 receptor, thus decreasing its biological activity. Therefore, a trade-off must be made between increased physiological stability and decreased biological activity as the degree of cross-linking increases. All these factors need to be considered when developing novel cross-linked hyaluronic acid formulations for biomedical applications. In some recent studies, polyethylene glycol diglycidyl ether (PEGDE) has been used as an alternative to BDDE, and results show that, compared to BDDE, PEGDE can generate elastic gels with less swelling at the same molar concentration, providing a new tool for optimizing the properties of cross-linked hyaluronic acid gels.

[0143] Compared to cross-linked hyaluronic acid (HA-XL) gels, gels containing or composed of linear hyaluronic acid fibers (HA) dissolve and / or degrade more rapidly. Due to the faster degradation of linear gels, theoretically, linear hyaluronic acid gels release peptides into the surrounding environment more quickly than cross-linked gels. As used herein, the terms "hyaluronic acid fibers" and "hyaluronic acid fibrous material" are used interchangeably. However, the interaction between the loaded peptide and the hydrogel must always be considered when calculating release rates. For example, sterically complex or charged peptides may become entangled in linear hyaluronic acid fibers, thus contradicting theoretical expectations.

[0144] Crosslinking of hyaluronic acid fibers makes the gel more stable, which slows down the degradation and release of peptides, as the peptides may have to diffuse out of the gel rather than be released during gel degradation. However, the specific release profile of peptides in the gel is a complex sum of factors including gel degradation, the physicochemical properties of the peptides, diffusion, and the gel hydration level. In some applications, rapid release of peptides and retention of hyaluronic acid CD44 receptor binding may be more desirable, while in others, slow release of peptides from the gel may be preferred. An intermediate between these two can be obtained by mixing crosslinked hyaluronic acid (HA-XL) and linear hyaluronic acid (HA), where one portion of the peptides is released more rapidly and the other less slowly. Furthermore, a stable level of swelling in the gel, without absorbing additional liquid from the surrounding environment, may also be desirable. This stability can be controlled by the amount of crosslinking used in the gel, which includes both the ratio between crosslinked and linear hyaluronic acid and the degree of crosslinking within the crosslinked hyaluronic acid itself. It should be assumed that more crosslinking generally results in a gel with higher mechanical stability and less water swelling. In applications such as wound healing, formulations with high mechanical stability and strength are preferred, as this allows the formulation to remain stable over a longer period of time, thereby promoting wound healing.

[0145] Example 11 shows that peptides diffuse more rapidly from gels formulated as a combination of linear hyaluronic acid and cross-linked (HA+HA-XL) gels or entirely HA-XL gels, while linear gels exhibit slower release (see Example 11). Figure 18 Furthermore, linear gels exhibited greater swelling over 24 hours (see...). Figure 18 The gel swells over time, which can be a disadvantage in some applications because swelling can lead to excessive pressure in the wound area, causing systemic discomfort, and also makes the gel more unstable. Example 5 and... Figure 4 The results show that, compared to linear gels, the biotinylated peptides of the present invention diffuse more slowly from HA-XL gels. This is likely due to the interaction of the biotinylated peptides with the HA-XL gel, resulting in slower release from the HA-XL gel than from the linear gel. Example 5 also demonstrates that the release profile of the hyaluronic acid gel can be further modified by conjugating the peptides with other substances.

[0146] As mentioned above, the release profile of peptides from the gel is highly dependent on various factors and is difficult to determine without experimentation and error. From these examples, it can be expected that a higher content of linear hyaluronic acid in the gel results in greater gel swelling, but a lower overall peptide release. The gels in the embodiments of this invention all contain a mixture of cross-linked and non-cross-linked hyaluronic acid. Although the ratio between cross-linked and non-cross-linked hyaluronic acid may vary, it is preferred that the gels of this invention contain both cross-linked and non-cross-linked hyaluronic acid to ensure sufficient physicomechanical stability and the desired release rate of the loaded peptides. In some embodiments, the gel may consist primarily of cross-linked hyaluronic acid.

[0147] In this invention, it was unexpectedly discovered that the use of hyaluronic acid increased the viscosity of formulations containing artificial peptides and / or formed a high-viscosity gel-like core. Crosslinking showed that it slowed the release of biotinylated artificial peptides after administration.

[0148] Furthermore, the release rate of peptides is highly dependent on the timing of their addition during the manufacturing process. For example, peptides can be added to a mixture of hyaluronic acid before cross-linking, meaning that once cross-linked, the peptides will ultimately be more firmly fixed in the gel, whereas adding peptides after cross-linking allows them to diffuse more freely from the gel into the surrounding environment. Therefore, adding peptides before cross-linking may lead to [unclear - possibly related to cross-linking or other processes]. Figure 4 A similarly slow release profile. On the other hand, adding peptides to a mixture of cross-linked and linear gels after cross-linking may result in faster release of the peptides from the gel, similar to... Figure 18 Similarly, this provides an additional avenue for customizing peptide release profiles from the gel. Therefore, it is also conceivable to add peptides in portions, with the first portion added before crosslinking and the second portion added after crosslinking, such as in the step of mixing HA and HA-XL.

[0149] Therefore, in some embodiments, the peptides are added before gel crosslinking. In other embodiments, the peptides are added after gel crosslinking. In further embodiments, a portion of the peptides are added before mixing the gel, and a portion of the peptides are added after gel crosslinking. In some embodiments, 50% of the peptides are added to HA-XL before crosslinking, and 50% of the peptides are added to HA. In some embodiments, 60% of the peptides are added to HA-XL before crosslinking, and 40% of the peptides are added to HA. In some embodiments, 70% of the peptides are added to HA-XL before crosslinking, and 30% of the peptides are added to HA. In some embodiments, 80% of the peptides are added to HA-XL before crosslinking, and 20% of the peptides are added to HA. In some embodiments, 90% of the peptides are added to HA-XL before crosslinking, and 10% of the peptides are added to HA. In some embodiments, 95% of the peptides are added to HA-XL before crosslinking, and 5% of the peptides are added to HA. In some embodiments, 40% of the peptides are added to HA-XL before crosslinking, and 60% of the peptides are added to HA. In some embodiments, 30% of the peptides are added to HA-XL before crosslinking, and 70% of the peptides are added to HA. In some embodiments, 20% of the peptides are added to HA-XL before crosslinking, and 80% of the peptides are added to HA. In some embodiments, 10% of the peptides are added to HA-XL before crosslinking, and 90% of the peptides are added to HA. In some embodiments, 5% of the peptides are added to HA-XL before crosslinking, and 95% of the peptides are added to HA. In some embodiments, the peptides are added only to HA-XL. In other embodiments, the peptides are added only to HA. In some embodiments, the peptides are added after mixing HA and HA-XL. In certain cases, a slower release profile is preferred, such as when the gel is used for chronic conditions, such as chronic wounds, chronic periodontitis, or chronic peri-implantitis. In such indications, it is preferred that a larger proportion of the peptides is released slowly from the gel, such as over days to months.

[0150] For other indications, faster release is preferred. For example, for more acute indications such as acute wounds (including surgical wounds), acute inflammation, and acute bone healing (e.g., post-operative and post-traumatic), faster peptide release is preferred. In such indications, it is preferred that a larger proportion of the peptide is released from the gel more rapidly, such as within hours to days.

[0151] It is well known that if a medication can be administered, for example, once or twice daily instead of three or four times, it can help improve adherence. However, it may also be beneficial to release at least a portion of the medication immediately, followed by sustained release.

[0152] In this paper, "hyaluronic acid" is defined as a non-sulfated glycosaminoglycan composed of repeating disaccharide units of N-acetylglucosamine (GIcNAc) and glucuronic acid 20 (GlcUA), linked together by alternating β-1,4 and β-1,3 glycosidic bonds. Hyaluronic acid is also known as hyaluronan, hyaluronic acid salt, or hyaluronic acid cellulose. The terms hyaluronic acid, hyaluronic acid, and hyaluronic acid cellulose are used interchangeably in this paper.

[0153] The hyaluronic acid content can be determined using a modified carbazole method (Bitter and Muir, 1962). Anal Biochem. 4: 330-334).

[0154] In a preferred embodiment, the hyaluronic acid used according to the present invention has a very high purity, especially the quality required by current Good Manufacturing Practices (cGMP).

[0155] The hyaluronic acid used in the formulations according to the present invention can be derived from a variety of sources. Rooster combs are an important commercial source of hyaluronic acid. Alternatively, microorganisms are an alternative source. US4,801,539 and EP0694616 disclose fermentation methods for preparing hyaluronic acid involving Streptococcus vesiculosus strains.

[0156] In embodiments, hyaluronic acid or its salts are derived from microorganisms, preferably recombinantly prepared. In one embodiment, hyaluronic acid or its salts are prepared by Gram-positive bacteria, such as, but not limited to, Bacillus and / or Streptococcus. In the currently preferred embodiment, hyaluronic acid or its salts are prepared by Bacillus subtilis. Hyaluronic acid or its salts can be prepared according to the method disclosed in WO 03 / 054163.

[0157] Hyaluronic acid synthases have been found in vertebrates, bacterial pathogens, and algal viruses (DeAngelis, PL, 1999, Cell. Mol. Life Sci. 56: 670-682). WO 99 / 23227 discloses a group I hyaluronic acid synthase from *Streptococcus equi*. WO 99 / 51265 and WO 00 / 27437 describe a group II hyaluronic acid synthase from *Pasteurella multocida*. Ferretti et al. disclosed a hyaluronic acid synthase operon from *Streptococcus pyogenes*, which consists of three genes: hasA、 hasB and hasCThe components encode hyaluronic acid synthase, UDP-glucose dehydrogenase, and UDP-glucose pyrophosphorylase, respectively (Proc. Natl. Acad. Sci. USA. 98, 4658-4663, 2001). WO 99 / 51265 describes a nucleic acid segment containing the coding region for Streptococcus equi hyaluronic acid synthase.

[0158] The host cell can be any Bacillus cell suitable for recombinant preparation of hyaluronic acid. The Bacillus host cell can be a wild-type Bacillus cell or a mutant thereof. Bacillus cells useful in the practice of this invention include, but are not limited to, Bacillus agalladus (…). Bacillus agaraderhens ), Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circinates ( Bacillus circulans ), Bacillus clausti ( Bacillus clausii Bacillus coagulans ( Bacillus coagulans ), Bacillus thuringiensis ( Bacillus firmus ), Bacillus laterosporus ( Bacillus lautus ), Bacillus tarda ( Bacillus lentus ), Bacillus licheniformis ( Bacillus licheniformis ), Bacillus megaterium ( Bacillus megaterium ), Bacillus pumilus ( Bacillus pumilus ), thermophilic fat bacillus ( Bacillus stearothermophilus Bacillus subtilis ( Bacillus subtilis ) and Bacillus thuringiensis ( Bacillus thuringiensis WO 98 / 22598 describes mutant Bacillus subtilis cells that are particularly well-suited for recombinant expression. Non-capsulated Bacillus cells are especially useful in this invention.

[0159] Since the hyaluronic acid in recombinant Bacillus cells is directly expressed in the culture medium, a simple process can be used to separate the hyaluronic acid from the culture medium.

[0160] hyaluronic acid salts

[0161] According to the present invention, any salt of hyaluronic acid can be used.

[0162] In a preferred embodiment, the salt of hyaluronic acid is an inorganic salt, preferably sodium hyaluronate, potassium hyaluronate, ammonium hyaluronate, calcium hyaluronate, magnesium hyaluronate, zinc hyaluronate, or cobalt hyaluronate.

[0163] Hyaluronic acid molecular weight

[0164] According to the present invention, any molecular weight can be used, but in a preferred embodiment, hyaluronic acid or a salt thereof has an average molecular weight of 0.2-6 Mda; more preferably 0.7-4.0 Mda; even more preferably 1.0-3.0 Mda; and even more preferably 1.25-1.75 Mda. The molecular weight can be determined according to methods known in the art. In a preferred embodiment, the molecular weight of hyaluronic acid is about 1.5 Mda.

[0165] The specific molecular weight of hyaluronic acid in the formulation described herein was chosen to provide a favorable viscosity relationship, namely, slow release due to high entanglement, as described in Falcone 2005 and WO 2013 / 030348A1, which show experiments demonstrating the effect of increasing hyaluronic acid concentration on release time.

[0166] The pharmaceutical and / or cosmetic formulations according to the present invention comprise cross-linked hyaluronic acid fibers (HA-XL) and linear hyaluronic acid fibers (HA).

[0167] Typically, pharmaceutical and / or cosmetic formulations according to the present invention contain 1-40 mg / mL of cross-linked hyaluronic acid fibers (HA-XL), such as 1.0, 2.5, 4, 10, 20, 25 or 40 mg / mL, and 1-40 mg / mL of linear hyaluronic acid fibers (HA), such as 1.0, 2.5, 4, 10, 20, 25 or 40 mg / mL.

[0168] The pharmaceutical and / or cosmetic formulations according to the present invention may comprise, or be composed of, cross-linked hyaluronic acid fibers (HA-XL), which comprise hyaluronic acid fibers cross-linked with 1,4-butanediol diglycidyl ether (HA-XL(BDDE)) and / or hyaluronic acid fibers cross-linked with polyethylene glycol diglycidyl ether (PEGDE) (HA-XL(PEGDE)).

[0169] Preferably, the formulation of the present invention comprises both cross-linked hyaluronic acid and linear hyaluronic acid. The ratio of cross-linked hyaluronic acid to linear hyaluronic acid may be varied depending on the specific purpose. Thus, in embodiments, the formulation of the present invention may comprise cross-linked hyaluronic acid and linear hyaluronic acid, wherein 87.5-92.5%, such as about 90%, of the hyaluronic acid is cross-linked, and 7.5-12.5%, such as about 10%, of the hyaluronic acid is linear.

[0170] In pharmaceutical and / or cosmetic formulations according to the present invention, the hyaluronic acid cellulose typically has a strength of 0.7-4.0 Mda, such as 1.0-2.0, 1.5-1.8, or 3.0-3.3 Mda. In one embodiment, the hyaluronic acid monomer has a strength of 3.0-3.3 Mda.

[0171] In the presently preferred embodiments, the pharmaceutical and / or cosmetic formulations according to the invention comprise a. 0.1-250 μg / mL of an artificial peptide, such as 50 µg / mL, and b. 1-40 mg / mL of hyaluronic acid fibers.

[0172] In the presently preferred embodiments, the pharmaceutical and / or cosmetic formulations according to the invention comprise a. 50 µg / mL of an artificial peptide, b. 20 mg / mL of hyaluronic acid fibers (HA-XL(BDDE)) crosslinked with 1,4-butanediol diglycidyl ether, and c. 2.5 mg / mL of linear hyaluronic acid fibers (HA).

[0173] In the presently preferred embodiments, the pharmaceutical and / or cosmetic formulations according to the invention comprise a. 50 µg / mL of an artificial peptide, b. 20 mg / mL of hyaluronic acid cellulose (HA-XL(PEGDE)) crosslinked with polyethylene glycol diglycidyl (PEGDE) ether, and c. 2.5 mg / mL of linear hyaluronic acid cellulose (HA). Preferably, the molecular weight (MW) of the poly(ethylene glycol) diglycidyl ether is 250-1500 Da, such as about 500 Da.

[0174] In the presently preferred embodiments, the pharmaceutical and / or cosmetic formulations according to the invention comprise a. 50 µg / mL of an artificial peptide having the amino acid sequences of SEQ ID NO: 4 and / or SEQ ID NO: 5, b. 20 mg / mL of hyaluronic acid fibers (HA-XL(BDDE)) crosslinked with 1,4-butanediol diglycidyl ether, and c. 2.5 mg / mL of linear hyaluronic acid fibers (HA).

[0175] In the presently preferred embodiments, the pharmaceutical and / or cosmetic formulations according to the present invention comprise a. 50 µg / mL of an artificial peptide having the amino acid sequences of SEQ ID NO: 4 and / or SEQ ID NO: 5, b. 20 mg / mL of hyaluronic acid fibers (HA-XL(PEGDE)) crosslinked with polyethylene glycol diglycidyl (PEGDE) ether, and c. 2.5 mg / mL of linear hyaluronic acid fibers (HA).

[0176] pharmaceutical preparations

[0177] This invention relates to a pharmaceutical and / or cosmetic formulation in gel form comprising: a. a proline sequence characterized by its ubiquitous presence in key stromal cell proteins involved in wound healing, bone and cartilage formation, and connective tissue maintenance in most vertebrates; b. cross-linked hyaluronic acid fibers (HA-XL); and c. linear hyaluronic acid fibers (HA). In embodiments, the pharmaceutical formulation further comprises MSC cells.

[0178] The pharmaceutical and / or cosmetic formulations according to the present invention are stable in RT for at least 1-2 years and / or stable in vivo in the form of a gel for at least 30 days.

[0179] The pharmaceutical and / or cosmetic formulations according to the invention are stable in RT / in gel form for at least 1-2 years, such as at least 1 year, for example at least 1.5 years, for example at least 1.7 years, for example at least 2 years, for example 1 year, 1.5 years, 1.7 years, 1.8 years, 2 years, 3 years or 5 years.

[0180] The pharmaceutical and / or cosmetic formulations according to the invention are stable in vivo in gel form under RT for at least 30 days, such as at least 31 days, such as at least 32 days, such as at least 35 days, such as at least 40 days, such as at least 50 days, such as at least 60 days, such as at least 90 days.

[0181] In one aspect, pharmaceutical and / or cosmetic formulations according to the invention release contained artificial peptides at a controlled rate. Hereinafter, the term "at a controlled rate" is used to describe a release that is not abrupt. Controlled release is characterized herein by the release of the drug at a predictable, rationally programmed rate to achieve optimal target-drug concentrations. This dosage form enhances the safety, efficacy, reliability, and convenience of drug therapy. While this is a slow-release system, unlike sustained-release, this process is designed to produce predictable, constant drug concentrations. For this approach, the concentration of the active ingredient in the target tissue is controlled, not merely the release of the drug.

[0182] In one aspect, the pharmaceutical and / or cosmetic formulations according to the invention release the contained artificial peptide at a controlled rate, such as about 2 μg per hour, such as up to 10, 5, 4, 3, 2, 1.5, 1.2, or 1 μg per hour, or such as about 10 ng, 100 ng, or 500 ng per hour, or such as 0.01, 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 μg per hour. In another aspect, the pharmaceutical and / or cosmetic formulations according to the invention release the contained artificial peptide at a controlled rate, such as 0.1-10 μg per hour, such as 1-8 μg per hour, or such as 1.5-5 μg per hour.

[0183] The pharmaceutical and / or cosmetic formulations according to the present invention may further comprise one or more fluoride sources, such as one or more fluoride sources selected from NaF, CaF2 and ZnF2. Preferably, the fluoride source is NaF.

[0184] The pharmaceutical and / or cosmetic formulations according to the present invention may further comprise one or more components selected from the following: sorbitol, xylitol, NaOH, HCl, phosphate buffer solution (PBS), and acetic acid.

[0185] In the currently preferred embodiments, the pharmaceutical and / or cosmetic formulations / compositions comprise sodium fluoride and / or citric acid.

[0186] In a preferred embodiment, the pharmaceutical and / or cosmetic formulation / composition comprises: a. 50 µg / mL of an artificial peptide having the amino acid sequence of SEQ ID NO 4 and / or SEQ ID NO 5, b. 20 mg / mL of hyaluronic acid cellulose (HA-XL(BDDE) or HA-XL(PEGDE)) crosslinked with 1,4-butanediol diglycidyl ether and / or polyethylene glycol diglycidyl ether, c. 2.5 mg / mL of linear hyaluronic acid cellulose (HA), d. 97 wt% water, e. 0.2 wt% sodium fluoride (NaF), f. 0.2 wt% citric acid, g. 0.11 wt% sodium hydroxide, h. 0.3 wt% sodium chloride, i. 0.36 wt% disodium hydrogen phosphate, and j. 0.08 wt% sodium phosphate.

[0187] osmotic molar concentration

[0188] Maintaining an appropriate osmotic molar concentration ensures compatibility with human cells, prevents cell damage, and reduces tissue irritation and minimizes inflammation and other complications. Therefore, maintaining an appropriate osmotic molar concentration for the pharmaceutical and / or cosmetic formulations / compositions of the present invention is highly beneficial. Many buffer solutions (such as PBS) mimic the preferred osmotic molar concentrations of mammals and have an osmotic molar concentration of about 280-310 mOsm / L. Thus, in embodiments, the pharmaceutical and / or cosmetic formulations of the present invention have an osmotic molar concentration of 50-400 mOsm / L, such as 100-310 mOsm / L, or such as 125-175 mOsm / L, such as about 150 mOsm / L, or such as 275-325 mOsm / L, such as about 300 mOsm / L.

[0189] Sodium fluoride

[0190] Sodium fluoride is a common ingredient in dental products such as toothpaste and mouthwash, used to prevent tooth decay and promote oral health. It helps reduce the number of bacteria by inhibiting the growth of bacteria that cause cavities and promoting the remineralization of tooth enamel. Fluoride strengthens teeth by forming fluorapatite, a natural component of tooth enamel. It prevents tooth tissue damage by inhibiting acid production by symbiotic bacteria in the mouth.

[0191] Citric acid

[0192] Citric acid can be used as a preservative and pH adjuster in pharmaceutical and / or cosmetic formulations / compositions according to the present invention. Citric acid has an impact on product stability. It can create an acidic environment, which inhibits bacterial growth and reduces the risk of spoilage. This is because most bacteria prefer neutral or slightly alkaline environments and cannot survive in highly acidic conditions. Citric acid can also chelate or bind certain essential nutrients, such as iron, which are required for the growth of some bacteria.

[0193] Active ingredients

[0194] In embodiments, the pharmaceutical and / or cosmetic formulations according to the present invention may further contain another / additional active ingredient, such as, but not limited to, active ingredients selected from growth factors, plasma-rich fibrin / plasma and enamel matrix derivatives.

[0195] MSC

[0196] Mesenchymal stem cells (MSCs), also known as mesenchymal stromal cells or drug signaling cells, are pluripotent stromal cells that can differentiate into various cell types, including osteoblasts (bone cells), chondrocytes (chondroblasts), myocytes (muscle cells), and adipocytes (adipocytes, which can produce bone marrow adipose tissue).

[0197] MSCs can be isolated from various tissues, such as umbilical cord, endometrial polyps, menstrual blood, bone marrow, and adipose tissue. They exist in the peripheral circulation and home to the site of injury, where they differentiate and promote healing. One of the challenges in using MSCs in clinical applications relates to cell homing, ensuring that cells remain at the site of injury and differentiate to promote healing.

[0198] In some embodiments, the pharmaceutical formulation described herein further comprises MSC cells. In a further embodiment, the pharmaceutical formulation comprises 100,000 to 10,000,000 MSC cells (per milliliter), preferably about 1,000,000 MSC cells (per milliliter). In an additional embodiment, the pharmaceutical formulation promotes homing of mesenchymal stem cell (MSC) cells.

[0199] Medical use

[0200] In the experimental section, the pharmaceutical and / or cosmetic formulations according to the present invention have shown to induce wound healing in soft tissues both in vivo and in vitro, as well as to stimulate collagen production and orientation.

[0201] Therefore, one aspect of the present invention relates to novel pharmaceutical formulations according to the invention for medical use, particularly for soft tissue healing, such as, but not limited to, for wound healing, inducing angiogenesis, inducing re-epithelialization, stimulating collagen production, and / or promoting directed collagen formation.

[0202] In Example 10, the pharmaceutical formulation described herein was demonstrated to have beneficial effects on cartilage and bone healing processes, particularly in pathways such as heparan sulfate degradation, glycosaminoglycan degradation, collagen fiber and other polymer assembly, collagen biosynthesis and modification enzymes, ECM-receptor interactions, extracellular matrix organization, and mineral uptake. Furthermore, it was found that the pharmaceutical formulation accelerated the first stage of fracture healing by upregulating genes such as collagen biosynthesis and modification enzymes.

[0203] The novel pharmaceutical formulations according to the present invention can be further used or combined with the above uses for anti-inflammatory and / or antimicrobial treatment, for the treatment of periodontitis, mucositis, and / or peri-implantitis.

[0204] The novel pharmaceutical formulations according to the present invention can be further used, or combined with the above-described uses, for anti-inflammatory and / or antimicrobial treatment of periodontitis, mucositis, and / or peri-implantitis.

[0205] The novel pharmaceutical formulations according to the present invention can be further used, or combined with the above-described uses, for fracture healing and / or tissue healing.

[0206] As shown in Example 10, the pharmaceutical formulation disclosed herein can assist MSC cells to hom to the site of injury, and provides a formulation containing the common peptide and hyaluronic acid gel disclosed herein with unexpectedly beneficial synergistic effects. In addition to promoting healing, the formulation has been found to reduce local inflammation, induce beneficial epigenetic changes, downregulate key signaling pathways, and activate bone mineralization and BMP pathways.

[0207] Therefore, in embodiments, the pharmaceutical formulations disclosed herein alleviate local inflammation. In further embodiments, the pharmaceutical formulations disclosed herein lead to upregulation of genes involved in collagen biosynthesis and / or collagen-modifying enzymes. Furthermore, the pharmaceutical formulations disclosed herein can activate enzymes and pathways involved in tissue healing processes (such as cartilage). In embodiments, the pharmaceutical formulations alter the expression of genes involved in heparan sulfate degradation, glycosaminoglycan degradation, collagen fiber and other multimer assembly, collagen biosynthesis and modifying enzymes, ECM-receptor interactions, extracellular matrix tissue formation, and / or mineral uptake. In embodiments, altered expression is determined by a decrease or increase in expression exceeding ln(2)-fold compared to a comparison state. Therefore, an altered expression can be considered when the gene expression level increases or decreases by at least ln(2). Therefore, the comparison condition can be basal expression, i.e., expression before injury, expression after injury, expression after treatment with hyaluronic acid gel, or similar conditions.

[0208] In additional embodiments, the pharmaceutical formulations disclosed herein result in downregulation of IL-2, IL-5, IL-3, and / or B-cell receptor signaling pathways. In further embodiments, the pharmaceutical formulations activate bone mineralization and / or BMP pathways.

[0209] In embodiments, the pharmaceutical formulations disclosed herein are used as anti-inflammatory compositions. In further embodiments, the anti-inflammatory effect is measured by a reduction in pro-inflammatory cytokines. In further embodiments, the pro-inflammatory cytokines include one or more cytokines selected from IL-23, IL-1α, IL-1β, TNF-α, MCP-1, IL-12P70, IFN-γ, IFN-β, IL-6, IL-10, IL-27, IL-17A, and GM-CSF.

[0210] In embodiments, the pharmaceutical formulations disclosed herein comprise a common peptide (see Table 4 - HA+HA-XL(BDDE) and HA+HA-XL(BDDE)+P2) that causes altered expression of one or more of 12 key genes identified in cell division, cell cycle, and cell metabolism. In a further embodiment, the formulations disclosed herein (comprising the common peptide and optionally MSC stem cells) induce altered expression of one or more genes, enabling MSCs to interact with cells at the fracture site, activating cell-cell interactions via exosomes, thereby activating downstream metabolism (see Table 4 - HA+HA-XL(BDDE) and HA+HA-XL(BDDE)+P2).

[0211] Periodontal disease

[0212] The formulation disclosed for the first time in this article is intended for use in treating periodontal diseases, disorders and / or conditions, such as by inducing periodontal regeneration, but not limited to.

[0213] Periodontal disease encompasses many forms and symptoms, but the most common are gingivitis and periodontitis. With the increasing prevalence of dental implants in routine dental treatments, the incidence of an associated periodontal disease—peri-implantitis—is rising.

[0214] According to the National Institute of Dentistry and Craniofacial Research, periodontal disease is an infection of the tissues that hold teeth in place. It is usually caused by poor oral hygiene, which allows a sticky film of bacteria called plaque to build up on the teeth, eventually forming tartar.

[0215] Gingivitis is characterized by redness, swelling, and bleeding at the gum line when brushing. It is classified as either chronic or acute. Most adults will experience gingivitis at some point. Gingivitis does not necessarily develop into periodontal disease. It is a silent disease, and patients may only notice it when the condition has become irreversible.

[0216] Acute gingivitis is associated with specific infections, microorganisms, or trauma, while chronic inflammation of the gingival tissue surrounding the teeth is associated with bacterial biofilms covering the teeth and gums.

[0217] Periodontitis is a chronic condition involving bacterial degeneration of the soft and hard tissues surrounding and supporting the teeth. In its early stages, inflammation and irritation are observed in the gums. If left untreated, the gradual loss of bone around the teeth can lead to loose teeth and eventual loss. Tooth loss not only causes complex and expensive dental health problems but can also have a devastating personal impact on a patient's self-confidence.

[0218] Periodontitis is one of the leading causes of poor oral health worldwide. When the disease progresses to severe periodontitis, dentists can use mechanical debridement or bleaching solutions to remove the acidic bacterial layer. While this can temporarily stop the progression of the disease, it cannot reverse the damage. There are currently some surgical alternatives, such as flap surgery, soft tissue grafts, and bone grafts, but these methods are invasive and therefore expensive.

[0219] Periodontitis affects the alveolar bone and supporting tissues, characterized by the formation of periodontal pockets or "gaps" between the teeth and gums. The severity of an attack varies from person to person, depending on the virulence of the bacterial plaque and the efficiency of the patient's local and systemic immune inflammatory response. Undoubtedly, the host's response is influenced by environmental and genetic factors. Systemic diseases are another factor determining the severity of periodontal disease. These diseases may include diabetes, leukemia, Down syndrome, etc. Other factors associated with periodontal disease include smoking and stress.

[0220] Periodontal disease is believed to be linked to a variety of systemic health problems. Growing evidence suggests a link between gum disease and conditions including Alzheimer's disease, cancer (pancreatic cancer), respiratory diseases, diabetes, hypertension, and atherosclerosis. The connection between heart disease (myocardial infarction) and gum disease is also thought to be caused by bacteria. Bacteria in the gums can enter the bloodstream and spread to distant sites, including the heart. Other organs that may be affected include the lungs and reproductive organs, as well as erectile dysfunction.

[0221] "Peri-implantitis" and "peri-implantitis" are dental terms used to describe destructive inflammatory processes affecting the soft and hard tissues surrounding dental implants. Compared to peri-implantitis, the definition of peri-implantitis includes bone loss. Factors influencing this include smoking, bacterial biofilm (plaque) buildup, oral hygiene, and periodontal condition. In this article, the term "periodontal disease" includes peri-implant infections such as peri-implantitis and peri-implantitis.

[0222] The formulations according to the invention are, in one aspect, intended for use in treating periodontal diseases selected from gingivitis, periodontitis, peri-implantitis, perimucosal inflammation, gingivitis, pharyngitis, and other oral infections and / or inflammations.

[0223] wound healing

[0224] Wound healing refers to the process by which a living organism replaces damaged or destroyed tissue with newly generated tissue.

[0225] In undamaged skin, the epidermis (surface, epithelial layer) and dermis (deep layer, connective layer) form a protective barrier against the external environment. When this barrier is broken, a series of regulated biochemical events are initiated to repair the damage. This process occurs in several predictable phases: coagulation (hemostasis), inflammation, tissue growth (cell proliferation), and tissue remodeling (maturation and cell differentiation). Coagulation can be considered part of the inflammatory phase, rather than a separate phase. Example 9 discloses the wound healing effects of the formulations disclosed herein, particularly compositions comprising P2 (SEQ ID NO: 4) or P6 (SEQ ID NO: 5).

[0226] Wound healing is not only complex but also fragile, easily interrupted or failed, leading to chronic, non-healing wounds. Factors contributing to non-healing chronic wounds include diabetes, venous or arterial disease, infection, and age-related metabolic defects. During wound healing, the synthetic peptides in the formulations of this invention promote collagen production and wound filling or refilling, and prevent scar formation by modulating inflammatory signaling. The synthetic peptides in the formulations of this invention can further effectively inhibit wound inflammation.

[0227] As shown in the experimental section, the formulation according to the invention promotes wound healing; for example, in the presence of the P6 active peptide motif, the rate of epithelialization of skin aspiration wounds is twice as fast. Furthermore, it was unexpectedly found that certain embodiments of the formulation, particularly HA+HA-XL(BDDE)+P2, significantly improved wound area reduction compared to HA+HA-XL(BDDE) alone.

[0228] Furthermore, Example 10 discloses that the formulation according to the invention also reduces the levels of inflammatory markers, particularly 13 pro-inflammatory cytokines. In this example, 12 pro-inflammatory cytokines (IL-23, IL-1α, IL-1β, TNF-α, MCP-1, IL-12P70, IFN-γ, IFN-β, IL-6, IL-10, IL-27, IL-17A, and GM-CSF) were detected, and it was found that 12 of the 13 cytokines were reduced after application of the formulation according to the invention. Surprisingly, when measuring pro-inflammatory cytokines, the formulation containing the concordant peptide in the HA+HA-XL(BDDE) crosslinked gel significantly outperformed the HA+HA-XL(BDDE) gel crosslinked alone in almost all respects. This clearly demonstrates that the benefit of formulating the concordant peptide in the HA+HA-XL(BDDE) crosslinked gel is superior to using the HA+HA-XL(BDDE) gel or the concordant peptide alone, and clearly shows the unexpected synergistic effect of the formulation.

[0229] Therefore, the anti-inflammatory effect of the formulation according to the present invention can be measured by reducing pro-inflammatory cytokines. Such pro-inflammatory cytokines may be selected from IL-23, IL-1α, IL-1β, TNF-α, MCP-1, IL-12P70, IFN-γ, IFN-β, IL-6, IL-10, IL-27, IL-17A, and GM-CSF.

[0230] In these embodiments, pro-inflammatory cytokines are administered and measured by flow cytometry. Alternative methods for determining the amount of a specific cytokine will be apparent to those skilled in the art.

[0231] Furthermore, as shown in Example 10, studies have shown that Hyd+P2 significantly accelerates the first stage of tissue healing by upregulating genes such as collagen biosynthesis and modification enzymes, ECM-receptor interactions, and extracellular matrix tissue.

[0232] In this embodiment, administration of the pharmaceutical formulation disclosed herein upregulates tissue healing genes, such as those involved in collagen biosynthesis and modification enzymes, ECM-receptor interactions, extracellular matrix tissues, etc.

[0233] Example 10 further demonstrates that the presence of mesenchymal stromal cells (MSCs) also promotes tissue healing and reduces inflammation. MSCs are well known to proliferate and differentiate into skin cells to restore injured or dead cells, and to stimulate cell regeneration and wound healing through autocrine and paracrine pathways. MSCs participate to varying degrees in all three phases of wound healing and influence whether a wound can successfully transition through the inflammatory phase and avoid progressing to a chronic wound state. MSCs are released from the bone marrow and are generally present in various tissues. After tissue damage, MSCs are recruited to the damaged area through a mechanism known as “MSC homing.” MSC homing is generally considered to be a multi-step process involving injury-region-specific molecular interactions that affect the number of MSCs attached to the injury area, and it is estimated that even with a large number of MSCs present in the injury area, the proportion of cells ultimately efficiently recruited to that area is typically only a few percent.

[0234] Therefore, one of the characteristics of the pharmaceutical formulations disclosed herein is enhanced and / or improved MSC homing. As shown in the examples, the pharmaceutical formulations disclosed herein, particularly those formulated with peptide P2, improve MSC homing, manifested as increased tissue regeneration and higher and more uniform bone formation (see...). Figure 12-15 Furthermore, the presence of MSCs further indicated improved orientation, and smaller T parameters and platelet size, suggesting more mature bone and bone remodeling. Figure 16 and 17 ).

[0235] The induced epigenetic changes, downregulation of local inflammation, and improved fracture healing shown in Example 10 further highlight the beneficial properties of the pharmaceutical formulation disclosed herein for MSC homing. These results clearly demonstrate the synergistic advantage of using the HA+HA-XL(BDDE)+common peptide formulation in reducing inflammation and improving fracture healing in multiple traumatic events.

[0236] In the implementation of the method, administration of the pharmaceutical formulation disclosed herein resulted in epigenetic changes in genes associated with local inflammation downregulation (as shown by cytokine reduction and / or tissue healing) (see Table 4 - HA+HA-XL(BDDE) vs. HA+HA-XL(BDDE)+P2).

[0237] Structurally, the stromal cell polyproline motif of the artificial peptides contained in the formulations according to the present invention provides stability to the extracellular matrix. Biologically, they play important local functions in one-to-many signal transduction, signal transduction, transcription, cell motility, healing and immune regulation, chaperoning, membrane stability, mineral binding, surface recognition and attachment, and participate in various protein-protein interactions.

[0238] These motifs can support dermal healing by promoting EGF expression in connective tissue cells, and can also support angiogenesis and regulate inflammation by stimulating the expression of PDGF, VEGF and anti-inflammatory cytokines.

[0239] This motif is highly conserved in mammals and does not induce any immunogenic response in humans. It was non-toxic in all tested models.

[0240] The artificial peptides contained in the formulations according to the invention also promote local angiogenesis and regulate acute and chronic inflammation. They revitalize senescent cells (such as those senescent due to radiation damage) and reduce pain and swelling when applied topically.

[0241] During wound healing, the artificial peptides contained in the formulations according to the invention particularly promote collagen production and wound filling, and prevent scar formation by regulating inflammatory signal transduction. The artificial peptides contained in the formulations according to the invention stimulate regeneration and healing.

[0242] As shown in the experimental section, the formulation according to the invention provides faster full-thickness wound epithelialization in pigs. More importantly, it promotes collagen expression and angiogenesis, and provides significantly better wound filling than the hydrogel control. In this way, the artificial peptides contained in the formulation according to the invention also prevent scarring and disfigurement in the wound area. As an added value, the artificial peptides contained in the formulation according to the invention also modulate inflammation during the initial healing phase, reducing healing time, swelling, and pain. This effect has also been demonstrated in gingival tissue.

[0243] The experimental results also show that the formulation according to this disclosure promotes re-epithelialization while reducing granulation tissue formation and inflammation 6 days after wound initiation. Furthermore, the formulation according to this disclosure also reduces the wound cavity area 6 days after wound initiation.

[0244] In particular, formulations containing P2 have been found to reduce wound cavity area, promote re-epithelialization, and reduce granulation tissue formation and inflammation 6 days after the injury. Therefore, formulations according to this disclosure can be used to reduce wound cavity area. In other cases, formulations according to this disclosure can also enhance and / or promote wound re-epithelialization and / or reduce granulation tissue formation. In particular, formulations containing the artificial peptide P2 have been found to be especially suitable for enhancing and / or promoting wound re-epithelialization and / or reducing granulation tissue formation.

[0245] In additional cases, the formulations according to this disclosure can also reduce inflammation. In particular, formulations containing the artificial peptide P2 (SEQ ID NO: 4) or P6 (SEQ ID NO: 5) have been found to be particularly suitable for reducing inflammation.

[0246] In summary, the artificial peptides contained in the formulations according to the present invention are effective against connective tissue and can subsequently promote epithelial cell and angiogenesis.

[0247] At dermatological doses (micrograms / mL), the artificial peptides contained in the formulations according to the invention restore the normal activity (revitalize) of radiation-damaged (e.g., sunburn) cells, repair irradiated skin, and regulate inflammation (reduce redness, swelling, and pain).

[0248] In wound care, the formulations according to the invention can be used to promote collagen production in both acute wounds (such as those from oral surgery) and chronic wounds (such as those from periodontitis). Application of the formulations according to the invention modulates inflammation, improves angiogenesis, promotes collagen production and wound filling, and prevents scar formation. Experiments have shown that it improves graft absorption rates after skin and mucosal transplantation surgeries.

[0249] In embodiments, the pharmaceutical and / or cosmetic compositions described herein are used to treat soft tissue wounds and / or promote the healing of soft tissue wounds of the craniomaxillofacial complex (CMS) in patients in need.

[0250] In embodiments, the pharmaceutical and / or cosmetic compositions described herein are used to treat soft tissue wounds and / or promote the healing of soft tissue wounds in the throat, neck, hypopharynx, and / or pharynx of patients in need.

[0251] Obtaining a graft / Preparing a graft

[0252] The pharmaceutical and / or cosmetic compositions described herein can be used to obtain grafts, such as in the treatment of chronic venous leg ulcers.

[0253] Chronic, slow-healing venous ulcers are a debilitating and potentially life-threatening condition affecting millions worldwide. Current treatments for chronic venous leg ulcers aim to control underlying venous insufficiency and promote healing. However, bacterial colonization, complications, and chronic inflammation often complicate the process. Current treatment modalities rely on autologous transplantation and grafting, but success rates are often limited and pose significant risks to patients.

[0254] Current standards for managing non-healing wounds include repeated surgeries, specialized medical knowledge, and prolonged hospitalizations. Furthermore, chronic wounds can lead to serious complications, including amputation, and ultimately death.

[0255] Chronic ulcers are a pressing health problem with high morbidity, posing a challenge for both patients and doctors. These wounds require prolonged treatment, and the long-term adherence by patients often leads to frustration. Despite the availability of medications and surgical treatments, chronic wounds persist and heal poorly if the normal repair process is disrupted. Leg venous ulcers, in particular, are a significant burden on the healthcare system. Although pressure bandages and good wound care are widely considered fundamental treatments, up to a quarter to half of ulcers fail to heal.

[0256] The pharmaceutical and / or cosmetic compositions described herein can provide sustainable, safe, and effective combined or standalone treatments that benefit patients and contribute to the long-term sustainability of healthcare.

[0257] In skin grafting surgery, reticular layered thick skin autografts are widely recognized as the gold standard for treating chronic vascular leg ulcers due to their healing-promoting effects. This type of graft can be expanded using a mesh to allow blood and exudate to drain, thereby improving graft absorption. For wounds with limited vascular supply, such as chronic or refractory ulcers, reticular thick skin autografts are preferred because they contain less tissue and require vascularization after transfer. Full-thickness skin grafts containing the epidermis and the entire dermis pose a challenge for treating patients with impaired arterial circulation because they are too thick to allow for adequate angiogenesis. In such cases, reticular thick skin grafts have a higher survival rate than full-thickness skin grafts. Point grafts are another treatment option for small leg ulcers, but their application is limited by the size requirement of the donor site—the donor area must be approximately twice the size of the target ulcer.

[0258] In the embodiments, the pharmaceutical and / or cosmetic compositions described herein are used in combination with and / or as an alternative to traditional skin grafting techniques using layered thick skin, full-thickness skin, or dotted autologous grafts.

[0259] Furthermore, transplantation can be combined with cell-based therapies, thereby significantly reducing autologous transplantation and associated discomfort, treatment time, and morbidity.

[0260] In this paper, wound preparation has become an essential step in improving the efficacy of autologous skin patch therapy, as it removes damaged tissue and foreign bodies from the wound, promotes the growth of healthy tissue, and reduces the risk of infection. On another front, cell homing relies on growth factors and other biological agents that attract cells to the wound site, promoting wound healing and regeneration. This invention employs an advanced, bio-friendly, non-antibiotic debridement method for wound preparation and uses biomimetic molecules designed to promote connective tissue health to treat the wound, thereby providing a significantly improved combined ATMP skin patch, offering a superior solution for the treatment of chronic ulcers.

[0261] In embodiments, the pharmaceutical and / or cosmetic compositions according to the invention are wound care products containing biomimetic intrinsically disordered proteins (IDPs) delivered in a cross-linked hyaluronic acid gel. The efficacy of this product in wound care models has been demonstrated, particularly its positive impact on angiogenesis via the VEGF pathway. The IPDs are synthetic, possess good safety profiles, and exhibit high binding affinity and specificity to diverse molecular partners, including proteins, nucleic acids, and small molecules, making them ideal for the treatment of chronic venous ulcers. The IPDs can also serve as scaffolds for delivering bioactive peptides or growth factors that promote tissue regeneration and wound healing.

[0262] This new approach has the potential to reduce patient discomfort and treatment time, while improving treatment outcomes and lowering healthcare costs. By reducing the need for tissue engineering and / or autologous transplantation, this innovative approach can significantly improve the treatment of refractory ulcers, thereby accelerating healing time and ulcer healing.

[0263] Therefore, one aspect of the present invention relates to novel treatment methods applicable to specific chronic wounds, particularly those requiring treatment with layered thick-skin mesh grafts.

[0264] Cosmetic uses

[0265] In cosmetic dosages, the artificial peptides contained in the formulations according to the invention preserve and protect connective tissue cells and regulate inflammation. The artificial peptides contained in the formulations according to the invention also promote collagen production and have the potential for use in anti-wrinkle formulations.

[0266] In particular, the artificial peptides described in this application have antimicrobial and anti-inflammatory effects. Therefore, including artificial peptides in hyaluronic acid-based hydrogels adds anti-inflammatory, antimicrobial, antibacterial, and / or antibiotic components to the formulation, thereby making hyaluronic acid-based hydrogels for cosmetic purposes safer and less prone to infection and / or inflammation after implantation or filling.

[0267] In embodiments, the cosmetic compositions described herein are used to improve skin elasticity and hydration. Therefore, in one aspect, the present invention relates to the cosmetic use / application of the cosmetic compositions described herein for improving skin elasticity and hydration in mammals, and to improving skin elasticity and hydration in mammals by applying the cosmetic compositions described herein.

[0268] In one embodiment, the present invention therefore relates to a cosmetic formulation comprising a. 1-3 µg / mL (such as 2 µg / mL) of an artificial peptide having the amino acid sequence of SEQ ID NO: 4 and / or SEQ ID NO: 5; b. 20 mg / mL of hyaluronic acid fibers crosslinked with 1,4-butanediol diglycidyl ether (HA-XL(BDDE)) and / or hyaluronic acid fibers crosslinked with polyethylene glycol diglycidyl ether (HA-XL(PEGDE)); and c. 2.5 mg / mL of linear hyaluronic acid fibers (HA).

[0269] Medical treatment

[0270] In another aspect, the present invention relates to treating patients with soft tissue diseases, disorders, and / or conditions by administering a pharmaceutical preparation according to the invention, thereby inducing soft tissue healing, wound healing, angiogenesis, re-epithelialization, collagen production, and / or directed collagen formation. Examples 10 and 11 provide exemplary uses of the pharmaceutical preparation in wound healing and fracture healing.

[0271] The present invention also relates to, or in combination with, treating patients with soft tissue diseases, disorders and / or conditions by administering a pharmaceutical preparation according to the invention as an anti-inflammatory and / or antimicrobial treatment.

[0272] The present invention also relates to, or in combination with, treating patients suffering from periodontitis, mucositis, or peri-implantitis by administering a pharmaceutical preparation according to the invention to the patient.

[0273] In particular, the present invention relates to the use of pharmaceutical compositions in the preparation of medicaments for treating soft tissue diseases, disorders, and / or conditions. Specifically, the present invention relates to the use of pharmaceutical compositions according to the present invention in the preparation of medicaments for treating soft tissue wounds. In certain embodiments, the present invention also relates to the use of pharmaceutical compositions according to the present invention in the preparation of medicaments for treating periodontal diseases.

[0274] In one aspect, the invention also relates to the use of the pharmaceutical compositions according to the invention in the preparation of medicaments for inducing soft tissue healing, wound healing, angiogenesis, re-epithelialization, collagen production and / or directed collagen formation.

[0275] In one aspect, the invention also relates to the use of the pharmaceutical compositions according to the invention in the preparation of medicaments for anti-inflammatory and / or antibacterial treatment.

[0276] In one aspect, the invention also relates to the use of the pharmaceutical composition according to the invention in the preparation of a medicament for promoting bone mineralization.

[0277] In one aspect, the invention also relates to the use of the pharmaceutical composition according to the invention in the preparation of a medicament for promoting the formation of non-mineralized bone.

[0278] In one aspect, the invention also relates to the use of the pharmaceutical composition according to the invention in the preparation of a medicament for treating patients suffering from periodontitis, mucositis, and peri-implantitis.

[0279] application

[0280] The pharmaceutical and / or cosmetic formulations of the present invention can be administered to subjects in need via any suitable route, depending on the tissue to which the peptide is to be applied, such as by topical (skin), oral cavity, cheek, nasal cavity, ear, rectum or vaginal administration, or by body cavity (such as oral cavity, nasal cavity and vaginal cavity).

[0281] Its application in the field of dentistry / oral medicine is particularly important.

[0282] Furthermore, the compositions are also suitable for use in conjunction with surgical procedures, such as intraoperative incisions. The pharmaceutical and / or cosmetic formulations of the present invention can also be administered via local injection, gel application, or medical devices (such as medical prosthetic devices, such as grafts, scaffolds, or bioglass materials).

[0283] In one aspect, the pharmaceutical and / or cosmetic formulations of the present invention are administered via injection as a minimally invasive procedure.

[0284] In another aspect, the pharmaceutical and / or cosmetic formulations of the present invention are administered orally.

[0285] In one embodiment, the pharmaceutical and / or cosmetic formulation of the present invention is an oral gel product for the care of teeth and oral mucosa. This product prevents dental caries, gingival recession, and periodontitis by forming a protective layer on the teeth and mucosa, thus preventing the formation of plaque and tartar. In one embodiment, it is provided as a unit in a disposable syringe for efficient delivery to the desired area.

[0286] Therefore, one aspect of the present invention relates to the use of the pharmaceutical and / or cosmetic compositions according to the invention in oral application: particularly to the oral mucosa and tooth interface.

[0287] It is applied to the oral mucosa and tooth interface to provide protection for the tissue.

[0288] Methods for preparing pharmaceutical and / or cosmetic formulations

[0289] The present invention also relates to methods for preparing the pharmaceutical and / or cosmetic formulations disclosed herein.

[0290] In some aspects, the present invention relates to a method for preparing the pharmaceutical and / or cosmetic formulations disclosed herein, the method comprising: g. Provide the artificial peptides specified herein. h. Provide linear hyaluronic acid with a molecular weight of 0.7-4 MDa (such as 1.0-2.0, 1.5-1.8, or 3.0-3.3 MDa, such as 1.5 MDa), i. Use a cross-linking agent (such as BDDE or PEGDE) to cross-link hyaluronic acid to obtain cross-linked hyaluronic acid (HA-XL). j. Optionally, the cross-linked hyaluronic acid is dialyzed. k. Mix 0.1-250 µg / mL of the artificial peptide and 1-40 mg / mL of the cross-linked hyaluronic acid, and add 1-40 mg / mL of linear hyaluronic acid to obtain a mixture comprising: - Cross-linked hyaluronic acid, - Linear hyaluronic acid, and - Artificial peptides.

[0291] The peptides are released from a gel containing cross-linked hyaluronic acid, linear hyaluronic acid, and artificial peptides in the aforementioned amounts, and are expected to follow a single-order release profile, in which the peptides are released primarily from the gel via diffusion, with less release via gel degradation. Therefore, gels exhibiting the aforementioned relatively rapid release profile are expected to have positive effects on, for example, wound healing and acute indications requiring rapid peptide onset.

[0292] In other respects, the present invention relates to a method for preparing the pharmaceutical and / or cosmetic formulations disclosed herein, the method comprising: a. Provide the artificial peptides specified in this article. b. Provide linear hyaluronic acid with a molecular weight of 0.7-4 MDa (such as 1.0-2.0, 1.5-1.8, or 3.0-3.3 MDa, such as 1.5 MDa). c. Mix 0.1-250 µg / mL of the artificial peptide and 1-40 mg / mL of the hyaluronic acid. d. Crosslink the mixture using a crosslinking agent (such as BDDE or PEGDE) to obtain a mixture of internally and / or inter-crosslinked peptides with hyaluronic acid. e. Add linear hyaluronic acid (HA) to obtain a mixture of internally cross-linked and / or inter-cross-linked artificial peptides and hyaluronic acid, as well as linear hyaluronic acid.

[0293] In the implementation method, the method further includes: f. Mix 1-40 mg / mL of internally cross-linked and / or inter-cross-linked artificial peptides and hyaluronic acid with 0.1-250 µg / mL of additional artificial peptides to obtain a mixture comprising: - Artificial peptides and hyaluronic acid with internal and / or inter-crosslinking. - Linear hyaluronic acid, and - Artificial peptides.

[0294] Peptide release from gels containing internally and / or inter-crosslinked hyaluronic acid and peptides, linear hyaluronic acid (HA), and non-crosslinked artificial peptides is expected to follow a biphasic order release profile. This profile involves the release of peptides from the gel via diffusion of non-crosslinked peptides and partial release via degradation of crosslinked gels, resulting in a moderate level of peptide release from the gel as a whole. Therefore, gels exhibiting this moderate peptide release profile are expected to produce a dual effect: rapid onset of action and more sustained positive effects on acute indications (such as acute wounds, including surgical wounds, acute inflammation, and acute bone healing, such as post-operative and post-traumatic conditions) and more chronic conditions (such as chronic wounds, chronic periodontitis, or chronic peri-implantitis).

[0295] In an alternative embodiment, no additional artificial peptides are added, so the mixture contains cross-linked peptides and hyaluronic acid, as well as linear hyaluronic acid.

[0296] The release of peptides from gels containing internally and / or inter-crosslinked peptides and hyaluronic acid, as well as linear hyaluronic acid, is expected to follow a monophasic release profile, where the peptides are primarily released after the degradation of the crosslinked gel. Therefore, the aforementioned gels are expected to have a more lasting positive effect on chronic conditions such as chronic wounds, chronic periodontitis, or chronic peri-implantitis.

[0297] Therefore, a further aspect of the invention relates to a method for preparing the pharmaceutical and / or cosmetic formulations of the invention, the method comprising: a. providing an artificial peptide as defined herein; b. providing hyaluronic acid with a molecular weight of 0.7-4 MDa (such as 1.0-2.0, 1.5-1.8, or 3.0-3.3 MDa, such as 1.5 MDa); c. mixing 0.1-250 µg / mL of the artificial peptide and 1-40 mg / mL of the hyaluronic acid; and d. optionally, adding a fluoride source to the mixture, wherein the osmotic pressure of the pharmaceutical and / or cosmetic formulation is 50-400 mOsm / L, such as 100-310 mOsm / L, or such as 125-175 mOsm / L, such as about 150 mOsm / L, or such as 275-325 mOsm / L, such as about 300 mOsm / L. In an additional embodiment, the hyaluronic acid provided in step b. of the method comprises, or is composed of, BDDE and / or PEGDE crosslinked hyaluronic acid.

[0298] In an additional embodiment, the method may include a. providing an artificial peptide at a concentration of up to 100 mg / mL, b. adding hyaluronic acid fibers dissolved in 0.3 M NaOH at a concentration of 10% by weight, c. mixing a crosslinking agent, such as BDDE and / or PEGDE, d. heating the reaction vessel to 20-100°C and incubating for 1-2 hours to induce gelation, e. cooling and neutralizing the solution, f. homogenizing the gel, g. dialyzing the gel in sterile PBS for at least 18 hours, h. adding linear hyaluronic acid in an amount of about 10% of the crosslinked hyaluronic acid, i. homogenizing the formulation to ensure uniform distribution of the peptide, and j. optionally adding phosphate buffered solution (PBS).

[0299] In an additional embodiment, the method may include a. providing a hyaluronic acid fiber solution dissolved in 0.3M NaOH at a concentration of 10% by weight; b. mixing a cross-linking agent, such as BDDE and / or PEGDE, into the hyaluronic acid solution; c. heating the hyaluronic acid solution to 20-100°C and incubating for 1-2 hours to induce gelation; d. cooling and neutralizing the solution; e. homogenizing the gel; f. dialyzing the gel in sterile PBS for at least 18 hours; g. adding linear hyaluronic acid in an amount of about 10% of the cross-linked hyaluronic acid; h. adding an artificial peptide up to the desired concentration, the peptide being added in lyophilized powder form or in an aqueous solution with a concentration up to 100 mg / ml; i. homogenizing the formulation to ensure uniform peptide distribution; and j. optionally adding phosphate buffered solution (PBS).

[0300] In an embodiment of the method for preparing a pharmaceutical and / or cosmetic formulation according to the present invention, the artificial peptide in step a. can be dissolved in 1% acetic acid at a concentration of 10 mg / mL, then b. hyaluronic acid cellulose (3.0-3.3 MDa) dissolved in an alkaline solution (preferably NaOH) at a concentration of 10% by weight is added, c. 1,4-butanediol diglycidyl ether (BDDE) and / or poly(ethylene glycol) diglycidyl ether (PEGDE) are mixed, d. the reaction vessel is heated to about 40°C and incubated for about 4 hours, e. the solution is cooled and neutralized with an acidic solution (preferably HCl), and f. the gel is homogenized into particles of 100-400 µm.

[0301] In an embodiment, the method further includes g. dialysis of the gel in a biocompatible solution (such as PBS or physiological saline), h. optionally adding linear hyaluronic acid in an amount of about 10% by weight of the cross-linked hyaluronic acid, i. homogenizing the preparation to ensure uniform distribution of the peptides, and j. optionally adding phosphate buffer solution (PBS).

[0302] In an embodiment, a method for preparing a pharmaceutical and / or cosmetic formulation according to the present invention comprises: steps a. adding hyaluronic acid cellulose (3.0-3.3 MDa) dissolved in an alkaline solution (preferably NaOH) at a concentration of 10% by weight; b. mixing 1,4-butanediol diglycidyl ether (BDDE) and / or poly(ethylene glycol) diglycidyl ether (PEGDE); c. heating the reaction vessel to approximately 40°C and incubating for approximately 4 hours; d. cooling and neutralizing the solution with an acidic solution (preferably HCl); e. optionally, dialyzing the gel in a biocompatible solution (such as PBS or physiological saline); f. homogenizing the gel into particles of 100-400 µm; g. dissolving the artificial peptide in 1% acetic acid at a concentration of 10 mg / mL; and h. mixing the dissolved peptide into the homogenized gel.

[0303] In an embodiment, the method additionally includes i. optionally adding linear hyaluronic acid in an amount of about 10% by weight of the crosslinked hyaluronic acid, j. homogenizing the formulation to ensure uniform distribution of the peptides, and k. optionally adding phosphate buffered solution (PBS).

[0304] In one embodiment, the method additionally includes the step of terminally sterilizing the gel.

[0305] In one embodiment, the preparation method includes: 1. Dissolve the artificial peptide in distilled water to achieve a final concentration of approximately 0.1-250 g / mL, 1-10 mg / mL, or at least 0.1 g / mL; 2. Vortex mixing; 3. Prepare an aqueous solution containing water for injection, NaF, citric acid, NaCl, Na2HPO4, NaH2PO4, and artificial peptides (such as P2 and P6); 4. Add NaOH to adjust the pH; 3. Add sodium hyaluronate; 4. Optionally, a crosslinking agent, such as BDDE and / or PEGDE, may be added; 5. Mix gently for 24 hours; 6. Fill one or more syringes; 7. Pack into small bags or blister packs; 8. High-pressure sterilization of packaged products.

[0306] In one embodiment, the preparation method includes: 1. Dissolve the artificial peptide in distilled water to achieve a final concentration of approximately 0.1-250 g / mL, 1-10 mg / mL, or at least 0.1 g / mL; 2. Vortex mixing; 3. Prepare an aqueous solution containing water for injection, NaF, citric acid, NaCl, Na2HPO4, NaH2PO4, and artificial peptides (such as P2 and P6); 4. Add NaOH to adjust the pH; 5. Provided a mixture of crosslinked and linear hyaluronic acid fibers (HA+HA-XL) disclosed herein, preferably crosslinked using a crosslinking agent (such as BDDE and / or PEGDE), and the mixture comprising about 90% HA-XL and about 10% HA; 6. Mix the peptide aqueous solution with the mixture of HA-XL and HA.

[0307] In an embodiment, the preparation method further includes mixing the peptide aqueous solution with a mixture of HA-XL and HA for 24 hours under gentle stirring. The preparation method may further include filling the mixture into one or more syringes, optionally packaging the syringes into pouches or blister packs, and optionally autoclaving the packaged product.

[0308] In this embodiment, the syringe has a volume of approximately 0.5-1.5 mL.

[0309] Therefore, the present invention also relates to a kit for clinical use, comprising a sachet, wherein the sachet comprises one or more disposable syringes pre-filled with the pharmaceutical formulation disclosed herein, and instructions for use.

[0310] Compared to natural proteins, artificial peptides are shorter in length, thus making them easier to prepare, for example, through synthetic preparation or biosynthesis. The artificial peptides of this invention can be prepared by any known peptide preparation method, such as chemical synthesis. Synthetic preparation also allows the use of amino acid analogs, which can improve the stability of the prepared peptides. Those skilled in the art will know which methods can be used to synthesize amino acid sequences.

[0311] Preferably, bioprocessing can be used as a method for preparing peptides. Bioprocessing refers to the preparation of amino acid sequences in biological systems, such as cell cultures or microbial cells, such as bacterial cells. In order to perform bioprocessing, the corresponding nucleic acid sequence encoding the specific amino acid sequence must be constructed. Once the specific amino acid sequence to be synthesized is determined, those skilled in the art will readily know how to reverse-engineer the corresponding nucleic acid sequence, how to prepare the peptide, and how to purify it from the production system used (see, for example, Svensson J, Andersson C, Reseland JE, Lyngstadaas SP, Bulow L. Histidine tag fusion increase expression levels of active recombinant Amelogenin in Escherichia coli. Protein Expr Purif, 48; 134-41 (2006)).

[0312] In one embodiment, the product is filled into a glass syringe, such as, but not limited to, a BD Hylok™ pre-filled glass syringe, which is also autoclaved to reduce bacterial contamination. Prior to autoclaving, it may be filled into an E-line pouch to maintain the product's sterility. In addition to the syringe containing the gel, the pouch also contains an application tip (19G, blunt tip).

[0313] This allows for effective use in dental clinics when delivered with pre-filled, single-use syringes (e.g., 1 mL or 0.7 mL capacity). To prevent cross-contamination between users, each package consists of, for example, 2, 4, or up to 8 single-use units and a matching dispensing tip. The syringe and dispensing tip are packaged together in a single pouch, which is aseptically packaged.

[0314] The product can be sterile or non-sterile.

[0315] Pharmaceutical and / or cosmetic formulations obtained according to the method of the present invention

[0316] In one aspect, the present invention relates to pharmaceutical and / or cosmetic preparations obtained according to the method of the present invention, and their use as pharmaceuticals.

[0317] This invention also relates to pharmaceutical and / or cosmetic formulations comprising an artificial peptide as defined herein or a combination of two or more artificial peptides as defined herein. Such pharmaceutical and / or cosmetic formulations may also optionally comprise a pharmaceutically acceptable carrier, excipient, and / or diluent. Furthermore, such pharmaceutical and / or cosmetic formulations may also comprise a fluoride source, such as one or more selected from NaF, CaF2, and ZnF2.

[0318] In this document, compositions include pharmaceutical and cosmetic compositions, as well as compositions that fall in the so-called gray area between pharmaceuticals and cosmetics, namely cosmeceuticals.

[0319] The pharmaceutical composition is usually in gel form, preferably in hydrogel form.

[0320] In one aspect, the pharmaceutical and / or cosmetic formulations of the present invention are formulated for administration via minimally invasive injection.

[0321] In one aspect, the pharmaceutical and / or cosmetic formulations of the present invention are formulated for oral administration. In another aspect, the pharmaceutical and / or cosmetic formulations of the present invention are formulated for delivery via skin application, topical application, and / or transdermal patch delivery.

[0322] The composition can be formulated using conventional pharmaceutical methods, for example, see “Remington’s Pharmaceutical Sciences” and “Encyclopedia of Pharmaceutical Technology”, edited by Swarbrick, J. & JC Boylan, MarcelDekker, Inc., New York, 1988.

[0323] Pharmaceutically or cosmetically acceptable excipients, carriers, and / or diluents are substances that are substantially harmless to the individual to whom the composition is to be applied. Such excipients, carriers, and / or diluents generally comply with the requirements of national health authorities. Official pharmacopoeias, such as the British Pharmacopoeia, the United States Pharmacopoeia, and the European Pharmacopoeia, all specify standards for pharmaceutically acceptable excipients.

[0324] Whether a pharmaceutically acceptable excipient is suitable for use in a pharmaceutical composition generally depends on the chosen dosage form. Examples of suitable pharmaceutically acceptable excipients according to the present invention for use in different types of compositions are given below.

[0325] The selection of pharmaceutically acceptable excipients and their optimal concentrations in the compositions used according to the present invention is generally unpredictable and must be determined based on experimental evaluation of the final composition. However, those skilled in the art of pharmaceutical formulation can find guidance in publications such as "Remington's Pharmaceutical Sciences," 18th Edition, Mack Publishing Company, Easton, 1990.

[0326] Those skilled in the art will readily understand that the concentration of the artificial peptide in the pharmaceutical composition of this invention will vary depending on the intended use of the composition. Typically, the concentration of the peptide in the pharmaceutical composition is about 0.01-1 mg / mL. The amount typically administered to a subject in vivo is about 10 ng / mL. 2 Up to 0.1 mg / cm 2 The preferred concentration is approximately 1 ug / cm³. 2 .

[0327] In one embodiment, the pharmaceutical and / or cosmetic composition according to the invention comprises the following ingredients listed in Table 3.

[0328] Table 3. An exemplary list of ingredients in the final pharmaceutical and / or cosmetic composition according to the present invention, arranged in descending order of ingredient content, including ingredient supplier, purity grade and weight percentage.

[0329]

[0330] The pharmaceutical and / or cosmetic composition according to the present invention is based on hyaluronic acid gel, meaning that the two main components are water and hyaluronic acid. To provide effective oral protection, it also contains sodium fluoride and artificial peptides, both of which form a mineral protective layer on teeth and protect gums. It also contains citric acid for a fresh taste. The addition of sodium hydroxide adjusts the pH to 6.0-7.0, and the addition of sodium chloride, disodium hydrogen phosphate, and monosodium phosphate increases the osmotic molar concentration.

[0331] It should be understood that although the invention has been described in conjunction with its detailed description, the foregoing description is intended to illustrate rather than limit the scope of the invention, which is defined by the scope of the appended claims.

[0332] Other aspects, advantages, and modifications fall within the scope of the following claims.

[0333] The following non-limiting experiments further illustrate the present invention.

[0334] Example

[0335] The following embodiments are provided to guide those skilled in the art in practicing representative implementations of the subject matter of this disclosure. Given the invention and the general level of expertise in the art, those skilled will understand that the following embodiments are intended to be exemplary only, and many variations, modifications, and alterations can be made without departing from the scope of this disclosure.

[0336] Example 1

[0337] This embodiment aims to compare two crosslinked hyaluronic acid gels prepared using two different crosslinking agents, BDDE and PEGDE. For both agents, crosslinking is induced through a similar process; therefore, the objective is to determine whether the two agents possess different physicochemical properties using rheology, FTIR, NMR, and SEM. One objective is to understand the nature of the methodological steps, specifically by in-situ monitoring of crosslinking kinetics via rheology and employing experimental designs to investigate how variable hyaluronic acid concentration, time, and temperature affect rheological properties.

[0338] Materials and Methods

[0339] Material

[0340] Pharmaceutical grade high molecular weight (MW = 1.5 MDa, IV = 22.2 m) 3 Hyaluronic acid ( / kg) was supplied by Fidia Farmaceutici SpA (Abano Terme, Italy). 1,4-Butanediol diglycidyl ether (BDDE) and polyethylene glycol diglycidyl ether (PEGDE, M) were also supplied. n = 500 Da), anhydrous sodium hydroxide and sodium chloride were supplied by Merck KGaA, Germany. Potassium bromide (99%, IR grade) was purchased from J&K Scientific GmbH, Germany.

[0341] Gel preparation

[0342] 10 w / v% hyaluronic acid was dissolved in 0.3M NaOH solution with manual stirring. 1.6 v / v% BDDE or 3.0 v / v% PEGDE was added and stirred. The solution was incubated in a sealed container at 40°C for 4 hours. The gel was then transferred to a cellulose membrane (MWC = 14 kDa) and dialyzed against distilled water for 18 hours. The gel was granulated by extrusion through a 130 µm mesh, and then further distilled water was added to obtain a final concentration of 20 mg / mL.

[0343] Rheological properties of gel samples

[0344] Amplitude scanning was used to study the viscoelastic region of the gel samples by applying a logarithmic slope of shear strain from 0.01% to 100% at a frequency of 10 rad / s. Furthermore, frequency scanning analysis was applied to understand the crosslinking properties of the gel in the frequency range of 0.1–100 rad / s at 0.1% shear strain.

[0345] Real-time crosslinking analysis

[0346] The kinetics of gel crosslinking were investigated using rheology. Dissolved hyaluronic acid was mixed with the crosslinking agent and immediately transferred to a rheometer for measurement. Crosslinking kinetics at 40°C were observed by monitoring changes in viscoelastic properties over a period of 5 hours at a constant shear strain of 0.1% and a frequency of 10 rad / s.

[0347] Material characterization

[0348] FTIR

[0349] Infrared spectroscopy was performed on pure hyaluronic acid, BDDE, PEGDE, and synthesized HA-XL(BDDE) and HA-XL(PEGDE) hydrogels using a Varian 640-IR spectrometer (Agilent Technologies, Santa Clara, CA, ISA). KBr pellets were used to record the spectral density from 400 to 4000 cm⁻¹. -1 The spectrum is within the range, with a resolution of 4 cm. -1 Each scan was performed 50 times. The gel sample used 300 mg KBr and 20 mg of gel.

[0350] Morphological representation

[0351] After the crosslinking step, the samples were dehydrated in an ethanol bath with progressively increasing concentrations until absolute ethanol was reached, and then dried overnight at 60°C and 10% humidity. Environmental scanning electron microscopy analysis of the gold-sputtered samples was performed using an Evo 50 EP instrument (Zeiss, Jena, Germany).

[0352] NMR

[0353] Liquid NMR

[0354] NMR spectroscopy was used to study the hyaluronic acid functionalization and cross-linking degree of the gel, and to confirm the removal of unreacted cross-linking agents after dialysis.

[0355] The lyophilized gel was diluted with deuterated water to 4 mg / mL, and its concentration was collected using a Bruker Advance NMR spectrometer (operating frequency 400 MHz, temperature 298 K). 11H-NMR spectra. Deuterated solvent (D2O) is used as a deuterated interlock. Chemical shifts (δ) are reported in ppm (parts per million) with reference to residual D2O. Spectra were processed and visualized using MestReNova x64 (Mestrelab Research).

[0356] Integrating the 1.60 and 2.0 ppm signals (I δH1.60 and I δH2.0 The degree of modification (MoD) of HA-XL (BDDE) gel is measured using [a specific method], which is the number of moles of cross-linking agent bound to each disaccharide unit (usually expressed as a percentage).

[0357] For HA-XL (PEGDE) hydrogels, due to signal overlap, the peaks in the chemical shift range of 3.20–4.0 ppm include both hyaluronic acid protons and PEGDE protons. Although PEGDE protons can be partitioned to a peak at 3.68 ppm, they cannot be integrated independently; therefore, the integral in the 3.20–4.0 ppm region is <(I δH3.20-4.0 The integral of hyaluronic acid within that range must be subtracted. This is the integral of PEGDE, and the PEGDE residue contains an average of 40.52 protons. Therefore, the degree of modification can be calculated based on the subtracted integral and the integral of the signal at 2.20 ppm.

[0358] This value represents the average degree of modification, as cross-linked PEGDE exhibits a molar mass distribution.

[0359] Leaching studies

[0360] To investigate the removal of unreacted crosslinking agents, a leaching study was employed. Gels were prepared as described above. Before and after dialysis, the gels were removed, and the hyaluronic acid concentration was adjusted to 20 mg / mL. The samples were then granulated as described above. Subsequently, 2 g of each sample was transferred to a cellulose membrane (MWC = 14 kDa) and placed in a sealed vial containing 50 mL of dH₂O. After 7 days, 1.8 mL of solution was removed from each sample, transferred to an NMR tube, and the liquid phase was removed. The residue was then dissolved in 1.8 mL of deuterated water and analyzed by NMR as described above.

[0361] Cytotoxicity test

[0362] Cytotoxicity assays were performed to determine if any leached material would adversely affect cell proliferation. Gel preparation was similar to that described above, but sterile physiological saline was used instead of distilled water. Samples were transferred to syringes and autoclaved at 121°C for 15 minutes. Osteoblasts (MC3T3-E1) were seeded at a concentration of 40,000 cells per well with 1 mL of cell culture medium in 24-well plates. Approximately 0.1 mL of gel was added to the insert (0.4 µm PET membrane, Merck) and then to the wells. Six wells were used per group, including a positive group (cells killed with Triton X-100 1 hour prior to completion) and a negative control (cells only). Quantitative cytotoxicity was assessed using the LDH activity of the gel, and cell viability was calculated using a CCK8 assay. The LDH assay detected the amount of LDH leaking through the plasma membrane of damaged cells as a marker of cytotoxicity. Cytotoxicity levels below 30% and cell viability above 70% were considered acceptable according to ISO 10993-5.

[0363] result

[0364] Using the above method, cross-linked hyaluronic acid gels were prepared using BDDE (1.6 vol%) and PEGDE (3 vol%) as cross-linking agents. A schematic diagram of this method can be seen below. Figure 1 Then, the two different gels were characterized by physicochemical properties and cytotoxicity tests. Although different molar equivalents of the two cross-linking agents (BDDE and PEGDE) and hyaluronic acid were used, the different gels still showed similar rheological data, indicating that the cross-linking agent plays a crucial role in the gel properties.

[0365] Rheological analysis

[0366] Rheology is used to understand the mechanical properties of gels. Gels exhibit very similar properties, with a wide viscoelastic region and shear strains exceeding 1000%. Figure 2 A, left figure). Furthermore, their frequency scanning behavior is similar ( Figure 2 A, right figure). The selected PEGDE concentration (3.0 vol%) resulted in gel behavior similar to that of 1.6 vol% BDDE. Figure 2 The rheological results shown confirm this. Therefore, although the molar equivalent of hyaluronic acid to crosslinking agent in PEGDE gel is lower than that in BDDE gel, the results indicate that PEGDE-crosslinked HA-XL (HA-XL(PEGDE)) gel is more elastic than BDDE-crosslinked HA-XL (HA-XL(PEGDE)) gel.

[0367] In addition, real-time rheological measurements were performed on the in-situ crosslinking process of the gel. Figure 2B). It can be seen that the two gel types exhibit similar kinetic behavior, with HA-XL(PEGDE) reaching its gelation point (G' = G'') faster than HA-XL(BDDE), after approximately 30 minutes, while HA-XL(BDDE) gels take about 1 hour to gel. They do indeed appear to converge to G' values ​​of approximately 1 MPa (HA-XL(BDDE)) and 2 MPa (HA-XL(PEGDE)), respectively, indicating that HA-XL(PEGDE) has higher structural stability than HA-XL(BDDE), consistent with the shear strength results described above.

[0368] Chemical characterization

[0369] To characterize the material properties from a chemical perspective, FTIR and NMR were employed. Based on FTIR analysis, several expected peaks of cross-linked hyaluronic acid were detected. Figure 2 C). In Figure 2 In C, 1650 cm -1 The peak at 1050-1150 cm⁻¹ represents the carboxylic acid group (C=O) of hyaluronic acid, while the peak at 1050-1150 cm⁻¹ represents the carboxylic acid group (C=O). -1 The broad bimodal peaks correspond to the typical C–O–C and C–O stretching of the ether bonds formed during the crosslinking process. This indicates that crosslinked hyaluronic acid gels were successfully prepared regardless of whether BDDE or PEGDE was used as the crosslinking agent. The peaks at 2900-3000 cm⁻¹... -1 Weak peaks were observed at 3050–3300 cm⁻¹, which correlated with the epoxy end groups bound to the HA hydroxyl groups in the crosslinking agent; this indicates that unreacted BDDE or PEGDE was successfully removed during the dialysis step. Finally, at 3050–3300 cm⁻¹ -1 There is a broad peak that is associated with intermolecular -OH groups and H bonds. These intermolecular bonds are crucial for the consistency of the gel after granulation and give it a "sticky" property, which is very beneficial for clinical treatment.

[0370] Morphological representation

[0371] Following the crosslinking step, the samples were dehydrated in an ethanol bath with progressively increasing concentrations until absolute ethanol was achieved, and then dried overnight at 60°C and 10% humidity. Environmental scanning electron microscopy analysis of the gold-sputtered samples was performed using an Evo 50 EP instrument (Zeiss, Jena, Germany).

[0372] Leaching studies

[0373] To investigate the removal of unreacted crosslinking agents, a leaching study was employed. Gels were prepared as described above. Before and after dialysis, the gels were removed, and the hyaluronic acid concentration was adjusted to 20 mg / mL. The samples were then granulated as described above. Subsequently, 2 g samples were transferred to a cellulose membrane (MWC = 14 kDa) and placed in a sealed bottle containing 50 mL of pure H₂O. After 7 days, 1.8 mL of solution was removed from each sample, transferred to an NMR tube, and the liquid phase was removed. The residue was then dissolved in 1.8 mL of deuterated water and subjected to the above-described process. 1 H NMR analysis.

[0374] discuss

[0375] The inventors characterized two different crosslinked hyaluronic acid hydrogels using BDDE or PEGDE as crosslinking agents. Crosslinking was induced using a similar method in both cases, and a granulation step was also performed to ensure compatibility with needle injection in a clinical setting. The method begins with the dissolution of hyaluronic acid. Since its solubility limit is typically around 4 mg / mL (depending on salt and molecular weight), it is necessary to dissolve it in an alkaline environment, which is ensured using sodium hydroxide (NaOH). The alkaline environment deprotonates the hydroxyl groups of the hyaluronic acid molecules, thereby increasing its reactivity and promoting its reaction with the epoxy groups of BDDE to achieve crosslinking. Since crosslinking is almost impossible at hyaluronic acid concentrations of 5% by weight or lower, a hyaluronic acid concentration of 10% by weight was used. Once the hyaluronic acid is dissolved, the crosslinking agent can be readily incorporated. The crosslinking process is time-dependent and can be accelerated by increasing the temperature. This is advantageous because a high pH accelerates the degradation of hyaluronic acid, leading to a competitive reaction between BDDE or PEGDE crosslinking and hyaluronic acid degradation. In real-time crosslinking studies, gel points were observed in PEGDE and BDDE gels after incubation at 40°C for approximately 30 and 60 minutes, respectively. Both gels appeared to follow an S-shaped crosslinking kinetic curve, where the increase in crosslinking decreased over time.

[0376] From an industrial perspective, limiting the crosslinking time may be beneficial for economic reasons. In our experimental setup, crosslinking was stopped after 4 hours. This can be achieved by neutralizing the pH with an acid (such as HCl) or by transferring the gel to a dialysis buffer. The dialysis step is intended to allow unreacted crosslinking agents to diffuse out of the gel.

[0377] To understand the efficacy of the dialysis step, leaching studies were performed on the gel before and after dialysis, and the leaching composition was characterized using NMR. Before dialysis, peaks associated with cross-linking agents appeared in the range of 3.3–3.9 ppm (for BDDE) and 3.5–3.75 ppm (for PEGDE) (Figure 3). After 18 hours of dialysis, these peaks had diminished, indicating that diffusion effectively removed unreacted BDDE and PEGDE from the gel. Further removal of unreacted cross-linking agents can be achieved, for example, by increasing the dialysis fluid volume or changing the dialysis medium. The latter was implemented by changing the dialysis medium after two hours of dialysis and then continuing dialysis for another 16 hours. Diffusion is also time-dependent, so extending the time also contributes to removal. For in vivo applications, it is desirable to perform dialysis in a solution suitable for in vivo use (e.g., PBS buffer or physiological saline) to obtain appropriate osmolarity.

[0378] Example 2

[0379] Preparation of formulations comprising BDDE-crosslinked hyaluronic acid and linear hyaluronic acid and co-peptides.

[0380] 1. While stirring, add hyaluronic acid (3.1 mg / mL) 3 ( / kg) is dissolved in 0.3M NaOH to obtain a concentration of 100 mg / mL.

[0381] 2. Add 16 µL / mL of 1,4-butanediol diglycidyl ether (BDDE) to the hyaluronic acid solution.

[0382] 3. Incubate the solution in a sealed container at 40°C for 4 hours to allow it to crosslink.

[0383] 4. Neutralize the gel with 1M HCl and gently shake overnight.

[0384] 5. Transfer the gel to a cellulose membrane with a molecular weight cutoff of 14 kDa, and then dialyze it in sterile phosphate-buffered saline (PBS) for 18 hours.

[0385] 6. The gel is granulated by extruding it through a stainless steel mesh with a pore size of 200µm.

[0386] 7. Prepare solutions of phosphate-buffered saline, hyaluronic acid, sodium fluoride, and the concomitant peptide separately using the following methods: a. Add hyaluronic acid equivalent to 10% by weight of the final solution to PBS and stir for 1 hour to hydrate.

[0387] b. Simultaneously, the peptide (P2 or P6) is dissolved in sterile water with a concentration of 10 mg / mL to prepare a peptide (P2 or P6) solution.

[0388] c. Add the peptide solution to the PBS-hyaluronic acid solution to achieve a final mixed concentration of 1-500 µg / mL gel.

[0389] 8. Add the PBS-hyaluronic acid solution to the granulated cross-linked hyaluronic acid-BDDE and linear hyaluronic acid and mix. Homogenize the solution with stirring for 6 hours.

[0390] 9. Transfer the gel to a suitable delivery system and sterilize it using autoclaving.

[0391] Example 3

[0392] A formulation was prepared consisting of BDDE-crosslinked hyaluronic acid, linear hyaluronic acid, and peptides, and doped with 2000 ppm sodium fluoride.

[0393] Hyaluronic acid gel crosslinked with BDDE and linear hyaluronic acid were prepared in a manner similar to that in Example 2. Then, a solution of PBS, hyaluronic acid, and peptides was prepared as in Example 2, with a slight modification: NaF was incorporated at a final concentration of 2000 ppm. The gel and solution were mixed together, transferred to a delivery system, and sterilized using autoclaving.

[0394] Example 4

[0395] Formulations containing linear hyaluronic acid, peptides, and sodium fluoride

[0396] 1. Dissolve 50 mg / mL of hyaluronic acid in 0.3 M NaOH using manual stirring.

[0397] 2. Adjust the concentration with sterile PBS so that the concentration of hyaluronic acid is 20 mg / mL at the end of the method.

[0398] 3. Add 100 ng / mL to 25 µg / mL of the common peptide and mix.

[0399] 4. Add 1 M HCl to neutralize the solution and stir to homogenize.

[0400] 5. Add 2000 PPM of NaF and mix.

[0401] Example 5

[0402] Release of biotin-labeled peptides from hyaluronic acid solutions and hyaluronic acid gels

[0403] Gels containing 500 µg / mL biotin-labeled P6 peptides as described in Examples 2-4 were prepared. 0.2 mL of gel was added to each well of a 24-well insert (0.4 µm, PET), with 1 mL of distilled water added to each well. At 3, 7, and 12 hours, 1 µL of water was collected, and its absorbance at 205 nm was measured using a NanoDrop One (Thermo Scientific, US) and compared with reference curves for 1, 10, 25, and 100 µg / mL P6-biotin distilled water. The reference curves were converted to mass release, and release percentage curves were plotted. The results showed that after 12 hours, the linear hyaluronic acid (HA) gel (Example 2) released 91% of its peptide loading, while the cross-linked HA-XL gel (Example 1) released 23% (e.g., ...). Figure 4 (As shown). This indicates that different methods of preparing gels can be used to regulate peptide release, and that the combination of cross-linked gels and linear gels (HA+HA-XL) provides more controlled and tunable peptide release compared to linear gels only (HA).

[0404] Example 6

[0405] Theoretical Examples of Treatment for Gingivitis

[0406] The gel prepared in Example 4 is filled into a syringe. The syringe is used to apply the gel directly to the gums of patients showing signs of gingival recession, or those prone to redness and other signs of inflammation. The gel is then spread evenly over the gums.

[0407] The expected results show that the gel composed of the shared peptides and fluoride has a better ability to reduce mucosal redness and other inflammatory symptoms compared to using either component alone. Furthermore, plaque formation at the gum and tooth junction will also be reduced. This is clinically significant because preventing inflammation and plaque formation will help prevent gingival recession and periodontitis.

[0408] Example 7

[0409] A study on the effect of NuPep gel on improving soft tissue wound healing after gingival tissue regeneration / periodontal surgery

[0410] This example aims to demonstrate the effect of an artificial peptide-loaded gel on improving soft tissue wound healing after periodontal surgery. In six pigs, gingival tissue from three premolars was dissected using a scalpel. The gel from Example 2 was used, wherein the concentration of P2 or P6 was 50 µg / mL. Experiments were conducted using peptide-free hyaluronic acid and Emdogain. (Straumann, Basel, CH) and a sham surgery control were used as controls. The gel was filled between the gum and the tooth, allowing it to close on its own.

[0411] Six days later, the pigs underwent scar removal treatment. In all gel cases, the gums were able to reattach to the molars, while the reattachment varied in the sham surgery cases. Significant pus and inflammation were observed when hyaluronic acid gel was used alone, but not when using peptide-loaded gel or in the sham surgery control.

[0412] Example 8

[0413] Preventive application

[0414] The purpose of this embodiment is to demonstrate how the formulation can exert a preventive effect on soft tissue using the gel described in Example 4.

[0415] Materials and Methods:

[0416] A 5% by weight hyaluronic acid solution in 0.3M NaOH was prepared. The solution was neutralized with a distilled aqueous solution of 11.65M HCl and 0.9% NaCl to obtain a final concentration of 2% by weight hyaluronic acid. Equal amounts of P2 and P6 were mixed into the acidic solution to obtain a final peptide concentration in the range of approximately 50 ng / mL to 500 ng / mL. The remaining preparation steps were performed as specified in Example 4.

[0417] Clinical use:

[0418] This solution can be used on the mucosal membranes of teeth in patients prone to periodontitis and gingivitis. When applied via syringe or tubing, the solution can be spread to evenly cover the patient's mucosal membrane surface. Prior to treatment, patients should undergo professional cleaning by a professional (e.g., a dentist or dental hygienist) to remove the mucosal membrane or any plaque between the mucosal membrane and tooth enamel.

[0419] Expected results: Patients should be less likely to exhibit less redness and swelling. Furthermore, patients should be less likely to experience the progression of periodontitis or gingival recession.

[0420] Example 9

[0421] Soft tissue wound healing effects of HA+HA-XL(BDDE) / co-peptides

[0422] Target

[0423] This embodiment aims to demonstrate the effect of artificial peptide-loaded gel on improving soft tissue wound healing after periodontal surgery.

[0424] method

[0425] In six pigs, eight rows of incisions were created along the spine on the back, with two incisions on each side of the spine (labeled L, LM, RM, R from left to right when viewed from the rear, with the spine located between LM and RM). The spacing between the incisions was 3 cm. The incisions were created using an 8 mm diameter histological punch, and then the skin was cut off with a scalpel.

[0426] A 10th row was created at the bottom, with only two wounds at the LM and RM positions, serving as a reference for wound care.

[0427] Bleeding from the back wound was stopped after a 10-minute pause, followed by treatment. The following treatments were used (abbreviations in parentheses): sham surgery (SHAM), HA+HA-XL(BDDE), HA+HA-XL(BDDE)+NuPep P2, HA+HA-XL(BDDE)+NuPep P6 (HA+P6), a mixture of HA+HA-XL(BDDE)+P2 and P6 (1:1 mixture), HA-XL(BDDE)+EMD (HA+HA-XL(BDDE)+EMD), Emdogain (EMD), NuBone Clean (after removal; NB Rem), and NuBone Clean (without removal; NB). A gel, comprising linear HA and cross-linked HA, was prepared as described in Example 2 and was designated HA+HA-XL(BDDE). Treatment was administered via syringe, except for the first three animals where a spatula was used for the HA+HA-XL(BDDE)+P2 and P6 mixture and HA+HA-XL(BDDE)+EMD (the application procedure was adjusted due to the greater convenience of syringe administration).

[0428] After wound treatment, a plastic covering is applied to the wound, followed by a dressing (with holes along the back). For row 10, the plastic covering is cut open to expose the wound, and VetricynVF Plus (standard care) is applied. Row 10, as standard treatment, allows for determination of when 50% wound closure has occurred.

[0429] Wound healing is tracked using histological staining (see, for example) Figure 8 (Example staining in the image), and wound healing is treated from the following variables: - Measured wound width (um) - Measured wound depth (µm) - Calculated wound void area (mm²) 2 ) - Re-epithelialization (observed by keratinization of the skin in the wound area) is scored from 0 to 3, among which... o 0: No epithelium; o 1: Partial reepithelialization; o 2: Most of it was completely re-epithelialized; o 3: Complete reepithelialization - Granulation tissue (observed by the presence / absence of proliferating cells and collagen fiber deposition) was scored from 0 to 3 (see Gupta, A and Kumar, P., 2015. Assessment of the histological state of the healing wound. Plastic and Aesthetic Research, 2, pp. 239-242), where o 0: Complete granulation tissue formation; o 1: Moderate granulation tissue formation; o 2: A small amount of granulation tissue forms; o 3: No granulation tissue formation - Inflammatory infiltration (observed by the presence of inflammatory cells around the wound area or necrotic tissue) is scored from 0 to 3, where o 0: Infected / necrotic tissue is present; o 1: Complete inflammation; o 2: Moderate inflammation; o 3: Does not exist result The ability of hydrogel compositions containing HA+HA-XL (BDDE) gel crosslinking and co-peptides P2 and / or P6 to promote wound healing was tested, measured by wound void area, reepithelialization score, presence of granulation tissue, and inflammation score (see [link to relevant documentation]). Figure 6 , 7 An evaluation will be conducted.

[0430] First, compositions containing P2 (SEQ ID NO: 4), P6 (SEQ ID NO: 6), or P2 and P6 were compared with HA+HA-XL (BDDE) alone and sham surgery controls, and different evaluation criteria were assessed 6 days after wound induction. It was observed that peptide P2 in BDDE-crosslinked HA (P2+ HA+HA-XL (BDDE)) was superior to sham surgery and HA+HA-XL (BDDE) alone in terms of wound void area, and the wound void area was significantly reduced compared to HA+HA-XL (BDDE) alone or sham surgery. Figure 6 a; Figure 6In this context, HA+HA-XL(BDDE) is represented as HA. Furthermore, compared to sham surgery, HA+HA-XL(BDDE)+P2 significantly improved reepithelialization; simultaneously, there is a trend indicating that both HA+HA-XL(BDDE)+P2 and HA+HA-XL(BDDE)+P6 can improve reepithelialization compared to HA+HA-XL(BDDE) alone. Figure 6 b; in Figure 6 In this context, HA+HA-XL(BDDE) is represented as HA. Furthermore, HA+HA-XL(BDDE)+P2 showed significant improvement in granulation tissue. Figure 6 c; in Figure 6 In the study, HA+HA-XL(BDDE) was designated as HA, with minimal to no granulation tissue formation (granulation score 2-3), while the remaining groups showed moderate to minimal granulation tissue formation (granulation score approximately 1-2). Finally, considering wound inflammation scores, sham surgery, HA+HA-XL(BDDE), and HA+HA-XL(BDDE)+P6 / P2 showed complete inflammation; while P2+HA+HA-XL(BDDE) and P6+HA+HA-XL(BDDE) showed moderate inflammation. Figure 6 d; in Figure 6 In this study, HA+HA-XL(BDDE) is represented as HA. Data indicate that P2+HA+HA-XL(BDDE) and P6+HA+HA-XL(BDDE) provide superior anti-inflammatory effects compared to HA+HA-XL(BDDE) alone.

[0431] Therefore, the first result indicates that the formulation of the co-peptide in the composite cross-linked-linear cross-linked HA gel (HA+HA-XL(BDDE)) has a beneficial effect; in addition, although P2 was shown to have a beneficial effect according to the evaluation criteria, P6 was shown to be beneficial in reducing inflammation, thus indicating that both peptides have a beneficial effect on wound healing.

[0432] As a second validation, compositions containing P2 or P6 were compared with Emdogain® and Emdogain® (EMD) formulated with HA+HA-XL (BDDE). Emdogain® is an enamel matrix extract containing amelogenin of varying molecular weights.

[0433] from Figure 7 It is evident that when assessing inflammation ( Figure 7d) Formulations containing P2 and P6 were superior to EMD and EMD+HA+HA-XL (BDDE); meanwhile, compositions containing P2 showed improved wound pore area compared to EMD and EMD+HA+HA-XL (BDDE), improved reepithelialization score compared to EMD, and improved granulation formation score compared to EMD and EMD+HA+HA-XL (BDDE).

[0434] In summary, these results indicate that peptide P2 performed better than HA+HA-XL(BDDE) crosslinked gels alone on all variables except reepithelialization. There is a trend suggesting that peptide P6 also leads to inflammation reduction in HA+HA-XL(BDDE) crosslinked gels, to a degree seemingly similar to that achieved by peptide P2 in HA+HA-XL(BDDE) crosslinked gels.

[0435] Furthermore, peptide P2, in gels intercalated with HA+HA-XL(BDDE), exhibited improved wound healing and re-epithelialization compared to EMD, with similar granulation formation and infiltration responses. HA-XL(BDDE)+P6 provided a similar response to EMD. HA-XL(BDDE)+P2 also outperformed HA-XL(BDDE)+EMD, demonstrating the efficacy of P2 compared to EMD.

[0436] In summary, this embodiment demonstrates that both peptides P2 and P6 have unexpectedly beneficial effects on wound healing, surpassing the effects of using HA+HA-XL (BDDE) gel alone. Furthermore, the beneficial effects of the co-peptides are more significant compared to using EMD alone or in formulation within HA+HA-XL (BDDE) gel.

[0437] Example 10

[0438] Target

[0439] The purpose of this embodiment is to illustrate that the formulation disclosed herein promotes the homing ability of MSCs to facilitate tissue healing.

[0440] method

[0441] Animal surgeries were performed in the Department of Orthopaedic Surgery at the University of California, Davis, following the ARRIVE guidelines and authorized by the Institutional Animal Care and Use Committee (IACUC). A total of 60 C57BL6J mice were used in this study (n=8 per group at each time point). A multiple trauma model (femoral osteotomy, 4 mm defect size, plus chest trauma) was established using a total of 60 C57BL6 / J mice, and inflammatory response and bone formation were investigated in three groups after three weeks of healing: 1) HA+HA-XL(BDDE), 2) HA+HA-XL(BDDE)+P2, and 3) HA+HA-XL(BDDE)+P2+MSC. Hydrogels were prepared as described in Example 2. For HA+HA-XL(BDDE)+P2+MSC, one million MSCs were encapsulated in each hydrogel. After creating a stable femoral osteotomy defect, the hydrogel was injected into the defect site, followed by a weight-drop device to induce chest trauma, resulting in bilateral hemopneumothorax. Three weeks after healing, fractured femur, serum, and total RNA from fractured femur tissue were collected. Biomineralization, inflammatory markers, and pathways were investigated using micro-CT (µCT), histology, immunohistochemistry, simultaneous accelerated SAXS / XRD, serum cytokine flow cytometry multiplex analysis, and bulk RNA sequencing as described below or known in the art.

[0442] In summary, micro-CT (µCT) was performed using a 1172 micro-CT imaging system (Bruker microCT, Kontich, Belgium) desktop X-ray CT scanner with a voxel resolution of 5.9 μm, an X-ray tube current of 169 μA, and a voltage of 55 kV, without any filters. The sample was mounted vertically on a plastic support and rotated 180° around its long axis (z-axis). Four absorption images were recorded for every 0.5850° rotation. These projected radiographic images of the porous structure were first reconstructed into continuous coronal orientation tomographic maps using a 3D cone-beam reconstruction algorithm (NRecon, Bruker microCT, Kontich, Belgium). Beam hardening was set to 20%, and annular artifact reduction was set to 12. 3D reconstruction of the internal pore morphology was performed using these axial bitmap images and analyzed using CTAn (Bruker microCT, Kontich, Belgium). The grayscale threshold was set to 18 to 255; additional noise was removed using a despeccary function. Therefore, all objects smaller than 300 voxels and not attached to the 3D model were removed before further analysis. Closed and open porosity were measured.

[0443] In summary, histology and IHC were performed after µCT imaging. Samples were cut in half to investigate bone formation and biomineralization in calcified and decalcified bone tissue. One half of the defect was embedded in methyl methacrylate (MMA) to preserve calcified tissue. The other half was decalcified using 10% EDTA for four months and then embedded in paraffin. Paraffin samples (eight samples per group) were cut transversely along the midline of the animal body to a thickness of 5 µm. Due to surgical difficulty, three MMA-embedded samples were randomly selected, longitudinally cut to a thickness of 5 µm along the midline of the pig, and placed on Kawamoto membrane (SECTION-LAB Co. Ltd., Hiroshima, Japan). The balance of bone mineralization / demineralization over time was assessed using Movat Pentachrome and Von Kossa / Van Gieson staining. Alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) enzyme histochemistry were performed to investigate the balance of osteoblasts and osteoclasts. Sirius Red was used to assess collagen fiber properties such as width, length, straightness, and angle.

[0444] Immunohistochemistry was performed using primary antibodies (Abcam Company, Cambridge, UK). The antibodies used were: rabbit monoclonal antibody (EPR53) against α-smooth muscle actin (α-SMA), rabbit monoclonal antibody (EPR14334) against Runt-related transcription factor 2 (Runx 2), rabbit polyclonal antibody (OAA100188) against osteopontin, mouse monoclonal antibody (LS-C83497-100) against osteocalcin, and rabbit monoclonal antibody (EPR7785) against type I collagen.

[0445] To study angiogenesis, α-SMA was diluted 1:1000 in DAKO-Diluent (S 0809). For bone formation in calcified sections, osteopontin, osteocalcin, and type I collagen were diluted 1:250, 1:800, and 1:2000 in DAKO-Diluent, respectively. Runx 2 was diluted 1:500 in DAKO-Diluent to study bone formation in decalcified sections. Goat anti-rabbit (BA-1000, Vector) and methyl green staining were used as secondary antibodies and counterstains, respectively.

[0446] Decalcified Movat pentachrome stained sections and calcified α-SMA stained sections were selected for descriptive studies of general tissue formation. In Movat pentachrome stained sections, two independent and calibrated examiners used a 3-point system (poor, fair, and good, corresponding to scores 1 to 3) to study tissue homogeneity, integrity, and defect closure in all groups. Additionally, α-SMA stained sections were used, employing a 3-point system to study vascular phenotype and its regularity over time. Round vessels were defined as regular type 1, small to medium oval vessels as regular type 2, and large oval or other undefined shapes as irregular type 3.

[0447] In summary, multiplex flow cytometry analysis of serum cytokines was performed using a 24-color antibody panel on an Aurora Spectroflow Cytometer (Cytek Biosciences, Inc, California). Ne-peak rainbow beads (Biolegend) provided the basis for voltage settings, and a compensation control was established using monochromatic control supercompensation beads (ThermoFisher Scientific) via automatic compensation. Voltage settings for each parameter minimized spillover between fluorophores. Data were acquired on the Aurora Spectroflow Cytometer (Cytek Biosciences, CA) and analyzed using SpectroFlo™ software. The resulting FCS files were examined using the Cytobank platform (Beckman Coulter Inc, CA).

[0448] In summary, synchrotron accelerated SAXS / XRD was performed on 70 µm thick calcified bone sections to investigate collagen / hydroxyapatite (HAp) orientation and hydroxyapatite plate size. SAXS / XRD scans were conducted at the Petra III synchrotron radiation beamline P12 of the Deutsche Electron Synchrotron (DESY) in Hamburg, Germany, operated by the European Molecular Biology Laboratory (EMBL). The photon energy used was 18 keV, and the beam size was 80 µm × 80 µm. The sample-to-detector distance was 1.5 m. A Pilatus 6 M detector (Dectris, Switzerland) with a pixel size of 172 µm was used. The exposure time was one second. Due to the energy setting, short sample-to-detector distance, and large detector size, SAXS and XRD signals could be recorded simultaneously on a single detector. Data averaging and reduction were performed using the EMBL analysis pipeline. Grid scanning was used with a step of 0.15 mm perpendicular to the sample and 10 mm parallel to the sample. The total size of the scanning area is adjusted according to the size of each sample.

[0449] Hydroxyapatite (HAp) platelet parameters and orientation data were evaluated using an internally written MATLAB® script. The T parameter is a measure of HAp platelet size, indicating HAp platelet thickening.

[0450] statistics

[0451] When normality tests failed, one-way ANOVA onranks was performed, with post-hoc comparisons using the Kruskal-Wallis test. Otherwise, a regular ANOVA was performed, with post-hoc comparisons using the Tukey test. All analyses were performed on GraphPad Prism 8 (GraphPad Software Company, San Diego, CA). Significant differences are expressed as follows: P < 0.05, P < 0.01, P < 0.001 and P < 0.0001.

[0452] result

[0453] µCT and histological staining of mineralized and non-mineralized tissues showed that HA+HA-XL(BDDE)+P2 enhanced bone mineralization at the fracture site compared with HA+HA-XL(BDDE) alone or HA+HA-XL(BDDE)+MSC. Figure 12-15 Furthermore, the HA+HA-XL(BDDE)+P2+MSCs group stimulated significantly higher and more uniform bone formation than other groups. Figure 12-15 ).

[0454] In addition, SAXS / XRD data ( Figure 16 and 17 This study confirmed that the HA+HA-XL(BDDE)+P2 and HA+HA-XL(BDDE)+P2+MSC groups exhibited higher degrees of orientation and smaller T parameters and platelet size, indicating better orientation / organization of the ultrastructure (see [link]). Figure 16 and 17 ), which means a more mature bone.

[0455] Analysis of 13 pro-inflammatory cytokines (IL1α, IL1β, TNFα, IL6, IL23, MCP1, IL12P70, IFNγ, IFNβ, IL27, IL10, IL17A, and GM CSF) showed that the HA+HA-XL(BDDE)+P2 and HA+HA-XL(BDDE)+P2+MSC groups reduced the systemic inflammatory response to the healthy baseline value three weeks after healing. Figure 9-10 ).

[0456] As mentioned above, batch sequencing was used to perform gene expression analysis to gain deeper insights into the shared peptide (P2) and the pathways affected by MSC cells.

[0457] Batch sequencing further confirmed that the HA+HA-XL(BDDE)+P2 group significantly accelerated the first stage of fracture healing by upregulating genes such as collagen biosynthesis and modification enzymes, ECM-receptor interactions, and extracellular matrix tissue (see Table 4). A total of seven genes showed significant gene expression changes (see Table 4). These genes are mainly involved in the healing process of cartilage and bone, such as heparan sulfate degradation, glycosaminoglycan degradation, assembly of collagen fibers and other multimer structures, collagen biosynthesis and modification enzymes, ECM-receptor interactions, extracellular matrix tissue, and mineral absorption. In addition, functional enrichment and gene set enrichment analysis (GSEA) showed that reactive oxygen species (ROS) and reactive nitrogen species (RNS) produced in phagocytes, PI3K-AKT, Ras, phagosomes, and lysosomes may lead to low expression of cytokines and reduced inflammation at the fracture site within 3 weeks.

[0458] Table 4 - HA and HA+HA-XL(BDDE)+P2

[0459] Table 5 - HA+HA-XL(BDDE) and HA+HA-XL(BDDE)+P2+MSC

[0460] Table 6 - HA+HA-XL(BDDE)+P2 and HA+HA-XL(BDDE)+P2+MSC

[0461] A comparison between the HA+HA-XL(BDDE)+P2+MSC group and the HA group revealed significant changes in the expression of a total of 14 genes (see Table 5). Surprisingly, compared to HA+HA-XL(BDDE)+P2, HA+HA-XL(BDDE)+P2+MSC significantly altered pathways involving more epigenetic changes, cell cycle, and protein expression at fracture sites. These included E2F-mediated DNA replication regulation, TP53-regulated metabolic and DNA repair genes, and snRNP assembly. The IL-2, IL-5, IL-3, and B-cell receptor signaling pathways were significantly downregulated in the HA+HA-XL(BDDE)+P2+MSC group. Similar to the HA+HA-XL(BDDE)+P2 group, the HA+HA-XL(BDDE)+P2+MSC group also activated bone mineralization and BMP pathways.

[0462] This demonstrates that the HA+HA-XL(BDDE)+P2+MSC formulation induced epigenetic changes, downregulation of local inflammation, and fracture healing.

[0463] These results indicate that the combined use of HA+HA-XL(BDDE)+P2 and HA+HA-XL(BDDE)+P2+MSC has a synergistic advantage in reducing inflammation and improving fracture healing in multiple traumas.

[0464] The results presented in this article indicate that HA+HA-XL(BDDE)+P2 (regardless of whether it contains MSC) are effective agents for reducing inflammatory responses to multiple traumas and guiding bone formation.

[0465] Furthermore, this embodiment demonstrates that the shared peptides embedded in the hydrogel promote MSC homing, thereby maintaining, for example, the ability of mesenchymal stem cells to invade the gel, proliferate and differentiate into osteocytes, and maintain sufficient metabolic activity of MSCs after implantation.

[0466] Example 11

[0467] Release of P2 peptide from hyaluronic acid solution and HA+HA-XL(BDDE).

[0468] Target

[0469] This example aims to illustrate the effect of the amount of cross-linked hyaluronic acid in the gel on gel swelling and the release of common peptides from the gel.

[0470] method

[0471] Hyaluronic acid gels HA-XL (BDDE) crosslinked with BDDE, hyaluronic acid solution (linear) HA, and gels prepared by mixing 90% HA-XL (BDDE) and 10% HA, all containing 500 µg / mL of P2 peptides (HA-XL (BDDE) + P2, HA + P2, HA + HA-XL (BDDE) + P2). 0.1 mL of each gel was added to 24-well inserts (0.4 µm, PET), with 1 mL of distilled water added to each well, and the mixture was kept for 12 h or 24 h. After the time point, the remaining distilled water in the wells was extracted, the swollen hydrogel in the insert was weighed, and its absorbance at 562 nm was measured using the Micro BCA™ Protein Assay Kit (Thermo Scientific, US) and a BioTek ELx800 microplate reader (BioTek Instruments, Inc., Winooski, VT, USA). The absorbance was compared with reference curves for albumin standards at concentrations of 0.5, 1, 2.5, 5, 10, 20, 40, and 200 µg / mL in distilled water. The absorbance was converted to protein concentration using the reference curves, and bubble charts were plotted to represent the percentage release and relative weight change over time.

[0472] result

[0473] Figure 18 The results showed that cross-linked HA-XL(BDDE) gel (12h: 27%; 24h: 28%) and the combination of cross-linked / linear gels (HA+HA-XL(BDDE)+P2 (12h: 25%; 24h: 24%)) exhibited higher peptide release compared to linear gel (12h: 14%; 24h: 18%). However, the swelling of cross-linked HA-XL(BDDE) gel (12h: 234%; 24h: 306%) and HA+HA-XL(BDDE) gel (12h: 249%; 24h: 342%) was reduced compared to linear non-cross-linked gel (HA) (12h: 468%; 24h: 484%). This suggests that the swelling and peptide release can be controlled by adjusting the gel preparation method.

[0474] Table 7: Relative weight change [%], peptide release percentage [µg / ml], and residual peptide concentration in gel [µg / ml] of P2 peptide-enhanced crosslinked HA-XL(BDDE) [HA-XL], linear HA [HA], and crosslinked HA-XL(BDDE) and linear HA combination [HA+HA-XL(BDDE)] gels over time.

[0475]

[0476] Example 12:

[0477] Target

[0478] This study aimed to understand the effects of novel proline-rich hyaluronic acid gels on early immune responses in periodontal regeneration compared to conventional biomaterials.

[0479] method

[0480] The gingiva of three premolars in each quartile of six conventional pigs was dissected using a scalpel. Using a blunt-tipped needle, the defects were treated as follows: none (sham surgery; n=6), HA+HA-XL(BDDE)+P2 (n=6), HA+HA-XL(BDDE)+P6 (n=6), HA+HA-XL(BDDE) (n=3), or Emdogain® (n=3). Hyaluronic acid was cross-linked with BDDE and mixed with 10% sodium hyaluronate. P2 and P6 are two distinct, amelogenin-mimicking proline-rich peptides. Animals were euthanized after 6 days, and local immune responses were investigated using histological staining (Masson Goldner Trichrome, expert-rated) and RT-PCR. In vitro biocompatibility was assessed using cell viability assays.

[0481] result

[0482] Histological scores showed that, compared with Emdogain®, HA+HA-XL(BDDE)+P2 and HA+HA-XL(BDDE)+P6 improved the immune response to cross-linked hyaluronic acid (see [link to article]). Figure 19 ).

[0483] In vitro survival tests showed that, compared with the control group, the cell viability of both peptide treatment groups was improved (median: HA+HA-XL(BDDE): 98%; HA+HA-XL(BDDE)+P2: 104%; HA+HA-XL(BDDE)+P6: 115%) (see... Figure 20 ).

[0484] RT-PCR results are expected to show changes in gene expression profiles caused by different treatment regimens.

[0485] Example 13

[0486] Preparation of formulations comprising BDDE-crosslinked hyaluronic acid and linear hyaluronic acid and co-peptides.

[0487] 1. While stirring, add hyaluronic acid (3.1 mg / mL) 3 ( / kg) is dissolved in 0.3M NaOH to obtain a concentration of 100 mg / mL.

[0488] 2. Simultaneously, the peptide (P2 or P6) is dissolved in sterile water with a concentration of 10-20 mg / mL to prepare a peptide (P2 or P6) solution.

[0489] 3. Optionally, the peptide solution is added to the hyaluronic acid solution to a final concentration of 1-2500 µg / mL gel.

[0490] 4. Add 16 µL / mL of 1,4-butanediol diglycidyl ether (BDDE) to the hyaluronic acid-peptide solution.

[0491] 5. Incubate the solution in a sealed container at 40°C for 4-20 hours to allow it to crosslink.

[0492] 6. Neutralize the gel with 1M HCl and gently shake overnight.

[0493] 7. Transfer the gel to a cellulose membrane with a molecular weight cutoff of 14 kDa, and then dialyze in sterile phosphate-buffered saline (PBS) for 18 hours.

[0494] 8. Optionally, the peptide concentration in the cross-linked gel is quantified using Micro BCA, as described in Example 11.

[0495] 9. The gel is granulated by extruding it through a stainless steel mesh with a pore size of 200µm.

[0496] 10. Add 10-90% by weight of hyaluronic acid to the PBS separately and stir for 1 hour to hydrate.

[0497] 11. Add PBS-hyaluronic acid solution to particulate cross-linked hyaluronic acid-BDDE and linear hyaluronic acid, mix to achieve the desired HA / HA-XL ratio, and optionally add peptide solution to achieve a final peptide concentration of 1-500 µg / mL gel.

[0498] 12. Homogenize the solution for 6 hours with stirring.

[0499] 13. Transfer the gel to a suitable delivery system and sterilize it.

Claims

1. A pharmaceutical and / or cosmetic formulation in gel form, comprising: a. An artificial peptide comprising or consisting of the following amino acid sequence: Pro-XX-Pro-YYY-Pro-XX-Pro-YY-Pro-XX-Pro-X-Pro-YYYYYY-Pro-YYYYY-Pro-XX-Pro-X-Pro-YYY-Pro-YY-Pro-Y-Pro-XX-Pro-Y-Pro-Y-Pro-XX-Pro-Y-Pro-Y-Pro-XX-Pro-Y-Pro-XX-Pro-Y-Pro-XX-Pro-Y-Pro-X-Pro-Pro-XXXXXXXX-Pro-XX-Pro-XXXX (SEQ ID NO 1), wherein i) Pro is proline; ii) X is an amino acid selected from Ala, Ile, Leu, Met, Phe, Trp, and Val; iii) Y is an amino acid selected from Asn, Cys, Gln, Ser, Thr, and Tyr, and b. Linear hyaluronic acid fibers (HA) at 1-40 mg / mL and cross-linked hyaluronic acid fibers (HA-XL) at 1-40 mg / mL.

2. A pharmaceutical and / or cosmetic formulation in gel form, comprising: a. Artificial peptides containing or composed of the following amino acid sequences: Pro-XX-Pro-YY-Pro-XX-Pro-YY-Pro-XX-Pro-YY-Pro-Y-Pro-Pro-X-Pro-Pro (SEQ ID NO 2), where i) Pro is proline; ii) X is an amino acid selected from Ala, Ile, Leu, Met, Phe, Trp, and Val; and iii) Y is an amino acid selected from Asn, Cys, Gln, Ser, Thr, and Tyr, and b. Linear hyaluronic acid fibers (HA) at 1-40 mg / mL and cross-linked hyaluronic acid fibers (HA-XL) at 1-40 mg / mL.

3. The pharmaceutical and / or cosmetic formulation according to claim 1 or 2, wherein the artificial peptide has at least 90% identity with an artificial peptide selected from the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, for example, wherein the artificial peptide is one or more artificial peptides selected from artificial peptides containing the amino acid sequences of SEQ ID NO: 4 and SEQ ID NO:

5.

4. A pharmaceutical and / or cosmetic formulation according to any one of the preceding claims, comprising: a. 0.1-250 µg / mL, for example, 0.1, 1.0, 5.0, 10, 50, 100, 200 or 250 µg / mL of the artificial peptide, b. Cross-linked hyaluronic acid fibers (HA-XL) at concentrations of 1-40 mg / mL, such as 1.0, 2.5, 4, 10, 20, 25, 30, or 40 mg / mL, and c. 1-40 mg / mL, such as 1.0, 2.5, 4, 10, 20, 25, 30 or 40 mg / mL linear hyaluronic acid fibers (HA).

5. The pharmaceutical and / or cosmetic formulation according to any one of the preceding claims, wherein the crosslinked hyaluronic acid fiber (HA-XL) comprises, or is composed of, hyaluronic acid fiber crosslinked with 1,4-butanediol diglycidyl ether (HA-XL(BDDE)) and / or hyaluronic acid fiber crosslinked with poly(ethylene glycol) diglycidyl ether (PEGDE) (HA-XL(PEGDE)).

6. A pharmaceutical and / or cosmetic formulation according to any one of the preceding claims, comprising: a. An artificial peptide with an amino acid sequence of SEQ ID NO: 4 and / or SEQ ID NO: 5, at a concentration of 50 µg / mL. b. 20 mg / mL of hyaluronic acid fibers crosslinked with 1,4-butanediol diglycidyl ether (HA-XL(BDDE)) and / or hyaluronic acid fibers crosslinked with poly(ethylene glycol) diglycidyl ether (HA-XL(PEGDE)), and c. 2.5 mg / mL linear hyaluronic acid fibers (HA).

7. A pharmaceutical and / or cosmetic formulation according to any one of the preceding claims, comprising: a. 2 µg / mL of an artificial peptide having the amino acid sequence SEQ ID NO: 4 and / or SEQ ID NO: 5, b. 20 mg / mL of hyaluronic acid fibers crosslinked with 1,4-butanediol diglycidyl ether (HA-XL(BDDE)) and / or hyaluronic acid fibers crosslinked with poly(ethylene glycol) diglycidyl ether (HA-XL(PEGDE)), and c. 2.5 mg / mL linear hyaluronic acid fibers (HA).

8. The pharmaceutical and / or cosmetic formulation according to any one of the preceding claims, wherein the hyaluronic acid fiber is 0.7-4.0 MDa, for example 1.0-2.0, 1.5-1.8 or 3.0-3.3 MDa, for example 1.5 MDa.

9. The pharmaceutical and / or cosmetic formulation according to claim 6, 7 or 8, further comprising: d. One or more buffers, e. Fluoride source, f. One or more salts, and g. Water.

10. The pharmaceutical and / or cosmetic formulation according to claim 6, 7 or 8, further comprising: d. Water e. Sodium fluoride (NaF) f. Citric acid, g. Sodium hydroxide, h. Sodium chloride, i. disodium hydrogen phosphate, and j. Sodium phosphate.

11. The pharmaceutical and / or cosmetic formulation according to any one of the preceding claims, wherein the release rate of the artificial peptide is controlled to be 0.01-10 ug / hour, for example about 0.01, 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 ug / hour.

12. A pharmaceutical and / or cosmetic formulation according to any one of the preceding claims, wherein the composition further comprises mesenchymal stromal cells (MSCs).

13. The pharmaceutical and / or cosmetic formulation according to claim 12, wherein the composition comprises 100,000-10,000,000 MSCs / ml, preferably about 1,000,000 MSCs / ml.

14. The pharmaceutical formulation according to any one of claims 1 to 13, wherein the composition promotes MSC homing.

15. A pharmaceutical preparation according to any one of the preceding claims, which is used as a medicine.

16. The pharmaceutical preparation according to any one of the preceding claims, for use in soft tissue healing, induction of angiogenesis, induction of re-epithelialization, stimulation of collagen production, promotion of directed collagen formation, post-transplant resorption, chronic wound healing, for anti-inflammatory and / or antimicrobial treatment and / or for treatment of periodontitis, peri-implantitis, perimucosal inflammation, gingivitis, aphthous stomatitis and / or infection and / or inflammation in the soft tissues of the craniofacial complex.

17. The pharmaceutical preparation according to any one of claims 1 to 13, which is used as an anti-inflammatory composition.

18. A pharmaceutical formulation for the use of claim 17, wherein the anti-inflammatory effect is measured by a reduction in pro-inflammatory cytokines.

19. The pharmaceutical formulation for use according to claim 18, wherein the pro-inflammatory cytokine comprises one or more cytokines selected from IL-23, IL-1α, IL-1β, TNF-α, MCP-1, IL-12P70, IFN-γ, IFN-β, IL-6, IL-10, IL-27, IL-17A and GM-CSF.

20. A method for preparing a pharmaceutical and / or cosmetic formulation according to any one of the preceding claims, the method comprising: a. To provide the artificial peptide as defined in any one of claims 1 to 3, b. Provide hyaluronic acid with a molecular weight of 0.7-4 MDa, such as 1.0-2.0, 1.5-1.8, or 3.0-3.3 MDa, such as 1.5 MDa. c. Mix 0.1-250 µg / mL of the artificial peptide and 1-40 mg / mL of the HA, and d. Optionally, a fluoride source is added to the mixture. The pharmaceutical and / or cosmetic formulations described herein have an osmotic molar concentration of 50-400 mOsm / L, for example 100-310 mOsm / L, or for example 125-175 mOsm / L, for example about 150 mOsm / L, or for example 275-325 mOsm / L, for example about 300 mOsm / L.

21. The method of claim 20, wherein the hyaluronic acid comprises, or is composed of, BDDE and / or PEGDE crosslinked hyaluronic acid and linear hyaluronic acid.

22. A method for preparing a pharmaceutical and / or cosmetic formulation according to any one of claims 1 to 19, the method comprising: a. To provide the artificial peptide as defined in any one of claims 1 to 3, b. Provide linear hyaluronic acid with a molecular weight of 0.7-4 MDa, such as 1.0-2.0, 1.5-1.8, or 3.0-3.3 MDa, such as 1.5 MDa. c. Use cross-linking agents, such as BDDE or PEGDE, to cross-link hyaluronic acid to obtain cross-linked hyaluronic acid (HA-XL). d. Optionally, dialyze cross-linked hyaluronic acid. e. Mix 0.1-250 µg / mL of the artificial peptide and 1-40 mg / mL of the cross-linked hyaluronic acid, and add 1-40 mg / mL of linear hyaluronic acid to obtain a mixture comprising: - Cross-linked hyaluronic acid, - Linear hyaluronic acid, and - Artificial peptides.

23. A method for preparing a pharmaceutical and / or cosmetic formulation according to any one of claims 1 to 19, the method comprising: a. To provide the artificial peptide as defined in any one of claims 1 to 3, b. Provide linear hyaluronic acid with a molecular weight of 0.7-4 MDa, such as 1.0-2.0, 1.5-1.8, or 3.0-3.3 MDa, such as 1.5 MDa. c. Mix 0.1-250 µg / mL of the artificial peptide and 1-40 mg / mL of the hyaluronic acid. d. Use cross-linking agents, such as BDDE or PEGDE cross-linking mixtures, to obtain a mixture of internally and / or inter-crosslinked peptides with hyaluronic acid. e. Add linear hyaluronic acid (HA) to obtain a mixture of internally cross-linked and / or inter-cross-linked artificial peptides and hyaluronic acid, as well as linear hyaluronic acid.

24. The method of claim 23, wherein the method further comprises: g. Mix 1-40 mg / mL of internally cross-linked and / or inter-cross-linked artificial peptides and hyaluronic acid with 0.1-250 µg / mL of additional artificial peptides to obtain a mixture comprising: - Artificial peptides and hyaluronic acid with internal and / or inter-crosslinking. - Linear hyaluronic acid, and - Artificial peptides.

25. A pharmaceutical and / or cosmetic formulation obtained by the method according to any one of claims 20 to 24.

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