Consensus peptides in hya

Abstract

Pharmaceutical and / or cosmetic formulations are described and their uses in medicine comprising artificial peptides characterized by a proline sequence prevalent in key matricellular proteins that partake in wound healing as well as in bone and cartilage formation and connective tissue maintenance in all vertebrates. The current invention relates to a pharmaceutical formulation in the form of a gel comprising a. an artificial peptide comprising the amino acid sequence of Pro-X-X-Pro-Y-Y-Y-Pro-X-X-Pro-Y-Y-Pro-X-X-Pro-X-Pro-Y-Y-Y-Y-Y-Y-Pro-Y-Y-Y-Y-Y-Y-Pro-X-X-Pro-X-Pro-Y-Y-Y-Pro-Y-Y-Pro-Y-Pro-X-X-Pro-Y-Pro-Y-Y-Pro-X-X- Pro-Y-Y-Pro-X-X-Pro-Y-Y- Pro-X-X-Pro-Y-Pro-Pro-X-Pro-Pro-X-X-X-X-X-X-X-X-Pro-X-X-Pro-X-X-X-X (SEQ ID NO 1 ) and / or an artificial peptide comprising or consisting of the amino acid sequence of Pro-X-X-Pro-Y-Y-Pro-X-X-Pro-Y-Y- Pro-X-X-Pro-Y-Y- Pro-Y-Pro-Pro-X-Pro-Pro (SEQ ID NO 2), wherein i) Pro is proline; ii) X is an amino acid selected from the group consisting of Ala, lie, Leu, Met, Phe, Trp and Vai; iii) Y is an amino acid selected from the group consisting of Asn, Cys, Gin, Ser, Thr and Tyr, b. cross-linked Hyaluronic fibres (HA-XL), and c. linear hyaluronic acid fibres (HA).

Description

[0001] CONSENSUS PEPTIDES IN HYA

[0002] TECHNICAL FIELD

[0003] The present invention relates to the field of soft tissue healing and / or improvement. Pharmaceutical and / or cosmetic formulations are described comprising artificial peptides characterized by a proline sequence prevalent in key matricellular proteins that partake in wound healing as well as in bone and cartilage formation and connective tissue maintenance in all vertebrates and their uses in medicine. The artificial peptides are herein formulated in hydrogels, comprising combinations of hyaluronic acids and cross- linked hyaluronic acids for minimally invasive administration and / or controlled release, for use in the treatment of soft tissue conditions, diseases and / or disorders, as well as for cosmetic uses, such as improved skin elasticity and / or hydration.

[0004] BACKGROUND

[0005] Soft tissue is a collective term for all the tissue in the body that is not hardened by the processes of ossification or calcification, such as bones and teeth. Soft tissue connects, surrounds or supports internal organs and bones, and includes muscle, tendons, ligaments, fat, fibrous tissue, lymph and blood vessels, fasciae, and synovial membranes. The term “soft tissue” is commonly used to describe muscles, tendons, ligaments and / or fascia, but several other tissue types and body systems contain soft tissue as well, including fat, skin, nerves, and blood vessels.

[0006] Soft tissue disorders, diseases and damages, are medical conditions affecting soft tissue. They include trauma, wounds and soft tissue injuries to connective and / or epithelial tissue.

[0007] In the craniomaxillofacial complex, soft tissue disorders include periodontitis, periimplantitis, peri-mucositis, gingivitis, aphthous stomatitis and other oral infections and / or inflammations. Skin and soft tissue infections remain among the most frequently encountered infections in surgery, and their severity ranges from mild cellulitis to severe, necrotizing infections with high incidences of morbidity and mortality. Most commonly, these disorders result from skin lesions in a susceptible host, but sometimes develop following hematogenous spread from a previously unknown focus.

[0008] Biomaterial scientists design in particular organic bone substitutes based on the biochemical properties of the mimicked tissue to achieve near native functionality. Several non-collagenous proteins in bone are known as intrinsically disordered proteins (IDPs), as they lack detectible ordered domains and a fixed 3D structure under physiological conditions.

[0009] EP2118136 discloses artificial IDPs peptides with improved properties for induction and / or stimulation of mineralization, in vivo and in vitro. Such peptides are provided which are easy to synthesize and methods of using the peptides for the induction and / or stimulation of mineral precipitation and / or biomineralization.

[0010] The characterising sequence of amino acids of the artificial peptides disclosed in EP2118136 is a proline rich sequence prevalent in key matricellular proteins that partake in wound healing as well as in bone and cartilage formation and connective tissue maintenance in all vertebrates. This core sequence of prolines is highly conserved in vertebrates, is intrinsically disordered and does not induce any immunogenic response in humans.

[0011] In contrast to the successful application of peptides for hard tissue healing, there is today still a largely unmet need for equally potent solutions for soft tissue healing and regeneration. In addition, because of the susceptibility of soft tissue to develop postsurgery and / or post-treatment complications and disorders, it would be preferable to find ways for minimally invasive administrations for the delivery of such advanced peptide- technology.

[0012] Hydrogels are an extensively investigated class of biomaterials, and an increasing number of products have reached the clinic. Hydrogels represent a group of biomaterials consisting of water- swollen polymer or colloidal networks. Hydrogels are viscoelastic materials that have attracted attention in regenerative medicine due to their ability to structurally mimic the extracellular matrix (ECM), thereby creating a conducive environment for cell proliferation and tissue regeneration. The viscoelastic properties of hydrogels allow them to function as stem cell carriers or scaffolds for controlled drug release. There is a need for widely different applications for diverse tissues with different loading modes and levels and different clinical requirements of the material.

[0013] Consequently, a one-fit-all hydrogel is an unlikely strategy.

[0014] The current invention addresses both the need for advanced peptide-technology to promote healing, growth and differentiation of soft tissue, as well as the need for novel means of minimally invasive administration of such active substances to and / or into the soft tissue affected, in particular the soft tissue of the craniomaxillofacial complex.

[0015] SUMMARY

[0016] The current inventors have shown that the artificial IDPs peptides disclosed in EP2118136 are surprisingly not only effective for stimulating regrowth of mineralized tissues, but can also promote growth and / or differentiation of connective tissue cells and, through them, have a secondary promotional effect on epithelial cells’ growth and spreading, e.g., to promote local vascularization and to modulate acute and chronic inflammation, to rejuvenate senescent cells (e.g., from irradiation damage) and to be able to reduce pain and swelling when applied locally.

[0017] To address the susceptibility of soft tissue to develop post-surgery and / or post-treatment complications, diseases and disorders, the current inventors have at the same time developed a novel hydro-gel formulation for the artificial IDPs peptides that enables minimally invasive administration of the same to soft tissue.

[0018] The current invention thus relates to a pharmaceutical and / or a cosmetical formulation in the form of a gel, such as a hydro-gel, comprising a. an artificial peptide characterized by a proline sequence prevalent in key matricellular proteins that partake in wound healing as well as in bone and cartilage formation and connective tissue maintenance in the majority of vertebrates, b. cross-linked hyaluronic acid fibres (HA-XL) and c. linear hyaluronic acid fibres (HA).

[0019] The current invention relates to a pharmaceutical and / or a cosmetic formulation in the form of a gel , such as a hydro-gel comprising or consisting of a. an artificial peptide comprising the amino acid sequence of Pro-X-X-Pro-Y-Y-Y-Pro-X-X-Pro-Y-Y-Pro-X-X- Pro-X- Pro-Y-Y-Y-Y-Y-Y-Pro-Y-Y-Y-Y-Y-Y-Pro-X-X-Pro-X-Pro-Y-Y-Y-Pro-Y-Y-Pro-Y-Pro-

[0020] X-X-Pro-Y-Pro-Y-Y-Pro-X-X- Pro-Y-Y-Pro-X-X-Pro-Y-Y-Pro-X-X-Pro-Y-Pro-Pro-X-Pro- Pro-X-X-X-X-X-X-X-X-Pro-X-X-Pro-X-X-X-X (SEQ ID NO 1), wherein i) Pro is proline; ii) X is an amino acid selected from the group consisting of Ala, lie, Leu, Met, Phe, Trp and Vai; iii) Y is an amino acid selected from the group consisting of Asn, Cys, Gin, Ser, Thr and Tyr, b. 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 cross-linked hyaluronic acid fibres (HA-XL), and c.1-40, such as 1 .0, 2.5, 4, 10, 20, 25 or 40 mg / mL or such as 1-30 mg / mL linear hyaluronic acid fibres (HA).

[0021] Typically, a pharmaceutical and / or a cosmetic formulation according to the current invention comprises or consists of a. 0.1-250 pg / mL, such as 0.1 , 1.0, 5.0, 10, 50, 100, 200 or 250 pg / mL of an artificial peptide comprising the amino acid sequence of Pro-X-X- Pro-Y-Y-Y-Pro-X-X-Pro-Y-Y-Pro-X-X-Pro-X- Pro-Y-Y-Y-Y-Y-Y-Pro-Y-Y-Y-Y-Y-Y-Pro-X-X-

[0022] Pro-X-Pro-Y-Y-Y-Pro-Y-Y-Pro-Y-Pro-X-X-Pro-Y-Pro-Y-Y-Pro-X-X- Pro-Y-Y-Pro-X-X-Pro-

[0023] Y-Y-Pro-X-X-Pro-Y-Pro-Pro-X-Pro-Pro-X-X-X-X-X-X-X-X-Pro-X-X-Pro-X-X-X-X (SEQ ID NO 1), b.1-40 mg / mL, such as 1.0, 2.5, 4, 10, 20, 25 or 40 mg / mL cross-linked hyaluronic acid fibres (HA-XL), and c.1-40, such as 1 .0, 2.5, 4, 10, 20, 25 or 40 mg / mL mg / mL linear hyaluronic acid fibres (HA).

[0024] Alternatively, or in addition, a pharmaceutical and / or a cosmetic formulation according to the current invention comprises or consists of an artificial peptide which comprises the amino acid sequence of Pro-X-X-Pro-Y-Y-Pro-X-X-Pro-Y-Y-Pro-X-X-Pro-Y-Y- Pro-Y-Pro-

[0025] Pro-X-Pro-Pro (SEQ ID NO 2).

[0026] Typically, a pharmaceutical and / or a cosmetic formulation according to the current invention comprises a. 0.1-250 pg / mL, such as 0.1 , 1.0, 5.0, 10, 50, 100, 200 or 250 pg / mL of an artificial peptide which comprises or consist of the amino acid sequence of Pro-X-X-Pro-Y-Y-Pro-X-X-Pro-Y-Y-Pro-X-X-Pro-Y-Y- Pro-Y-Pro-Pro-X-Pro-Pro (SEQ ID NO 2), b.1-40 mg / mL, such as 1.0, 2.5, 4, 10, 20, 25 or 40 mg / mL cross-linked hyaluronic acid fibres (HA-XL), and c.1-40, such as 1.0, 2.5, 4, 10, 20, 25 or 40 mg / mL mg / mL linear hyaluronic acid fibres (HA).

[0027] In embodiments, a pharmaceutical and / or a cosmetic formulation according to the current invention comprises one or more artificial peptide(s) selected from the group consisting of artificial peptides comprising the amino acid sequence of SEQ ID NO 4 (P2) (peptide 2) and SEQ ID NO 5 (P6), or wherein the artificial peptide is at least 90% identical to an artificial peptide selected from the group consisting of the amino acid sequences of SEQ ID NO 4 and SEQ ID NO 5.

[0028] A pharmaceutical and / or a cosmetic formulation according to the current invention can comprise cross-linked hyaluronic acid fibres (HA-XL) which comprise or consist of 1 ,4- butanediol diglycidyl ether cross-linked hyaluronic acid fibres (HA-XL(BDDE)) and / or diglycidyl ether cross-linked hyaluronic acid fibres (HA-XL(BDDE) or HA-XL(PEGDE)).

[0029] In a currently preferred embodiment, a pharmaceutical and / or a cosmetic formulation according to the current invention comprises a.50 pg / mL of an artificial peptide with the amino acid sequence of SEQ ID NO 4 (P2) and / or SEQ ID NO 5 (P6), b. 20 mg / mL 1 ,4- butanediol diglycidyl ether cross-linked hyaluronic acid fibres (HA-XL(BDDE)) and / or diglycidyl ether cross-linked hyaluronic acid fibres (HA-XL(BDDE) or HA-XL(PEGDE)), and c. 2.5 mg / mL linear hyaluronic acid fibres (HA).

[0030] In a pharmaceutical and / or a cosmetic formulation according to the current invention, the hyaluronic acid fibres are typically between 0.7-4.0 MDa, such as between 1.0-2.0, between 1 .5-1 .8, or between 3.0-3.3 MDa. In one embodiment, the hyaluronic acid fibres are between 3.0-3.3 MDa, such as 1.5 MDa.

[0031] A typical pharmaceutical and / or a cosmetic formulation according to the current invention is a hydrogel.

[0032] A pharmaceutical and / or a cosmetic formulation according to the current invention can further comprise, d. one or more buffering agent(s), e. a source of fluoride, f. one or more salt(s), and g. water.

[0033] In addition, a pharmaceutical and / or a cosmetic formulation according to the current invention can further comprise one or more of a component selected from the group consisting of d. Water, e. Sodium Fluoride (NaF), f. Citric Acid, g. Sodium Hydroxide, h. Sodium Chloride, i. Disodium Phosphate and j. Sodium Phosphate.

[0034] A pharmaceutical and / or a cosmetic formulation according to the current invention can further comprise one or more of a component selected from the group consisting of sorbitol, xylitol, NaOH, HCI, phosphate-buffered saline (PBS), acetic acid, citric acid and maleic acid.

[0035] A pharmaceutical formulation according to the current invention can further comprise Mesenchymal Stromal Cells (MSCs), such as between 100.000-10.000.000 MSC cells pr. ml, preferably about 1.000.000 MSCs pr. ml. Additionally, the pharmaceutical formulation according to the current invention may promote MSC homing.

[0036] A pharmaceutical and / or a cosmetic formulation according to the current invention is stable / in gel form at RT for at least 1-2 years and / or in the body for at least 30 days.

[0037] In one aspect, a pharmaceutical and / or a cosmetic formulation according to the current invention releases the comprised artificial peptide at a controlled rate, such as of 1pg per hour.

[0038] In embodiments, a pharmaceutical and / or a cosmetic formulation according to the current invention can further comprise another active ingredient, such as, but not limited to, an active ingredient selected from the group consisting of growth factors, plasma rich fibrin / plasma and enamel matrix derivatives.

[0039] One aspect relates to the novel pharmaceutical formulation according to the current invention for use in medicine, in particular, for use in soft tissue healing, such as, but not limited to, for use in wound healing, for inducing neovascularization, for inducing reepithelization, for stimulating collagen production, and / or for promoting oriented collagen formation.

[0040] The pharmaceutical formulation according to the present invention comprises antiinflammatory properties. Anti-inflammatory properties may be measured by a reduction in proinflammatory cytokines. In embodiments the proinflammatory cytokines comprise one or more cytokines selected from the group consisting of IL-23, IL-1 alpha, IL-1 beta, TNF- alpha, MCP-1, IL-12P70, IFN-Y, IFN-beta, IL-6, IL-10, IL-27, IL-17A and GM-CSF.

[0041] The novel pharmaceutical formulation according to the current invention can be for use in an anti-inflammatory and / or antimicrobial treatment, for use in treating periodontitis, mucositis, periimplantitis, and / or cartilage regeneration. In the treatment of conditions, disease and / or disorders in the craniomaxillofacial complex, the novel pharmaceutical formulation according to the current invention can be used for treating periodontitis, periimplantitis, peri-mucositis, gingivitis, aphthous stomatitis, as well as other oral infections and / or inflammations.

[0042] One aspect of the invention relates to a method for producing a pharmaceutical and / or cosmetic formulation according to the current invention, said 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 between 1.0-2.0, between 1 .5-1 .8, or between 3.0-3.3 MDa, such as 1.5 MDa, wherein said Hyaluronic acid comprises or consists of a mixture of linear hyaluronic acid and BDDE and / or PEGDE cross-linked hyaluronic acid, c. mixing 0.1-250 pg / mL of said artificial peptide and 1-40 mg / mL of said hyaluronic acid mixture and d. optionally, adding a source of fluoride to said mixture, wherein said pharmaceutical and / or cosmetic formulation has an osmolarity of 50-400 mOsm / L, such as between 100 and 310 mOsm / L, or such as between 125 and 175 mOsm / L, such as about 150 mOsm / L, or such as between 275 and 325 mOsm / L, such as about 300 mOsm / L.

[0043] In additional embodiments, said method comprises a. providing the artificial peptide at a concentration of up to 100 mg / mL, such as between 0.01 ug / mL and 100 mg / mL, b. adding hyaluronic acid fibres dissolved at a concentration of 10 wt.% in 0.3M NaOH, c. mixing in a cross-linking agent such as e.g., BDDE and / or PEGDE, d. heating the reaction vessel to 20-100 °C and incubating for between 1- 2 hours to induce gelification, e. cooling and neutralizing the solution, f. homogenizing the gel, g. dialyzing the gel for at the least 18 hours in sterile PBS, h. adding linear hyaluronic acid in an amount of about 10% of the cross-linked hyaluronic acid, i. homogenizing the formulation to reassure uniform distribution of the peptide, and j. optionally adding Phosphate Buffer Solution (PBS).

[0044] In embodiments of a method for producing a pharmaceutical and / or a cosmetic formulation according to the current invention, the artificial peptide in step a. is dissolved in 1% acetic acid at a concentration of 10 mg / mL, followed by b. adding hyaluronic acid fibres (3.0-3.3 MDa) dissolved at a concentration of 10 wt.% in an alkaline solution, preferably NaOH, c. admixing 1 ,4-butanediol diglycidyl ether (BDDE) and / or poly(ethylene glycol) diglycidyl (PEGDE), d. heating the reaction vessel to about 40 °C and incubating for approximately 4 hours, e. cooling and neutralizing the solution with an acidic solution, preferable HCI, f. homogenizing the gel into particles of 100-400 pm. In embodiments, the method further comprises g. dialyzing the gel in a biocompatible solution such as e.g., PBS or saline, h. adding linear hyaluronic acid, in an amount of about 10 wt.% of the cross-linked hyaluronic acid, i. homogenizing the formulation to reassure uniform distribution of the peptide, and j. optionally adding Phosphate Buffer Solution (PBS).

[0045] In a further aspect, a method is disclosed for producing a pharmaceutical and / or a cosmetic formulation according to any of the preceding claims, comprising a. providing the artificial peptide at a concentration of up to 100 mg / mL, b. adding hyaluronic acid fibres dissolved at a concentration of 10 wt.% in 0.3M NaOH, c. mixing in a cross-linking agent, d. heating the reaction vessel to 20-100 °C and incubating for between 1-2 hours, e. stopping the heating and neutralizing the solution e.g., using HCI, f. homogenizing the gel, g. dialyzing the gel in e.g., sterile water, sterile PBS or sterile Saline, h. adding linear hyaluronic acid e.g., about 10% compared to amount of cross-linked hyaluronic acid, i. homogenizing the formulation to reassure uniform distribution of the peptide, and j. optionally adding sterile water, sterile PBS or sterile Saline.

[0046] In additional as aspects a method is disclosed for producing a pharmaceutical and / or cosmetic formulation according to the present disclosure, the method comprises, a. providing an artificial peptide as defined in any one of claims 1-3, b. providing linear hyaluronic acid with a molecular weight of 0.7-4 MDa, such as between 1 .0-2.0, between 1.5-1 .8, or between 3.0-3.3 MDa, such as 1.5MDa, c. cross-linking the linear hyaluronic acid using a crosslinking agent, such as e.g., BDDE or PEGDE to obtain a crosslinked hyaluronic acid (HA-XL), d. optionally, dialysing the crosslinked hyaluronic acid, e. mixing 0.1-250 pg / mL of said artificial peptide and 1-40 mg / mL of said crosslinked hyaluronic acid, and adding 1-40 mg / mL of linear hyaluronic acid to obtain a mixture comprising crosslinked hyaluronic acid, linear hyaluronic acid, and artificial peptide. In additional as aspects a method is disclosed for producing a pharmaceutical and / or cosmetic formulation according to the present disclosure, the method comprises a. providing an artificial peptide as defined in any one of claims 1-3, b. providing linear hyaluronic acid with a molecular weight of 0.7-4 MDa, such as between 1 .0-2.0, between 1.5-1 .8, or between 3.0-3.3 MDa, such as 1.5MDa, c. mixing 0.1-250 pg / mL of said artificial peptide and 1-40 mg / mL of said hyaluronic acid, d. cross-linking the mixture of using a crosslinking agent, such as e.g., BDDE or PEGDE to obtain a mixture of intra- and / or inter-crosslinked peptide and hyaluronic acid, e. adding linear hyaluronic acid (HA) to obtain a mixture of intra- and / or intercrosslinked artificial peptide and hyaluronic acid, and linear hyaluronic acid.

[0047] In embodiments, the method further comprises f. mixing 1-40mg / mL of the intra- and / or inter-crosslinked artificial peptide and hyaluronic acid, with 0.1-250 pg / mL additional artificial peptide to obtain a mixture comprising intra- and / or inter-crosslinked artificial peptide and hyaluronic acid, linear hyaluronic acid, and artificial peptide.

[0048] Consequently, the current invention also relates to a pharmaceutical formulation obtained by a method according to the current invention, as well as to its use in medicine, as well as to a cosmetic formulation and its uses.

[0049] DEFINITIONS AND ABBREVIATIONS

[0050] In the present context, an "artificial peptide" refers to a peptide that is a non-natural peptide in the sense that it does not normally occur in nature but is the product of amino acids put together and selected in an order, amount and manner generating peptides suitable for use in the context of the present invention. An "artificial peptide" is still a peptide embraced by the present invention even though it might encompass parts of or a whole peptide which happens to be present in nature. "Artificial" may be used interchangeably with terms such as "synthetic" or "non-natural".

[0051] In the present context "Pro" denotes the amino acid proline. In the present context "X" denotes a hydrophobic amino acid. A hydrophobic amino acid is, in the present context, defined as an amino acid selected from the group consisting of: Ala, lie, Leu, Met, Phe, Trp and Vai.

[0052] In the present context "Y" denotes a polar amino acid. A polar ("hydrophilic") amino acid is, in the present context, defined as an amino acid selected from the group consisting of: Asn, Cys, Gin, Ser, Thr and Tyr. In the present context, common nomenclature is used for denoting amino acids.

[0053] Therefore, for example, A is Ala (hydrophobic), C is Cys (polar), F is Phe (hydrophobic), H is His, I is lie (hydrophobic), L is Leu (hydrophobic), M is Met (hydrophobic), N is Asn (polar), Q is Gin (polar), S is Ser (polar), T is Thr (polar), V is Vai (hydrophobic), W is Trp (hydrophobic), Y is Tyr (polar).

[0054] In the present context "surface" refers to any surface which may be of interest to provide with an artificial peptide of the invention, such as a metal surface, e.g. a titanium, zirconium, tantalum, aluminium, gold, surgical steel or a nickel surface, or an alloy thereof, or a metal oxide surface thereof, or a metal hydroxide or metal hydride surface thereof, or a hydroxyl apatite, aragonite, bioglass, glass, or polyurethane surface. Another example of a surface according to the invention is a biological surface such as a graft surface, a wound surface etc. Additional examples of surfaces comprise mucosal surfaces, skin surfaces and other equivalent surfaces. In the present context "subject" relate to any vertebrate animal, such as bird, reptiles, mammals, primates and humans.

[0055] Hyaluronic acid; Hyaluronic acid (often abbreviated as HA) is a natural and linear carbohydrate polymer belonging to the class of non-sulphated glycosaminoglycans. As used herein, linear hyaluronic acid relates to non-cross-linked (linear) hyaluronic acid and is referred to as HA. It is composed of beta-1 ,3-N-acetyl glucosamine and beta-1 , 4- glucuronic acid repeating disaccharide units with a molecular weight (MW) up to 6 MDa. hyaluronic acid is present in hyaline cartilage, synovial joint fluid, and skin tissue, both dermis and epidermis. BDDE: 1 ,4-butanediol diglycidyl ether is a class of crosslinking agents, with a molecular weight which may be designed for the particular use, e.g., a molecular weight (Mw) of about 200 Da.

[0056] PEGDE: poly(ethylene glycol) diglycidyl ether, is a class of crosslinking agents with a repetitive poly(ethylene glycol) motif, with a molecular weight which may be designed for the particular use, e.g., a molecular weight (Mw) of about 500 Da.

[0057] HA-XL(BDDE) is in the present context used to illustrate that the hyaluronic acid is crosslinked using BDDE as defined above.

[0058] HA-XL(PEGDE) is in the present context used to illustrate that the hyaluronic acid is cross-linked using PEGDE as defined above.

[0059] The term “biocompatible” as used herein refers to causing no clinically relevant tissue irritation, injury, toxic reaction, or immunological reaction to living tissue.

[0060] The term “cell” is used herein to refer to the structural and functional unit of living organisms and is the smallest unit of an organism classified as living.

[0061] The term “compatible” as used herein means that components of a composition are capable of being combined with each other in a manner such that there is no interaction that would substantially reduce the efficacy of the composition under ordinary use conditions.

[0062] The term “component” as used herein refers to a constituent part, element, or ingredient.

[0063] The term “condition” as used herein refers to a variety of health states and is meant to include disorders or diseases caused by any underlying mechanism or disorder, injury, and the promotion of healthy tissues and organs. The term “differentiation” as used herein refers to the process of development with an increase in the level of organization or complexity of a cell or tissue, accompanied with a more specialized function.

[0064] The terms “disease” and “disorder” as used herein refer to an impairment of health or a condition of abnormal functioning.

[0065] The term “mucosa” as used herein refers to a mucous tissue lining various tubular structures consisting of epithelium, lamina propria, and, in the digestive tract, a layer of 45 smooth muscle. The term “mucosal graft” as used herein refers to a graft of mucus membrane.

[0066] The term “patient” as used herein refers to any mammal. Examples of mammals are humans, farm animals and domestic animals.

[0067] The term “peptide” is used herein to refer to two or more amino acids joined by a peptide bond.

[0068] It must be noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise.

[0069] As used herein, the term “comprising” means the presence of the stated features, integers, steps, or components as referred to in the claims, but that it does not preclude the presence or addition of one or more other features, integers, steps, components or groups thereof. The term “comprising” is intended to include embodiments encompassed by the terms “consisting essentially of’ and “consisting of. Similarly, the term “consisting essentially of is intended to include embodiments encompassed by the term “consisting of.

[0070] As used herein, the term “about” modifying the quantity of an ingredient or reactant employed refers to variation in the numerical quantity that can occur, for example, through typical measuring and liquid handling procedures used for making concentrates or use solutions in the real world; through inadvertent error in these procedures; through differences in the manufacture, source, or purity of the ingredients employed to make the compositions or carry out the methods; and the like. The term “about” also encompasses amounts that differ due to different equilibrium conditions for a composition resulting from a particular initial mixture. Whether or not modified by the term “about”, the claims include equivalents to the quantities. In the current context, the abbreviation “u”, e.g., as used in “uM” or “ug” or “uL”, is used interchangeably with “micro”, “mcg” or “p”. E.g., a microgram or microgramme is a unit of mass equal to one millionth (1 x10-6) of a gram.

[0071] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges which may independently be included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding both of those included limits are also included in the invention.

[0072] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

[0073] Unless defined otherwise, 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 belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, exemplary methods and materials have been described. All publications mentioned herein are incorporated to disclose and describe the methods and / or materials in connection with which the publications are cited. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] Figure 1: Schematic representation of the production steps for the manufacturing of a hyaluronic acid gel cross-linked (XL) with either BDDE or PEGDE. Figure 2: A, Rheology shear and frequency sweeps of the HA-XL(BDDE) and HA- XL(PEGDE) gel. B, Real-time crosslinking monitoring of the two gels over a 5h time period at 40 °C. C FTIR analysis of the two gels. D SEM analysis of the cross-section of the two gels.

[0075] Figure 3:1H NMR of leaching medium of A. HA-XL(BDDE) or B. HA-XL(PEGDE), before (top panels in A and B) and after 18h dialysis (bottom panels in A and B) in distilled water.

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

[0077] Figure 5: Comparison of wound healing properties of (a) Sham, (b) Emdogain® (Straumann, Basel, CH), (c) Crosslinked HA+HA-XL(BDDE) with P2. Figure 6: Wound healing properties of HA+HA-XL(BDDE) gels with P2, P6 or P2+P6 compared to Sham and “empty” HA+HA-XL(BDDE) gel. a) Wound Void Area, b) Re- epithilization score, c) Granulation Score, d) Infiltrate score.

[0078] Figure 7: Wound healing properties of HA+HA-XL(BDDE) gels with P2 or P6 compared to EMD and HA+HA-XL(BDDE) gels with EMD. a) Wound Void Area, b) Re-epithilization score, c) Granulation Score, d) Infiltrate score.

[0079] Figure 8: Exemplary images of histological wound stainings. Top) Re-epithelisation: 3 - Fully intact, Granulation: 3 - Absent, Infiltration: 2 - Some present around wound. Middle) Re-epithelisation: 3 - Fully intact, Granulation: 1 - Moderate around wound, Infiltration: 0 - Infection / necrotic tissue by scab + infiltrate surrounding wound. Bottom) Re- epithelisation: 3 - Fully intact, Granulation: 0 - Extensive, Infiltration: 1 - infiltrate surrounding wound. This scoring show the regenerative potential with HA+HA- XL(BDDE)+P2 compare to only HA+HA-XL(BDDE)

[0080] Figure 9: Inflammation reduction after three weeks of tissue healing addressed by Flow cytometry based on indicated biomarkers. Figure 10: Inflammation reduction after three weeks of tissue healing addressed by Flow cytometry based on indicated biomarkers.

[0081] Figure 11: Inflammation reduction after three weeks of tissue healing addressed by Flow cytometry based on indicated biomarkers.

[0082] Figure 12: Micro-computed tomography reconstruction of mice femur poly-trauma fracture healing 3 weeks after fracture with treatment with HA+HA-XL(BDDE) gel (Hyd), HA+HA- XL(BDDE)+P2 (Hyd+P2), HA+HA-XL(BDDE)+P2+MSC (Hyd+P2+MSC).

[0083] Figure 13: Parameter output after morphological analysis after micro-computed tomography reconstruction.

[0084] Figure 14: von Kossa / van Gieson staining of the fracture healing after treatment with HA+HA-XL(BDDE) (A), HA+HA-XL(BDDE)+P2 (C), HA+HA-XL(BDDE)+P2+MSC (E), and Movat’s staining of HA+HA-XL(BDDE) (B), HA+HA-XL(BDDE)+P2 (D), HA+HA- XL(BDDE)+P2+MSC (F) 3 weeks after fracture.

[0085] Figure 15: Histomorphometric analysis of the histological data.

[0086] Figure 16: Small angle X-ray scattering / X-ray diffraction (SAXS / XRD) analysis of the osteogenesis and biomineralization processes after healing. The full width half max (fwhm) 002 HAp reflects the degree of orientation and T parameter of the defect site are shown in the top panel.

[0087] Figure 17: Boxplot of Small angle X-ray scattering / X-ray diffraction (SAXS / XRD) analysis.

[0088] Figure 18: Comparison of %P2-Peptide release from cross-linked HA-XL(BDDE) [HA-XL], linear hyaluronic acid [HA] and combination of cross-linked and linear hyaluronic acid [HA+HA-XL(BDDE)] gels over time, with bubble size indicating the %-relative weight change. Figure 19: Representative Masson goldner trichrome stain slides with dashed line circling in regions of active inflammation (Sham: necrotic / encapsulated tissue; HA+HA- XL(BDDE): Inflammatory infiltrate; Emdogain®: Active infection) and dotted line illustrating oedema (a-e). All histology images, except the Emdogain® (pig 6), are from pig 2. Scalebar = 1 mm. The graph shows the mean inflammation score for each of the groups (f) [1 = compromised, 3 = normal]. The inflammation score of each sample was an average of the three indicators of epithelium physiology, oedema, and inflammatory infiltrate / active infection. n=6 for sham, HA+HA-XL(BDDE)+P2, HA+HA-XL(BDDE)+P6, and n=3 for HA+HA-XL(BDDE) and Emdogain®.

[0089] Figure 20: Cell viability measured using CCK8 assay. n=8, *p<0.5, **p<0.01, ***p<0.001.

[0090] DETAILED DESCRIPTION The current invention relates to a pharmaceutical and / or a cosmetic formulation in the form of a gel, such as a hydrogel, comprising a. an artificial peptide characterized by a proline sequence prevalent in key matricellular proteins that partake in wound healing as well as in bone and cartilage formation and connective tissue maintenance in the majority of vertebrates, b. cross-linked hyaluronic acid fibres (HA-XL), and c. linear hyaluronic acid fibres (HA).

[0091] In one aspect, the formulation of the current invention comprises one or more artificial peptides loaded into a hydrogel carrier. The one or more artificial peptide(s) comprised in the formulation of the current invention is characterized by comprising or consisting of the amino acid sequence of Pro-X-X-Pro-Y-Y-Y-Pro-X-X-Pro-Y-Y-Pro-X-X-Pro-X- Pro-Y-Y-Y-

[0092] Y-Y-Y-Pro-Y-Y-Y-Y-Y-Y-Pro-X-X-Pro-X-Pro-Y-Y-Y-Pro-Y-Y-Pro-Y-Pro-X-X-Pro-Y-Pro-Y-

[0093] Y-Pro-X-X- Pro-Y-Y-Pro-X-X-Pro-Y-Y-Pro-X-X-Pro-Y-Pro-Pro-X-Pro-Pro-X-X-X-X-X-X-X-

[0094] X-Pro-X-X-Pro-X-X-X-X (SEQ ID NO 1 ), wherein i) Pro is proline; ii) X is an amino acid selected from the group consisting of Ala, lie, Leu, Met, Phe, Trp and Vai; iii) Y is an amino acid selected from the group consisting of Asn, Cys, Gin, Ser, Thr and Tyr.

[0095] Alternatively, or in addition, the one or more artificial peptide(s) comprised in the formulation of the current invention is characterized by comprising or consisting of the amino acid sequence of Pro-X-X-Pro-Y-Y-Pro-X-X-Pro-Y-Y- Pro-X-X-Pro-Y-Y- Pro-Y-Pro- Pro-X-Pro-Pro (SEQ ID NO 2), wherein i) Pro is proline; ii) X is an amino acid selected from the group consisting of Ala, lie, Leu, Met, Phe, Trp and Vai; and iii) Y is an amino acid selected from the group consisting of Asn, Cys, Gin, Ser, Thr and Tyr. In one embodiment, the formulation is delivered via a syringe in a minimally invasive manner. In vitro and in vivo trials suggest that the herein described gel-formulation with peptides stimulates periodontal tissue formation in a similar or better manner than the active ingredients of the market-leading products. Additionally, the formulation of the current invention is fully synthetic, which will e.g., allow scalability and price competitiveness. Moreover, the herein disclosed formulation does not require the use of animal tissue. The use of hydrogel carrier of the current invention uses covalently crosslinked hyaluronic acid, thereby overcoming limitations, such as, but not limited to, rapid clearance and gum flap collapse after administration. The artificial peptide

[0096] In one aspect, the current invention relates to the surprising insight that the artificial peptides disclosed in EP2118136 can be used to promote connective tissue cells and to, through them, have a secondary promotional effect on epithelial cell growth and spreading, e.g., to promote local vascularization and to modulate acute and chronic inflammation, to rejuvenates senescent cells (e.g., damaged from irradiation damage) and to be able to reduce pain and swelling when applied locally.

[0097] The ability of the peptides to aid in inflammation is also highlighted herein, in examples 11 and 12, wherein it is shown that the formulation as disclosed herein is capable of reducing the level of several inflammatory cytokines, thereby aiding the inflammatory response following injurie. Furthermore, it is also shown herein that the pharmaceutical formulation provided herein also leads to acceleration of the first stages of healing by upregulating genes such as collagen biosynthesis and modifying enzymes, in addition to improving the cartilage and bone healing process through e.g., heparan sulfate degradation, glycosaminoglycan degradation, assembly of collagen fibrils and other multimeric structures, collagen biosynthesis and modifying enzymes, ECM-receptor interaction, extracellular matrix organization and mineral absorption.

[0098] The artificial peptides according to the current invention are biomimetic peptides inspired by the motifs found in amelogenins. Due to intrinsic disorder, they are flexible peptides that dynamically adopt to the local environment. They are thereby able to interact with other structural polymers such as collagen, giving an improved appearance. This is e.g., done by the peptides folding into a temporary extracellular matrix that can e.g., bind to surfaces of the mucous membrane. This protects the soft tissue of the mucous membrane providing a prophylactic function. The peptides have also been demonstrated to nucleate calcium phosphate and to orient the crystallite growth into lamella-like platelets, thereby having the ability to form a protective mineral layer on the dental enamel.

[0099] The artificial peptides according to the current invention are biomimicking, intrinsically disordered proteins (IDPs).

[0100] Under physiological conditions i.e., conditions of the external or internal milieu that may occur in nature for an organism, such as a human being, the peptides are generally intrinsically disordered. The release of peptides from a gel matrix is usually tied to the structure of the peptides / proteins, wherein intrinsically disordered proteins / peptides are generally more easily released from the gel than structured proteins and / or peptides, due to their high degree of conformational flexibility. This feature makes the artificial peptides of the current disclosure highly useful as constituent to be released from a gel, such as a cross-linked HA-XL gel and / or a combined HA-HA-XL gel.

[0101] One of the advantages of using synthetic peptides is that it is not always practical to use natural peptides and / or proteins for medical and / or cosmetic uses. For example, natural proteins are often long, which means that they are difficult to synthesize, both chemically and by cellular expression systems. Typically, artificial peptides are easier to synthesize than full, larger proteins, as peptides often lack the complicated higher order structure that make large proteins difficult to synthesize. Also, a natural protein only contains natural amino acids and may therefore be susceptible to rapid degradation. Also, if purified from a natural environment, such as developing teeth, there is always a risk of contamination of other products which e.g., may cause allergic reactions. In addition, a long natural protein normally has many roles in a living body and may therefore not be optimized for the intended use.

[0102] Furthermore, the active motif of the synthetic peptides in the formulation of the current invention can be designed to specifically stimulate healing and / or growth of soft tissue and skin. The artificial peptide(s) comprised in the formulation of the current invention is / are characterized by a proline sequence prevalent in key matricellular proteins that partake in wound healing as well as in bone and cartilage formation and connective tissue maintenance in the majority of vertebrates.

[0103] The artificial peptide(s) comprised in the formulation of the current invention is / are characterized by comprising or consisting of the amino acid sequence of Pro-X-X-Pro-Y- Y-Y-Pro-X-X-Pro-Y-Y-Pro-X-X-Pro-X- Pro-Y-Y-Y-Y-Y-Y-Pro-Y-Y-Y-Y-Y-Y-Pro-X-X-Pro-X-

[0104] Pro-Y-Y-Y-Pro-Y-Y-Pro-Y-Pro-X-X-Pro-Y-Pro-Y-Y-Pro-X-X- Pro-Y-Y-Pro-X-X-Pro-Y-Y- Pro-X-X-Pro-Y-Pro-Pro-X-Pro-Pro-X-X-X-X-X-X-X-X-Pro-X-X-Pro-X-X-X-X (SEQ ID NO 1 ), wherein i) Pro is proline; ii) X is an amino acid selected from the group consisting of Ala, lie, Leu, Met, Phe, Trp and Vai; iii) Y is an amino acid selected from the group consisting of Asn, Cys, Gin, Ser, Thr and Tyr. Alternatively, or in addition, the one or more artificial peptide(s) comprised in the formulation of the current invention is characterized by comprising or consisting of the amino acid sequence of Pro-X-X-Pro-Y-Y-Pro-X-X-Pro-Y-Y- Pro-X-X-Pro-Y-Y- Pro-Y-Pro- Pro-X-Pro-Pro (SEQ ID NO 2), wherein i) Pro is proline; ii) X is an amino acid selected from the group consisting of Ala, lie, Leu, Met, Phe, Trp and Vai; and iii) Y is an amino acid selected from the group consisting of Asn, Cys, Gin, Ser, Thr and Tyr.

[0105] Furthermore, the peptides according to the invention may comprise between 20 and 120 amino acids, such as, but not limited to, between 20-25, 25-30, 30-35, 35-40, 40-45, 45- 50, 50-60, 60-70, 70-80, 80-90, 90-100, or between 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 comprises between 20-50 amino acids.

[0106] The amino acids in an artificial peptide of the invention may further be modified in terms of chemistry, isometry or in any other way, as long as the sequences of the peptides are intact. Modifications of the amino acids of the artificial peptides of the invention may increase the activity, stability, biocompatibility, or clinical performance of the peptides, or reduce toxicity and adverse reactions to the peptides. Examples of chemical modifications include, but are not limited to, glycosylation and methylation. The amino acids may also be of all different types of stereoisomeric forms, such as D or L forms of amino acids, or S or R isomers. The amino acids in an artificial peptide of the invention may also be replaced by synthetic analogues thereof. The use of synthetic analogues may e.g., result in a peptide that is more stable and less prone to degradation. Examples of unnatural amino acids include; alpha* and alpha-disubstituted* amino acids, N-alkyl amino acids*, lactic acid*, halide derivatives of natural amino acids such as trifluorotyrosine*, p-CI- phenylalanine*, p-Br-phenylaianine*, p-l-phenylatanine*, L-allyl-glycine*, β-alanine*, L-a- amino butyric acid*, L-g-amino butyric acid*, L-a-amino isobutyric acid*, L-e-amino caproic acid#, 7-amino heptanoic acid*, L-methionine sulfone#*, L-norleucine*, L-norvaline*, p- nitro-L-phenylalainine*, 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-tetrahydr- oisoquinoline-3-carboxyl acid)*, L- diaminopropionic acid # and L-Phe (4-benzyl)*. The notation * is herein utilised to indicate the hydrophobic nature of the derivative whereas # is utilised to indicate the hydrophilic nature of the derivative, #* indicates amphipathic characteristics.

[0107] The above identified peptides are artificial (synthetic) peptides comprising a poly-proline consensus sequence, further comprising hydrophobic ("X") and polar amino acids ("Y"). It induces and / or stimulates soft tissue healing in biological systems, and that also may be used clinically, industrially, chemically or otherwise to stimulate the formation of soft tissue. The protein sequences used for constructing the artificial peptide included the sequences for collagen 1 and 2 (human, mouse and rat), amelogenin (human, mouse, rat, rabbit, pig and cow), ameloblastin (human, rat), bone sialoprotein (human, mouse), enamelin (human, mouse). The artificial peptides comprised in the formulation of the current invention are particularly suitable for the induction and / or stimulation of soft tissue healing, as the amino acid sequences are optimised for this purpose. The use of an artificial peptide according to the invention is advantageous due to its shorter length compared to natural peptides, which facilitates the synthesis thereof and allows for the use of amino acid analogues as explained herein. Also, the use of an artificial peptide allows modifications of the amino acid sequence to enable the peptides to bind to e.g., metal surfaces or being easily purified, such as by the choice of amino acid sequences of the peptide itself or the use of N- and / or C-terminal tags. Therefore, in one aspect, the present invention relates to a formulation comprising an artificial peptide comprising an amino acid sequence of SEQ ID NO 1 and / or SEQ ID NO 2, which is able to induce and / or stimulate soft tissue growth and / or differentiation. Preferably such an artificial peptide consists of an amino acid sequence as shown in SEQ ID NO 1 or SEQ ID NO 2.

[0108] One embodiment of the invention relates to a formulation comprising a shorter consensus peptide sequence comprising or consisting of an amino acid sequence of Pro-X-X-Pro-Y- Y-Pro-X-X-Pro-Y-Y- Pro-X-X-Pro-Y-Y-Pro-Y-Pro-Pro-X-Pro-Pro (SEQ ID NO 2), where- in i) Pro is proline; ii) X is an amino acid selected from the group consisting of Ala, lie, Leu, Met, Phe, Trp and Vai, preferably lie, Leu, Vai and Met; iii) Y is an amino acid selected from the group consisting of Asn, Cys, Gin, Ser, Thr and Tyr, preferably Ser and Gin.

[0109] SEQ ID NO 2 is constructed by the assembly of amino acids 47-50, 53-66 and 70-76 of SEQ ID NO 1 , i.e., amino acids underlined in SEQ ID NO 1 above. One preferred embodiment of the invention relates to a formulation comprising an artificial peptide comprising an amino acid sequence as shown in SEQ ID NO 2, more preferably consisting of an amino acid sequence as shown in SEQ ID NO 2. Another preferred embodiment relates to a formulation comprising an artificial peptide consisting of an amino acid sequence as shown in SEQ ID NO 2, which is able to induce and / or stimulate soft tissue growth and / or differentiation. Due to its short length, SEQ ID NO 2 is advantageous for synthetic production.

[0110] The invention also relates to a formulation according to the current invention comprising other artificial peptides with a specified amino acid sequence comprised in a consensus sequence of the invention. In a first aspect, such an artificial peptide is PLV PSY PLV PSY PLV PSY PYP PLPP (SEQ ID NO 3). Another preferred amino acid sequence is PLV PSQ PLV PSQ PLV PSQ POP PLPP (SEQ ID NO 4). These two sequences are the two sequences of the invention that represent the most conserved sequences.

[0111] In embodiments, an artificial peptide comprised in the formulation according to the current invention comprises an amino acid sequence as shown in SEQ ID NO 4 (peptide 2 or P2) or SEQ ID NO 5 (peptide 6 or P6). As exemplified in the examples, in embodiments, an artificial peptide comprised in the formulation according to the current invention consists of an amino acid sequence as shown in SEQ ID NO 4 (peptide 2 or P2) or SEQ ID NO 5 (peptide 6 or P6).

[0112] Table 1 Exemplified peptide sequences

[0113] In one aspect, the present invention relates to a formulation according to the current invention comprising an artificial peptide comprising an amino acid sequence of SEQ ID NO 4, which is able to induce and / or stimulate soft tissue growth and / or differentiation. Preferably such an artificial peptide consists of an amino acid sequence as shown in SEQ ID NO 4.

[0114] In one aspect, the present invention relates to a formulation according to the current invention comprising an artificial peptide comprising an amino acid sequence of SEQ ID NO 5, which is able to induce and / or stimulate soft tissue growth and / or differentiation. Preferably such an artificial peptide consists of an amino acid sequence as shown in SEQ ID NO 5.

[0115] Further aspects of the invention relate to a formulation according to the current invention comprising one or more artificial peptides having between 80-100% identity with any one of the sequences 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 NO1- 5.

[0116] In preferred embodiments, a formulation according to the current invention comprises one or more artificial peptides disclosed herein which consist of any one of SEQ ID NO 1-5, respectively.

[0117] In a formulation according to the current invention, a peptide may further comprise N- and / or C-terminal tags comprising the amino acids His and / or Met. Met contains sulphur, which as previously explained facilitates binding to metal surfaces. His has a strong affinity for e.g., Ni and other metals. The use of these tags therefore has the advantage of enabling the peptides to attach to metal surfaces like titanium, zirconium, aluminium, tantalum, gold, surgical steel and nickel, or a metal oxide hydroxide and / or hydride surface etc. The C- and / or N-terminal tags are also useful in the process of purification of produced peptides, as is well known to the skilled person. The use of an N-terminal and / or C-terminal tag also allows the peptide to be fully exposed, i.e. , the tag is used for binding the peptide to a surface and the rest of the peptide is free for interactions with e.g., atoms, molecules, cells and tissue. The use of one tag in each end of a peptide may be useful during production of the peptide, allowing one end of the peptide to be attached to a column during the purification of the peptide of interest from incomplete peptide products, while the other end of the peptide may be used for binding to a surface of interest.

[0118] Consequently, one preferred embodiment of the invention relates to a formulation according to the current invention comprising an artificial peptide as defined herein, further comprising an N-terminal and / or a C-terminal histidine tag. Such a tag may, as previously mentioned, comprise methionine and / or histidine residues, which have been attached to an artificial peptide according to the invention. In a preferred embodiment, this tag comprises 3 or more residues, such as between 3-5 or 5-10 residues. A tag can comprise any number of residues attached to an artificial peptide according to the invention, which still provides for a stable composition together with the artificial peptide according to the invention not affecting the secondary structure of the artificial peptide in a negative manner. Preferably this 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, a peptide of the invention comprises a methionine tag in its C- or N-terminal end and a histidine tag in the other end.

[0119] SEQ IDS

[0120] In embodiments, a pharmaceutical and / or a cosmetic formulation according to the current invention comprises one or more artificial peptide(s) which comprises or consists of one or more of the amino acid sequences selected from the following table (Table 2):

[0121] Table 2 peptide sequences

[0122] In the current context, Pro is Proline (Pro); L is Leucine (Leu), V is Valine (Vai), S is Serine (Ser), Q is Glutamine (Gin), H is Histidine (His), M is Methionine (Met), C is

[0123] Cysteine (Cys), X is an amino acid selected from the group consisting of A (Alanine, Ala),

[0124] I (Isoleucine, lie), L (Leucine, Leu), M (Methionine, Met), F (Phenylalanine, Phe), W

[0125] (Tryptophan, Trp) and V (Valine, Vai), preferably lie, Leu, Vai and Met; Y is an amino acid selected from the group consisting of N (Aspargine, Asn), C (Cysteine, Cys, CysH), Q

[0126] (Glutamine, Gin), S (Serine, Ser), T (Threonine, Thr) and Y (Tyrosine, Tyr), preferably Ser and Gin.

[0127] In embodiments, a pharmaceutical and / or a cosmetic formulation according to the current invention comprises one or more artificial peptide(s) selected from the group consisting of artificial peptides comprising the amino acid sequence 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, a pharmaceutical and / or a cosmetic formulation according to the current invention comprises one or more artificial peptide(s) selected from the group consisting of artificial peptides consisting of the amino acid sequence 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, a pharmaceutical and / or a cosmetic formulation according to the current invention comprises one or more artificial peptide(s) selected from the group consisting of artificial peptides which are at least 90% identical to the amino acid sequence 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, respectively, such as at least 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence 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, respectively.

[0130] Typically, a pharmaceutical and / or a cosmetic formulation according to the current invention comprises a. 0.1-250 pg / mL, such as 0.1 , 1.0, 5.0, 10, 50, 100, 200 or 250 pg / mL of an artificial peptide according to the current invention.

[0131] Typically, artificial peptides comprised in the formulations of the current invention are designed with constraints on length of no more than 30 amino acids, such as of no more than 29 amino acids, such as of no more than 28 amino acids, such as of no more than 27 amino acids, such as of no more than 26 amino acids, such as of no more than 25 amino acids, such as of no more than 24 amino acids, such as of no more than 23 amino acids, such as of no more than 22 amino acids, such as of no more than 21 amino acids, such as of no more than 20 amino acids, such as of no more than 19 amino acids, such as of no more than 18 amino acids, such as of no more than 17 amino acids, such as of no more than 16 amino acids, such as of no more than 15 amino acids, such as of no more than 14 amino acids, such as of no more than 13 amino acids, such as of no more than 12 amino acids. In addition, artificial peptides comprised in the formulations of the current invention are designed with conservation of proline positions and with systematic variation of other composing amino acids as is defined in SEQ ID NO 1 and SEQ ID NO 2.

[0132] The designed peptides (P2, P4, P5, and P6) differs in their physical, chemical, and structural properties due to the different amino acid compositions, except at the conserved proline positions in the sequence. A recently developed Web-based method of peptide design was used to explore possible correlation between the structure and biological response to the particular peptide. Hyaluronic acid

[0133] The current invention relates to a pharmaceutical and / or a cosmetic formulation in the form of a gel comprising a. an artificial peptide characterized by a proline sequence prevalent in key matricellular proteins that partake in wound healing as well as in bone and cartilage formation and connective tissue maintenance in the majority of vertebrates, and b. hyaluronic acid fibres, wherein the hyaluronic acid fibres comprise cross-linked hyaluronic acid (HA-XL) and non-cross-linked (linear) hyaluronic acid (HA). A gel referred to as HA+HA-XL is a gel comprising a mixture of linear hyaluronic acid (HA) and crosslinked hyaluronic acid (HA-XL). Ways to prepare such a mixture are exemplified in Example 2, wherein 10% HA is used together with 90% HA-XL. It is to be understood that the amount of HA and HA-XL may be adapted to the specific intended use.

[0134] Hyaluronic acid is a natural polymer that is natively found in the human extracellular matrix. It facilitates an environment with high water content where cells can attach and proliferate. Moreover, it is proven to exhibit intrinsic anti-bacterial and anti-inflammatory properties which are favourable for a prophylactic function. Hyaluronic acid is already used frequently for pharmacological formulations, in medical devices and in cosmetics. Therefore, it is readily available as pharma-grade raw material derived through bacterial fermentation.

[0135] Due to the unique physical and biological properties of hyaluronic acid (including biocompatibility and biodegradability), hyaluronic acid is employed in a wide range of current and developing applications.

[0136] WO 03 / 061626 discloses an injectable, insertable, or implantable drug delivery system that form hydrogels when implanted, injected, or inserted comprising hyaluronic acid and a pharmaceutically effective compound.

[0137] Hyaluronic acid is a long-chain polysaccharide composed of repeating units of glucuronic acid and N-acetylglucosamine. It is a natural polymer with significant clinical adoption within medical fields such as ophthalmology, aesthetics (dermal fillers), and orthopaedics (viscosupplementation). The hyaluronic acid is naturally occurring in the body as part of the extracellular matrix which has inspired its use in medical devices, and it does indeed demonstrate high bioactivity. Initially, the hyaluronic acid was animal derived from roosters which caused the polysaccharide to be expensive and challenging to bring through the regulatory hurdles. However, the last couple of decades, hyaluronic acid production from bacterial fermentation has become the gold standard, which gives high reproducibility, low cost, and is regulatory favourable. A considerable limitation is that hyaluronic acid is rapidly cleared from the body from cleavage by the enzyme hyaluronidase, typically with a half-life of a few hours up to a couple of days. Covalently crosslinking has become a popular strategy to overcome this and to increase the therapeutic effect of the hyaluronic acid gel. A popular strategy has been to crosslink hyaluronic acid with e.g., 1 ,4-butanediol diglycidyl ether (BDDE) or poly(ethylene glycol) diglycidyl ether (PEGDE), where covalent bonds are formed through ring-opening esterification by means of nucleophilic attach of the reactive hydroxyl groups found in hyaluronic acid.

[0138] Crosslinked hyaluronic acid gels can also be achieved by base-catalyst reactions in an organic solvent where the carboxyl groups are activated and subsequently undergo nucleophilic acyl substitution with the hydroxyl groups. Similarly, carbodiimides such as 1- ethyl-3-(3-dimethyl aminopropyl) (EDC) can be used as a catalyst to induce crosslinking between hyaluronic acid polymers in a two-step process wherein the carboxylic group of the hyaluronic acid is first activated by the EDC in an acidic environment, then an ester bond is formed between the hyaluronic acid’s carboxylic acid group and the hydroxyl group of the adjacent hyaluronic acid, and the EDC is recovered. The crosslinking can also be induced by means of photopolymerization, for instance by functionalizing the carboxylic acid groups with methacrylate end-groups.

[0139] Although there are multiple alternative strategies for crosslinking of hyaluronic acid groups, where both the carboxylic and hydroxy end-groups can be functionalized, hyaluronic acid cross-linked BDDE (referred to herein as HA-XL(BDDE) remains most popular in clinic. It has been adopted primarily for use in dermal filling.

[0140] The biomedical application of hyaluronic acid is diverse, and hyaluronic acid-based products obtain its therapeutic effect through different means. Dermal fillers require the material to fill a volume and maintain volume stability over a prolonged period. In ophthalmology hyaluronic acid can be used both to lubricate and retain water in patients prone to dry eye syndrome. In the dental domain, hyaluronic acid cross-linked with BDDE has been used to fill periodontal pockets and function as a scaffold for gingival regeneration. Although the application and mechanism of action of the hyaluronic acid is diverse, a commonality is that it requires a high biocompatibility. Hyaluronic acid is a native component of the human extra cellular matrix and has high affinity to cells through binding with the CD44 receptors. This renders the hyaluronic acid with intrinsic anti-inflammatory properties through attenuating the release of NF-kp. When hyaluronic acid is cross-linked, this does occupy some of the carboxylic acid and hydroxide groups which will reduce its ability to bind to the CD44 receptor, thus reducing its bioactivity. Consequently, there is a trade-off between the increased physiological stability and reduction in bioactivity as the degree of crosslinking is increased. All of this is something that needs to be considered when developing a new formulation for cross-linked hyaluronic acid for biomedical application. In some recent work, poly(ethylene glycol) diglycidyl ether (PEGDE) has been used as an alternative to BDDE with results suggesting that for the same number of moles, PEGDE can give a more elastic gel with less swelling compared to BDDE, which provides a new tool for optimizing the properties of cross-linked hyaluronic acid gels.

[0141] Gels comprising or consisting of linear hyaluronic acid fibres (HA) dissolve and / or degrade faster than cross-linked hyaluronic acid (HA-XL) gels. Due to the faster degradation, of the linear gel peptides are theoretically released more quickly to the surrounding from a linear hyaluronic acid gel than from a cross-linked gel. The terms “hyaluronic acid fibres” and “hyaluronic fibres” are herein used interchangeably. Still, there is always the interaction between the loaded peptide and the hydrogel to be taken into account for calculations of release-rates. E.g., a sterically complex, or charged peptide might get entangled in the linear hyaluronic fibres and not behave as theoretically expected.

[0142] Since the crosslinking of the hyaluronic fibres makes the gel more stable, it will slow down the degradational release of the peptide, as the peptide may have to diffuse out of the gel, rather than being released upon degradation of the gel. However, the specific release profile of the peptides from the gel is a complex sum of degradation of the gel, physiochemical properties of the peptide, diffusion, hydration level of the gel etc. For some applications it may be favoured to obtain a fast release of the peptide and maintain the hyaluronic acid CD44 receptor binding, while for other applications it may be favoured to obtain a slow release of the peptide form the gel. An intermediate between the two is herein shown to be obtained by mixing cross-linked hyaluronic acid (HA-XL) and linear hyaluronic acid (HA), such that a portion of the peptide is released faster, while another portion of the peptide is released at a slower rate. Additionally, it may also be favoured that the gel has a stable swelling level and does not take up additional fluid upon administration from the surroundings. Such stability may be controlled based on the amount of crosslinking used in the gel, both in terms of the ratio between the cross-linked and linear hyaluronic acid, but also with the degree of crosslinking in the cross-linked hyaluronic acid as such. It is to be assumed that more crosslinking in general results in a more mechanically stable and less water-bloated gel. For applications such as wound healing it is preferred that the formulation has a high mechanical stability and strength, making the formulation stable for longer periods of time, which allows for healing of the wounds.

[0143] Example 11 shows that the peptide diffuses faster from the gels which are formulated either as a combination of linear hyaluronic acid and cross-linked (HA+HA-XL), or complete HA-XL gel, while the linear gel shows a slower release (See figure 18). Additionally, the linear gel shows a higher degree of swelling over a period of 24 hrs (see figure 18). Swelling of the gel over time may for some applications be a disadvantage since swelling can lead to excessive pressure in the wound area and general discomfort while at the same time making the gel more unstable. Example 5 and figure 4 shows that a biotinylated version of a peptide of the invention may diffuse slower from the HA-XL gel compared to the linear gel. This is thought to be due to the biotinylated peptide interacting with the HA-XL gel, thereby leading to a slower release from the HA-XL gel compared to from the linear gel. Example 5 also serves to show that the release profile from the hyaluronic acid gel may be modified by conjugation of the peptides to a further substance in order to adapt the release profile further.

[0144] As mentioned above, the release profile of the peptide from the gels is highly dependent on different factors which are difficult to establish without a certain amount of trial and error. From the examples it is to be expected that a higher content of linear hyaluronic acid in the gel results in more swelling of the gel but results in a lower overall peptide release. The gels presented in the examples of the present invention all comprise a mixture of cross-linked and non-cross-linked hyaluronic acid. While the ratio between cross-linked and non-cross-linked hyaluronic acid may differ, it is preferable that the gel of the present invention comprises both cross-linked and non-cross-linked hyaluronic acid, so as to guarantee a tailored gel with enough physio-mechanical stability and a desired rate of release of the loaded peptide. In some embodiments, the gel may essentially consist of cross-linked hyaluronic acid.

[0145] In the current invention, it was surprisingly found that the use of hyaluronic acid increases the viscosity and / or forms a highly viscous gel-like core in the formulation comprising the artificial peptide. The crosslinking was shown to slow down the release of the biotinylated artificial peptide after administration.

[0146] Additionally, the release rate of the peptide also largely depends on when in the manufacturing process the peptide is added. In example the peptide may be added initially into the mixture of hyaluronic acid before crosslinking, meaning that once crosslinked, the peptide will eventually be more fixated in the gel contra when the peptide is added after the crosslinking, where the peptide is freer to diffuse from the gel and into the surroundings. Accordingly, the addition of the peptide before the crosslinking is likely to result in a slower release profile similar to what is seen in figure 4. On the other hand, addition of the peptide after crosslinking, in the mixing of the crosslinked and linear gel is likely to result in a faster release of the peptide from the gel, similar to what is seen in figure 18. This provides an additional handle which may be used to tailor the release profile of the peptide from the gel. Thus, it can also be imagined that the peptide is added in portions, where a first portion is added before crosslinking and a portion is portion is added after the crosslinking, such as in the mixing step of the HA and HA-XL.

[0147] Accordingly, in embodiments, the peptide is added before crosslinking of the gel. In other embodiments the peptide is added after the crosslinking of the gel. In a further embodiment, a portion of the peptide is added before the mixing of the gel and a portion of the peptide is added after the crosslinking of the gel. In some embodiments, 50% of the peptide is added in the HA-XL before crosslinking and 50% of the peptide is added to the HA. In some embodiments, 60% of the peptide is added in the HA-XL before crosslinking and 40% of the peptide is added to the HA. In some embodiments, 70% of the peptide is added in the HA-XL before crosslinking and 30% of the peptide is added to the HA. In some embodiments, 80% of the peptide is added in the HA-XL before crosslinking and 20% of the peptide is added to the HA. In some embodiments, 90% of the peptide is added in the HA-XL before crosslinking and 10% of the peptide is added to the HA. In some embodiments, 95% of the peptide is added in the HA-XL before crosslinking and 5% of the peptide is added to the HA. In some embodiments, 40% of the peptide is added in the HA-XL before crosslinking and 60% of the peptide is added to the HA. In some embodiments, 30% of the peptide is added in the HA-XL before crosslinking and 70% of the peptide is added to the HA. In some embodiments, 20% of the peptide is added in the HA-XL before crosslinking and 80% of the peptide is added to the HA. In some embodiments, 10% of the peptide is added in the HA-XL before crosslinking and 90% of the peptide is added to the HA. In some embodiments, 5% of the peptide is added in the HA-XL before crosslinking and 95% of the peptide is added to the HA. In some embodiments, the peptide is only added to the HA-XL. In other embodiments, the peptide is only added to the HA. In embodiments, the peptide is added after mixing HA and HA- XL. For some indication it is preferable that the release profile is slower, such as when the gel is used for chronic conditions, such as chronic wounds, chronic periodontitis or chronic periimplantitis. In such indications it is preferable that a larger portion of the peptide is released slowly from the gel, such as over several days to months.

[0148] For other indications a faster release is preferable. In example for more acute indications, such as acute wounds, including surgical wounds, acute inflammation and acute bone healing e.g., after surgy and traumas, a faster peptide release is preferred. In such indications it is preferable that a larger portion of the peptide is released faster from the gel such as within hours to days.

[0149] It is known in the art that it contributes to a better compliance if a drug can be dosed, e.g., 1-2 times per day instead of 3-4 times per day. However, it may also be favourable that there is an immediate release of at least a portion of the drug followed by a sustained release of the drug.

[0150] In the current context, “Hyaluronic acid” is defined herein as an unsulphated glycosaminoglycan composed of repeating disaccharide units of N-acetylglucosamine (GIcNAc) and glucuronic acid 20 (GlclIA) which are linked together by alternating beta-1 ,4 and beta-1 ,3 glycosidic bonds. Hyaluronic acid is also known as hyaluronan, hyaluronate, or hyaluronic acid fibres. The terms hyaluronan, hyaluronic acid, and hyaluronic acid fibres are in the current context used interchangeably. The level of hyaluronic acid may be determined according to the modified carbazole method (Bitter and Muir, 1962, Anal Biochem. 4: 330-334).

[0151] In a preferred embodiment the hyaluronic acid used according to the invention is of a very pure quality, in particular current Good Manufacturing Practice (cGMP) quality. The hyaluronic acid used in the formulations according to the current invention can be derived from a variety of sources. Rooster combs are a significant commercial source for hyaluronic acid. Alternatively, microorganisms are an alternative source. US 4,801 ,539 and EP 0694616 disclose fermentation methods for preparing hyaluronic acid involving a strain of Streptococcus zooepidemicus.

[0152] In embodiments, the hyaluronic acid or salt thereof is of microbial origin, preferably recombinantly produced. In one embodiment, the hyaluronic acid or salt thereof is produced by a Gram-positive bacterium, such as, but not limited to Bacillus and / or Streptococcus. In a currently preferred embodiment, the hyaluronic acid or salt thereof is produced by Bacillus subtilis. The hyaluronic acid or salt thereof may be produced as disclosed in WO 03 / 054163.

[0153] Hyaluronan synthases have been described from vertebrates, bacterial pathogens, and algal viruses (DeAngelis, P. L„ 1999, Cell. Mol. Life Sci. 56: 670-682). WO 99 / 23227 discloses a Group I hyaluronate synthase from Streptococcus equisimilis. WO 99 / 51265 and WO 00 / 27437 describes a Group II hyaluronate synthase from Pasturella multocida. Ferretti et al. disclose the hyaluronan synthase operon of Streptococcus pyogenes, which is composed of three genes, hasA, hasB, and hasC, that encode hyaluronate 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 having a coding region for a Streptococcus equisimilis hyaluronan synthase.

[0154] The host cell may be any Bacillus cell suitable for recombinant production of hyaluronic acid. The Bacillus host cell may be a wild-type Bacillus cell or a mutant thereof. Bacillus cells useful in the practice of the present invention include, but are not limited to, Bacillus agaraderhens, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis,

[0155] 20 Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells. Mutant Bacillus subtilis cells particularly adapted for recombinant expression are described in WO 98 / 22598. Non encapsulating Bacillus cells are particularly useful in the present invention. Since the hyaluronan of a recombinant Bacillus cell is expressed directly to the culture medium, a simple process may be used to isolate the hyaluronan from the culture medium. Sa / ts of hyaluronic acid

[0156] Any salt of hyaluronic acid may be used according to the present invention.

[0157] 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. hyaluronic acid Molecular weight

[0158] Any molecular weight may be used according to the invention, but in a preferred embodiment the hyaluronic acid or the salt thereof has an average molecular weight in the range of 0.2-6 MDa; more preferably an average molecular weight in the range of 0.7-4.0 MDa; more preferably an average molecular weight in the range of 1.0-3.0 MDa; and even more preferably an average molecular weight in the range of 1 .25 to 1 .75 MDa. The molecular weight may be determined as known in the art. In preferred embodiments, the molecular weight of the hyaluronic acid is about 1.5 MDa.

[0159] The specific MWs of the hyaluronic acid in the currently described formulation are chosen to provide a beneficial viscosity relationship, i.e., a slow release due to higher entanglement as is described in e.g., Falcone 2005, as well as in WO 2013 / 030348 A1 , showing experiments of increasing hyaluronic acid concentrations vs release times.

[0160] A pharmaceutical and / or cosmetic formulation according to the current invention comprises cross-linked hyaluronic acid fibres (HA-XL), and linear hyaluronic acid fibres (HA).

[0161] Typically, a pharmaceutical and / or cosmetic formulation according to the current invention comprises 1-40 mg / mL, such as 1.0, 2.5, 4, 10, 20, 25 or 40 mg / mL cross-linked hyaluronic acid fibres (HA-XL), and 1-40, such as 1.0, 2.5, 4, 10, 20, 25 or 40 mg / mL mg / mL linear hyaluronic acid fibres (HA).

[0162] A pharmaceutical and / or cosmetic formulation according to the current invention can comprise cross-linked hyaluronic acid fibres (HA-XL) which comprise or consist of 1 ,4- butanediol diglycidyl ether cross-linked hyaluronic acid fibres (HA-XL(BDDE)) and / or poly(ethylene glycol) diglycidyl (PEGDE) ether cross-linked hyaluronic acid fibres (HA- XL(PEGDE)).

[0163] Preferably, the formulation of the current invention comprises both cross-linked and linear hyaluronic acid. The ratio of cross-linked to linear hyaluronic acid may be varied for specific purposes. Accordingly, in embodiments, the formulation of the current invention may comprise cross-linked and linear hyaluronic acid, wherein 87.5-92.5%, such as about 90% of the hyaluronic acid is cross-linked and between 7.5% and 12.5%, such as about 10% of the hyaluronic acid is linear.

[0164] In a pharmaceutical and / or a cosmetic formulation according to the current invention, the hyaluronic acid fibres are typically between 0.7-4.0 MDa, such as between 1.0-2.0, between 1 .5-1 .8, or between 3.0-3.3 MDa. In one embodiment the Hyaluronic acid monomers are between 3.0-3.3 MDa.

[0165] In a currently preferred embodiment, a pharmaceutical and / or a cosmetic formulation according to the current invention comprises a. 0.1-250ug / mL such as 50 pg / mL of an artificial peptide and b. 1-40 mg / mL hyaluronic acid fibres.

[0166] In a currently preferred embodiment, a pharmaceutical and / or a cosmetic formulation according to the current invention comprises a. 50 pg / mL of an artificial peptide, b. 20 mg / mL 1 ,4-butanediol diglycidyl ether cross-linked hyaluronic acid fibres (HA-XL(BDDE)), and c. 2.5 mg / mL linear hyaluronic acid fibres (HA).

[0167] In a currently preferred embodiment, a pharmaceutical and / or a cosmetic formulation according to the current invention comprises a. 50 pg / mL of an artificial peptide, b. 20 mg / mL poly(ethylene glycol) diglycidyl (PEGDE) ether cross-linked hyaluronic acid fibres (HA-XL(PEGDE)), and c. 2.5 mg / mL linear hyaluronic acid fibres (HA). Preferably the, poly(ethylene glycol) diglycidyl ether has a molecular weight (MW) of 250-1500 Da, such as about 500 Da.

[0168] In a currently preferred embodiment, a pharmaceutical and / or a cosmetic formulation according to the current invention comprises a. 50 pg / mL of an artificial peptide with the amino acid sequence of SEQ ID NO 4 and / or SEQ ID NO 5, b. 20 mg / mL 1 ,4-butanediol diglycidyl ether cross-linked hyaluronic acid fibres (HA-XL(BDDE)), and c. 2.5 mg / mL linear hyaluronic acid fibres (HA). In a currently preferred embodiment, a pharmaceutical and / or a cosmetic formulation according to the current invention comprises a. 50 pg / mL of an artificial peptide with the amino acid sequence of SEQ ID NO 4 and / or SEQ ID NO 5, b. 20 mg / mL poly(ethylene glycol) diglycidyl (PEGDE) ether cross-linked hyaluronic acid fibres (HA-XL(PEGDE)), and c. 2.5 mg / mL linear hyaluronic acid fibres (HA).

[0169] A pharmaceutical formulation

[0170] The current invention relates to a pharmaceutical and / or a cosmetic formulation in the form of a gel comprising a. an artificial peptide characterized by a proline sequence prevalent in key matricellular proteins that partake in wound healing as well as in bone and cartilage formation and connective tissue maintenance in the majority of vertebrates, b. cross-linked hyaluronic acid fibres (HA-XL), and c. linear hyaluronic acid fibres (HA). In embodiments, the pharmaceutical formulation further comprises MSG cells.

[0171] A pharmaceutical and / or a cosmetic formulation according to the current invention is stable / in gel form at RT for at least 1-2 years and / or in the body for at least 30 days.

[0172] A pharmaceutical and / or a cosmetic formulation according to the current invention is stable / in gel form at RT for at least 1-2 years, such as for at least 1 year, such as for at least 1 .5 years, such as for at least 1 .7 years, such as for at least 2 years, such as for 1 year, 1 .5, 1 .7, 1 .8, 2, 3, or 5 years.

[0173] A pharmaceutical and / or a cosmetic formulation according to the current invention is stable / in gel in the body for at least 30 days, such as for at least 31 days, such as for at least 32 days, such as for at least 35 days, such as for at least 40 days, such as for at least 50 days, such as for at least 60 days, such as for at least 90 days.

[0174] In one aspect, a pharmaceutical and / or a cosmetic formulation according to the current invention releases the comprised artificial peptide at a controlled rate. In the current context, the term “at a controlled rate” is used to describe the release to not be a burst release. Controlled release is herein characterized by releasing drugs according to a predictable and rational programmed rate to achieve the optimal target-drug concentration. This dosage form enhances the safety, efficacy, reliability, and convenience of drug therapy. Although this is a slow releasing system, unlike sustained release, this process is designed to produce predictable, constant concentrations of the drug. For this approach, the concentration of the active ingredient in the target tissue is controlled, not just the release of the drug. In one aspect, a pharmaceutical and / or a cosmetic formulation according to the current invention releases the comprised artificial peptide at a controlled rate, such as of about 2 ug per hour, such as at the most 10, 5, 4, 3, 2, 1.5, 1.2 or 1 ug per hour or such as about 10ng, 100ng, 500ng per hour, or such as 0.01 , 0.1 , 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 ug per hour. In one aspect, a pharmaceutical and / or a cosmetic formulation according to the current invention releases the comprised artificial peptide at a controlled rate, such as of between 0.1-10ug per hour, such as of between 1-8ug per hour, such as of between 1.5- 5ug per hour.

[0175] A pharmaceutical and / or a cosmetic formulation according to the current invention can further comprise one or more sources of fluoride, such as e.g., one or more fluoride sources selected from the group consisting of NaF, CaF2 and ZnF2. Preferably the fluoride source is NaF.

[0176] A pharmaceutical and / or a cosmetic formulation according to the current invention can further comprise one or more of a component selected from the group consisting of:

[0177] Sorbitol, Xylitol, NaOH, HCL, Phosphate Buffer Solution (PBS) and Acetic Acid.

[0178] In a currently preferred embodiment, the pharmaceutical and / or cosmetic formulation / composition comprises Sodium fluoride and / or Citric Acid.

[0179] In preferred embodiments, the pharmaceutical and / or cosmetic formulation / composition comprises a. 50 pg / mL of an artificial peptide with the amino acid sequence of SEQ ID NO 4 and / or SEQ ID NO 5, b. 20 mg / mL 1 ,4-butanediol diglycidyl ether and / or poly(ethylene glycol) diglycidyl ether cross-linked hyaluronic acid fibres (HA-XL(BDDE) or HA-XL(PEGDE)), c. 2.5 mg / mL linear hyaluronic acid fibres (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 Phosphate and j. 0.08 wt.% Sodium Phosphate. Osmolarity

[0180] Maintaining an appropriate osmolarity ensures compatibility with the body's cells and prevents cellular damage, and solutions with similar osmolarity to body fluids reduce tissue irritation, minimize inflammation and other complications. Accordingly, maintaining a suitable osmolarity of the pharmaceutical and / or cosmetic formulation / composition of the current invention is highly beneficial. Many buffers, such as PBS, mimic the preferred osmolarity of mammals, and have an osmolarity of about 280-310 mOsm / L. Accordingly, in embodiments, the pharmaceutical and / or cosmetic formulation of the present invention has an osmolarity of 50-400 mOsm / L, such as between 100 and 310 mOsm / L, or such as between 125 and 175 mOsm / L, such as about 150 mOsm / L, or such as between 275 and 325 mOsm / L, such as about 300 mOsm / L.

[0181] Sodium Fluoride

[0182] Sodium fluoride is a commonly used ingredient in dental products, such as toothpaste and mouthwash to prevent cavities and promote oral health. It can help to reduce the number of bacteria that causes tooth decay by inhibiting their growth and promoting remineralization of the tooth enamel. The fluoride enhances the strength of teeth by the formation of fluorapatite, a naturally occurring component of tooth enamel. It can prevent damage to dental tissue by inhibiting acid production from commensal oral bacteria.

[0183] Citric Acid

[0184] Citric Acid is used for product preservation and for pH adjustment of the pharmaceutical and / or cosmetic formulation / composition according to the current invention. The citric acid has an impact on the stability of the product. Citric acid can create an acidic environment that can inhibit bacterial growth and reduce the risk of spoilage. This is because most bacteria prefer a neutral or slightly alkaline environment and cannot survive in highly acidic conditions. Citric acid can also chelate, or bind to, certain essential nutrients like iron, which some bacteria require for growth.

[0185] Active ingredient

[0186] In embodiments, a pharmaceutical and / or a cosmetic formulation according to the current invention can further comprise another / additional active ingredient, such as, but not limited to, an active ingredient selected from the group consisting of growth factors, plasma rich fibrin / plasma and enamel matrix derivatives. MSCs

[0187] Mesenchymal stem cells (MSCs) also known as mesenchymal stromal cells or medicinal signalling cells, are multipotent stromal cells that can differentiate into a variety of cell types, including osteoblasts (bone cells), chondrocytes (cartilage cells), myocytes (muscle cells) and adipocytes (fat cells which give rise to marrow adipose tissue).

[0188] MSCs can be isolated from a variety of tissues, such as umbilical cord, endometrial polyps, menses blood, bone marrow, adipose tissue, etc. and are found circulating peripherally and are homed in at sites of injurie, where they undergo differentiation and promote healing. One of the challenges with using MSCs in clinical application is related to problems with the homing of the cells, i.e., making the cells stay at the injured area and therefrom differentiate to promote healing.

[0189] In embodiments, the pharmaceutical formulation described herein further comprises MSC cells. In further embodiments, the pharmaceutical formulation comprises between 100.000-10.000.000 MSC cells pr. ml, preferably about 1.000.000 MSC cells pr. ml. In additional embodiments, the pharmaceutical formulation promotes Mesenchymal Stromal Cell (MSC) cell homing. Medical uses

[0190] In the experimental section, a pharmaceutical and / or a cosmetic formulation according to the current invention is shown to in vivo and in vitro induce wound healing in soft tissue, as well as to stimulate collagen production and orientation. One aspect of the current invention thus relates to the novel pharmaceutical formulation according to the current invention for use in medicine, in particular for use in soft tissue healing, such as, but not limited to, for use in wound healing, for inducing neovascularization, for inducing reepithelization, for stimulating collagen production, and / or for promoting oriented collagen formation.

[0191] In example 10 it is shown that the pharmaceutical formulation as described herein has beneficial effects in cartilage and bone healing process, in particular in pathways such as heparan sulfate degradation, glycosaminoglycan degradation, assembly of collagen fibrils and other multimeric structures, collagen biosynthesis and modifying enzymes, ECM- receptor interaction, extracellular matrix organization, mineral absorption. And it was found that the pharmaceutical formulation accelerated the first stages of fracture healing by upregulating genes such as collagen biosynthesis and modifying enzymes.

[0192] The novel pharmaceutical formulation according to the current invention can, further or in combination with the above-mentioned uses, be for use in an anti-inflammatory and / or antimicrobial treatment, for use in treating periodontitis, mucositis and / or periimplantitis.

[0193] The novel pharmaceutical formulation according to the current invention can, further or in combination with the above-mentioned uses, be used as an anti-inflammatory and / or antimicrobial treatment for periodontitis, mucositis, and / or periimplantitis.

[0194] The novel pharmaceutical formulation according to the current invention can, further or in combination with the above-mentioned uses, be for use in bone fracture healing and / or hard tissue healing.

[0195] As is shown in example 10, the pharmaceutical formulation as disclosed herein can aid in the homing of MSC cells to sites of injury, and the provision of a formulation comprising both the consensus peptide and the hyaluronic acid gel as disclosed herein has a surprisingly beneficial synergistic effect in the since that besides promoting healing it was also found that the formulation reduced local inflammation, resulted in beneficial epigenetic changes, downregulation of essential signalling pathways and activation of the bone mineralization and BMP pathway.

[0196] Accordingly, in embodiments the pharmaceutical formulation as disclosed herein reduces local inflammation. In further embodiments, the pharmaceutical formulation as disclosed herein leads to an upregulation of genes involved in collagen biosynthesis and / or collagen modifying enzymes. Additionally, the pharmaceutical formulation as disclosed herein may activate enzymes and pathways involved in the tissue healing process e.g., cartilage. In embodiments the pharmaceutical formulation modifies the expression of genes involved in heparan sulfate degradation, glycosaminoglycan degradation, assembly of collagen fibrils and other multimeric structures, collagen biosynthesis and modifying enzymes, ECM- receptor interaction, extracellular matrix organization and / or mineral absorption. In embodiments, the modified expression is determined by a reduced or increased expression which is more than ln(2)-fold different from the comparative state. Accordingly, a modified expression may be considered when the expression level of a gene is at least ln(2)-fold increased or decreased. As such the comparative condition may be a basal expression i.e., before injurie, expression following injurie, expression after treatment with and hyaluronic acid gel, or similar conditions. In additional embodiments, the pharmaceutical formulation as disclosed herein, leads to a down regulation of IL-2, IL-5, IL-3 and / or B cell receptor signalling pathways. In further embodiments, the pharmaceutical formulation activates the bone mineralization and / or BMP pathways. In embodiments, the pharmaceutical formulation as disclosed herein is for us as an antiinflammatory composition. In further embodiments, the anti-inflammatory effect is measured as a reduction in proinflammatory cytokines. In further embodiments, the proinflammatory cytokines comprises one or more cytokines selected from the group consisting of IL-23, IL-1 alpha, IL-1 beta, TNF-alpha, MCP-1 , IL-12P70, IFN-Y, IFN-beta, IL-6, IL-10, IL-27, IL-17A and GM-CSF.

[0197] In embodiments, the pharmaceutical formulation as disclosed herein, which comprises the consensus peptide (see Table 4 - HA+HA-XL(BDDE) vs. HA+HA-XL(BDDE)+P2), leads to a modified expression of one or more of the genes 12 significant genes identified in cell division, cell cycle and cell metabolism. In further embodiments, the formulation disclosed herein, comprising the consensus peptide and optionally MSC cells, induces a modified expression of one or more of the genes to allow MSC to interact with cells in the fracture sites to activate the cell- cell interactions by exosomes to active the downstream metabolisms (see Table 4 - HA+HA-XL(BDDE) vs. HA+HA-XL(BDDE)+P2).

[0198] Periodontal diseases

[0199] The herein for the first-time disclosed formulation is in one aspect intended for treating periodontal diseases, disorders and / or conditions, such as by, but not limited to, inducing periodontal regeneration.

[0200] Periodontal diseases include several forms and symptoms; however, the most common are gingivitis and periodontitis. As dental implants are becoming more common in the routine treatment in dentistry, an additional periodontal disease, peri-implantitis, is increasing in prevalence. According to the US National Institute of Dental and Craniofacial Research, periodontal disease is an infection of the tissue that holds one's teeth in place. It is typically caused by poor oral hygiene that allows the sticky film of bacteria, known as plaque, to build up on the teeth and later develop into calculus.

[0201] Gingivitis is characterized by the redness of the gum margins, swelling and bleeding on brushing. Gingivitis occurs in both chronic and acute forms. The majority of adults are affected by gingivitis. Gingivitis does not always develop into periodontal disease. It is a silent disease and a patient suffering from the disease might only begin to notice when it is already irreversible.

[0202] Acute gingivitis is associated with specific infections, micro-organisms, or trauma, and chronic inflammation of the gum tissue surrounding the teeth is associated with the bacterial biofilm that covers the teeth and gums.

[0203] Periodontitis is a chronic condition involving bacterial degradation of the soft and hard tissues that surround and support teeth. In the early stages, inflammation and irritation is observed in the gums. Progressive loss of the bone around the teeth may lead to loosening and eventual loss of teeth if left untreated. The loss of teeth leads not only to complicated and costly dental health problems, but also a devastating personal impact for the patient in terms of self-confidence.

[0204] Periodontitis is one of the leading causes of poor oral health globally. When the disease is allowed to progress into severe periodontitis, the acidic bacteria layer can be removed by the dentist using mechanical debridement or bleach-based solutions. Although this momentarily stops the progress of the disease, it does not revert the damage. There are currently some surgical treatment alternatives, e.g., flap surgery, soft tissue grafting and bone grafting, but these are invasive and thereby costly.

[0205] Periodontitis affects the bone and supporting tissue and is characterized by the formation of pockets or ‘spaces’ between the tooth and gums. The severity of the attack varies from one individual to another, depending on the virulence of the bacterial plaque and on the efficiency of the local and systemic immune inflammatory response of the person. No doubt the host response is influenced by both environmental and genetic factors. Systemic diseases are an additional factor in determining the severity of periodontal disease. These may include diabetes, leukemia, Down syndrome and others. Other factors associated with periodontal disease are smoking and stress.

[0206] Periodontal disease is believed to be associated with various systemic health issues. Increasing evidence points to the fact that diseases, including Alzheimer's, cancer (pancreatic cancer), respiratory diseases, diabetes, hypertension, atherosclerosis and others are associated with gum diseases. It is also believed that the link between heart (myocardial infarction) and gum disease may be due to bacteria. Bacteria in the gums can enter the blood supply and be transferred to distant destinations, including the heart. Other organs which may be affected are the lungs and genital organs, as well as erectile disfunction.

[0207] “Peri-implantitis” or “periimplantitis” and “perimucositis” is dental terms used to describe the destructive inflammatory process affecting the soft and hard tissues surrounding dental implants. Compared to mucositis, the definition of peri-implantitis includes bone loss. Among others, smoking, accumulation of bacterial biofilms (plaque), oral hygiene and periodontal status are influential factors. In the present context, the term “periodontal diseases” encompasses peri-implant infections, such as periimplantitis and perimucositis. The formulation according to the current invention is in one aspect intended for treating periodontal diseases selected from the group consisting of gingivitis, periodontitis, periimplantitis, peri-mucositis, gingivitis, aphthous stomatitis and other oral infections and / or inflammations. Wound healing

[0208] Wound healing refers to a living organism's replacement of destroyed or damaged tissue by newly produced tissue.

[0209] In undamaged skin, the epidermis (surface, epithelial layer) and dermis (deeper, connective layer) form a protective barrier against the external environment. When the barrier is broken, a regulated sequence of biochemical events is set into motion to repair the damage. This process is divided into predictable phases: blood clotting (hemostasis), inflammation, tissue growth (cell proliferation), and tissue remodelling (maturation and cell differentiation). Blood clotting may be considered to be part of the inflammation stage instead of a separate stage. Example 9 discloses the wound healing effect of the formulations as disclosed herein, in particular compositions comprising P2 (SEQ ID NO: 4) or P6 (SEQ ID NO: 5).

[0210] The wound healing process is not only complex but fragile, and it is susceptible to interruption or failure leading to the formation of non-healing chronic wounds. Factors that contribute to non-healing chronic wounds are diabetes, venous or arterial disease, infection, and metabolic deficiencies of old age. During wound healing, the synthetic peptides in the formulation of the current invention e.g., promote collagen production and wound filling or refilling, and prevent scarring through modulation of inflammation signalling. The synthetic peptides in the formulation of the current invention can further be effective in suppressing inflammation in the wound.

[0211] As is shown in the experimental section, the formulation according to the current invention is promotes wound healing, e.g., split skin suction wounds were epithelialized twice as fast with P6 active peptide motif present. Additionally, it was surprisingly found that some embodiments of the formulation, in particular HA+HA-XL(BDDE)+P2, significantly improved the reduction in wound area, compared to HA+HA-XL(BDDE) alone.

[0212] Additionally, Example 10 discloses that a formulation according to the current invention also reduces the level of inflammatory markers, in specific 13 proinflammatory cytokines. In the example 12 proinflammatory cytokines were assayed (IL-23, IL-1 alpha, IL-1 beta, TNF-alpha, MCP-1 , IL-12P70, IFN-Y, IFN-beta, IL-6, IL-10, IL-27, IL-17A and GM-CSF), and out of the 13 cytokines, 12 was found to be reduced following administration of the a formulation according to the current invention. It is surprising that the formulation comprising the consensus peptide in the HA+HA-XL(BDDE)cross-linked gels so clearly exceeds the effect of the HA+HA-XL(BDDE) gels cross-linked alone on almost all accounts, when measuring the proinflammatory cytokines. This clearly shows the benefit of formulating the consensus peptide in the HA+HA-XL(BDDE) cross-linked over just providing the HA+HA-XL(BDDE) gel or the consensus peptide alone and clearly suggests that there is a surprising synergistic effect of the formulation.

[0213] As such, the anti-inflammatory effect of a formulation according to the current invention may be measured as a reduction in proinflammatory cytokines. Such proinflammatory cytokines may be selected from the group consisting of IL-23, IL-1 alpha, IL-1 beta, TNF- alpha, MCP-1 , IL-12P70, IFN-Y, IFN-beta, IL-6, IL-10, IL-27, IL-17A and GM-CSF. In the examples the proinflammatory cytokines were measured using flow cytometry, and alternative methods for determining the amount of particular cytokines will be evident to the skilled person.

[0214] Furthermore, as is shown in Example 10, it was shown that Hyd+P2 significantly accelerated the first stages of tissue healing by upregulating genes such as collagen biosynthesis and modifying enzymes, ECM-receptor interaction, extracellular matrix organization.

[0215] In embodiments, administration of the pharmaceutical formulation disclosed herein upregulates tissue healing genes such as genes involved in e.g., collagen biosynthesis and modifying enzymes, ECM-receptor interaction, extracellular matrix organization.

[0216] Example 10 further shows that the presence of mesenchymal stromal cells (MSCs) also promotes tissue healing and reduces inflammation. MSCs largely are known to proliferate and differentiate into skin cells to restore injured or dead cells, but also perform by an autocrine and paracrine pathway to stimulate cell regeneration and wound healing. MSCs are involved in all three phases of wound healing to varying degrees and also influence the wound's ability to progress beyond the inflammatory phase and not regress to a chronic wound state. MSCs are released from bone marrow and are generally found to be present in several tissues. Upon infliction of tissue damages, MSCs are recruited to the area of infliction by the mechanism known as “MSC homing”. It is generally recognised that MSC homing is a multi-step process involving specific molecular interactions in the inflicted area which influences the amount of MSCs which will stick to the inflicted area, and estimates are that only few percent of cells are efficiently recruited to the infliction area even when high amounts of cells are present in the area of the infliction.

[0217] Accordingly, one of the properties of the pharmaceutical formulation disclosed herein is to enhance and / or improve MSC homing. As the examples show, the pharmaceutical formulation as disclosed herein, specifically one formulated with the peptide P2, improves the MSC homing, which shows as improved tissue regeneration and higher and more uniform bone formation (See figures 12-15). Furthermore, the presence of MSCs further showed an improved orientation and smaller T parameter and platelet sizes indicating an more mature bone and bone remodeling (Figure 16 and 17). The beneficial properties of the pharmaceutical formulation disclosed herein on MSC homing were further underlined by the induced epigenetic changes, local inflammation downregulation, and fracture healing shown in example 10. These results clearly show the synergistic advantage of using the HA+HA-XL(BDDE)+consensus peptide formulation to reduce inflammation and improve fracture healing in polytrauma.

[0218] In embodiments, administration of the pharmaceutical formulation disclosed herein leads to epigenetic changes in genes associated with (see Table 4 - HA+HA-XL(BDDE) vs. HA+HA-XL(BDDE)+P2], local inflammation downregulation as seen by cytokine reduction and / or tissue healing.

[0219] Structurally the matricellular poly-proline motifs of the artificial peptides comprised in the formulation according to the current invention provide stability for the extracellular matrix. Biologically they play important local functions in “one-to-many” signalling, signal transduction, transcription, cell motility, healing and immune modulation, chaperoning, membrane stabilization, mineral-binding, surface recognition and attachment and participate various protein-protein interactions.

[0220] The motifs support dermal healing by promoting EGF expression in connective tissue cells and can also support vascularisation and modulate inflammation through stimulating the expression of PDGF, VEGF and anti-inflammatory cytokines.

[0221] This motif is highly conserved in mammals and does not induce any immunogenic response in humans. It is non-toxic in all models tested. The artificial peptides comprised in the formulation according to the current invention also promote local vascularization and modulate acute and chronic inflammation. They rejuvenate senescent cells (e.g., from irradiation damage) and reduce pain and swelling when applied locally. During wound healing the artificial peptides comprised in the formulation according to the current invention in particular promote collagen production and wound filling and prevents scarring through inflammation modulation signalling. The artificial peptides comprised in the formulation according to the current invention stimulates regeneration and healing. As is shown in the experimental section, the formulation according to the current invention provides a faster epithelialization of full thickness wounds in pigs. More importantly, it promotes collagen expression and vascularisation and gives a much better wound-fill than the hydrogel control. In this way the effect of the artificial peptides comprised in the formulation according to the current invention also prevents scarring and disfiguration of the wound area. As an added value, the artificial peptides comprised in the formulation according to the current invention also modulate inflammation and reduce healing time, swelling and pain during the initial healing phase. This effect has also been proven in gingival tissues.

[0222] As is also shown in the experimental section, the formulation according to the current disclosure was also shown to promote re-epithelisation while reducing granulation and inflammation 6 days after wound infliction. Additionally, the formulation according to the current disclosure also reduced the wound void area 6 days after wound infliction.

[0223] In particular formulations with P2 were found to reduce the wound void area, promote re- epithelisation while reducing granulation and inflammation 6 days after wound infliction. Accordingly, the formulation according to the current disclosure may be used to reduce the wound void area. In further instances the formulation according to the current disclosure may also enhance and / or promote wound re-epithelisation, and / or reduce granulation. In particular, the formulations comprising the artificial peptides P2 was found to be especially suited for enhancing and / or promoting wound re-epithelisation, and / or reducing granulation.

[0224] In additional instances the formulation according to the current disclosure may also reduce inflammation. Particularly, the formulations comprising the artificial peptides P2 (SEQ ID NO: 4) or P6 (SEQ ID NO:5) were found to be especially suited for reducing inflammation.

[0225] In summary, the artificial peptides comprised in the formulation according to the current invention are effective on connective tissue with a secondary promotion of epithelial cells and vascularization.

[0226] In dermatological dosages (micrograms / mL) the artificial peptides comprised in the formulation according to the current invention restore normal activity in radiation (e.g., sun burn) senescent cells (rejuvenation), repair irradiated skin and modulate inflammation (reduces redness, swelling and pain).

[0227] In wound care, the formulation according to the current invention can be used to promote collagen production, for both acute (e.g., oral surgery) and chronic wounds (e.g., periodontitis) where its application modulates inflammation, improves vascularization, promotes collagen production and wound fill and prevents scarring. It has also been proven experimentally to improve graft-take after skin and mucosal grafting procedures. In embodiments, a pharmaceutical and / or cosmetic composition described herein is used for treating soft tissue wounds and / or promoting soft tissue wound healing in the craniomaxillofacial complex (CMS) of a patient in need thereof.

[0228] In embodiments, a pharmaceutical and / or cosmetic composition described herein is used for treating soft tissue wounds and / or promoting soft tissue wound healing in the larynx, soft tissue of the neck, hypopharynx and / or pharynx of a patient in need thereof.

[0229] Taking grafts / Graft preparation

[0230] The pharmaceutical and / or cosmetic composition described herein can be used for taking grafts, such as in therapy for chronic venous leg ulcers.

[0231] Chronic, hard-to-heal venous ulcers are a debilitating and potentially life-threatening condition affecting millions worldwide. Current treatment modalities for chronic venous leg ulcers aim to manage underlying venous insufficiency and promote healing. Still, the presence of bacterial colonisation, comorbidities, and chronic inflammation often complicate the process. The current treatment paradigm relies on auto-grafts and transplants, which often have limited success rates and pose significant risks to patients.

[0232] The current standard of care for non-healing wounds involves repeated surgical procedures, medical expertise, and lengthy hospital stays. Moreover, chronic wounds can lead to severe complications, including amputations and, eventually, mortality.

[0233] Chronic ulcers are a pressing health issue that causes significant morbidity and pose a challenge to both patients and physicians. These wounds require lengthy treatments, and the need for long periods of patient compliance often leads to frustration. Despite the availability of numerous medical and surgical treatments, chronic wounds persist when the normal reparative process is disrupted, leading to poor healing outcomes. Venous leg ulcers, in particular, are a significant burden on the healthcare system. Although compression bandages and good wound care are widely accepted as fundamental treatments, at best, between a quarter and a half of all ulcers still remain unhealed.

[0234] A pharmaceutical and / or cosmetic composition described herein can provide sustainable, safe and effective combined or stand-alone treatments that benefit patients and contribute to the long-term sustainability of health care.

[0235] Among skin grafting procedures, meshed split-thickness autografts are recognised as the standard golden treatment for chronic vascular leg ulcers due to their efficacy in promoting healing. This type of graft can be expanded using meshing, which improves graft take by allowing blood and exudates to escape through the mesh. Split-thickness autografts are preferred for wounds with limited vascular supply, such as chronic or recalcitrant ulcers, as they contain less tissue requiring vascularisation after transfer. Fullthickness skin grafts containing the epidermis and entire dermis pose a challenge for treating patients with compromised arterial circulation, as they are too thick to be adequately vascularised. In such cases, split-thickness grafts have a higher chance of survival than full-thickness grafts. Pinch grafting is another treatment option for small leg ulcers, but its application is limited by the donor site's required size, which is approximately twice the size of the target ulcer.

[0236] In embodiments, a pharmaceutical and / or cosmetic composition described herein is used in combination and / or replacing conventional skin transplantation using split-thickness, full-thickness or pinch autografts.

[0237] The grafting can in addition be combined with a cell-based therapy that can significantly reduce autografting and associated discomfort, treatment times, and morbidity.

[0238] In this context, wound preparation has emerged as one essential step to improve the efficacy of autologous skin patch treatment for wound debridement involving the damaged tissue and foreign material to promote healthy tissue growth and reduce the risk of infections. On the other hand, cell homing relies on growth factors and other biological agents that attract cells to the wound site, promoting healing and regeneration. By preparing the wound with an advanced, bio-friendly, non-antibiotic debridement and wound priming with biomimicking molecules designed to promote connective tissue health, the current invention provides a combined ATMP skin patch which is significantly improved, offering a superior solution for chronic ulcer treatment.

[0239] In embodiments, a pharmaceutical and / or cosmetic composition according to the current invention is wound care product that contains biomimicking, intrinsically disordered proteins (IDPs) delivered in a cross-linked hyaluronic acid gel. The product has proven effects in wound care models, specifically by affecting vascularisation through the VEGF pathway positively. The IPDs are synthetic and have a favourable safety profile, a high binding affinity and specificity for different molecular partners, including proteins, nucleic acids, and small molecules, making them an ideal option for priming chronic venous ulcers. The IPDs can act as scaffolds for presenting bioactive peptides or growth factors that promote tissue regeneration and wound healing.

[0240] This novel approach can potentially reduce patient discomfort and treatment time while improving patient outcomes and reducing healthcare costs. By reducing the need for tissue engineering and / or autografting, this innovative approach can significantly improve the treatment of hard-to-heal ulcers, leading to faster healing times and ulcers.

[0241] In consequence, the current invention in one aspect relates to a novel treatment indicated for a specific subset of chronic wounds, in particular, those that require treatment with split-thickness mesh grafts.

[0242] Cosmetic uses

[0243] In cosmetic dosages, the artificial peptides comprised in the formulation according to the current invention conserve and protect connective tissue cells and modulate inflammation. The artificial peptides comprised in the formulation according to the current invention also promote collagen production and have potential for use in an anti-wrinkle formulation.

[0244] In particular, the artificial peptides described in the current application are antimicrobial as well as anti-inflammatory, thus, the inclusion of the artificial peptide in the hyaluronic acidbased hydrogel adds an active anti-inflammatory, antimicrobial, antibacterial and / or antibiotic component to the formulation, which renders the use of hyaluronic acid-based hydrogels for cosmetic purposes safer and less prone to infections and / or inflammations after implanting or filling. In embodiments, a cosmetic composition as described herein is used for improving skin elasticity and hydration. Thus, in one aspect, the current invention relates to the cosmetic use / application of the cosmetic composition as described herein for improving skin elasticity and hydration in a mammal, as well as the improvement of skin elasticity and hydration of skin in a mammal by applying a cosmetic composition as described herein.

[0245] In one embodiment, the current invention thus relates to a cosmetic formulation comprising a. 1-3 pg / mL, such as 2 pg / mL of an artificial peptide with the amino acid sequence of SEQ ID NO 4 and / or SEQ ID NO 5, b.20 mg / mL 1 ,4-butanediol diglycidyl ether cross-linked hyaluronic acid fibres (HA-XL(BDDE)) and / or poly(ethylene glycol) diglycidyl ether cross-linked hyaluronic acid fibres (HA-XL(PEGDE)), and c. 2.5 mg / mL linear hyaluronic acid fibres (HA).

[0246] Medical treatment

[0247] In another aspect the invention relates to treating a patient suffering from a disease, disorder and / or condition in soft tissue by administering a pharmaceutical formulation according to the current invention and thus inducing in said patient soft tissue healing, wound healing, neovascularization, for reepithelization, collagen production, and / or oriented collagen formation. Examples 10 and 11 provides exemplary uses of the pharmaceutical formulation in both wound healing and in fracture healing.

[0248] The invention also or in combination relates to treating a patient suffering from a disease, disorder and / or condition in soft tissue by administering a pharmaceutical formulation according to the current invention as an anti-inflammatory and / or antimicrobial treatment.

[0249] The invention also or in combination relates to treating a patient suffering from periodontitis, mucositis, periimplantitis by administering a pharmaceutical formulation according to the current invention to said patient.

[0250] In particular, the invention relates to the use of the pharmaceutical composition for the preparation of a medicament for the treatment of a disease, disorder and / or condition in soft tissue. In particular, the invention relates to the use of a pharmaceutical composition according to the current invention for the preparation of a medicament for the treatment of a wound in soft tissue. In a particular embodiment, the invention also relates to the use of a pharmaceutical composition according to the current invention for the preparation of a medicament for the treatment of a periodontal disease.

[0251] In one aspect, the invention also relates to the use of a pharmaceutical composition according to the current invention for the preparation of a medicament for inducing soft tissue healing, wound healing, neovascularization, for reepithelization, collagen production, and / or oriented collagen formation.

[0252] In one aspect, the invention also relates to the use of a pharmaceutical composition according to the current invention for the preparation of a medicament for use in an antiinflammatory and / or antimicrobial treatment.

[0253] In one aspect, the invention also relates to the use of a pharmaceutical composition according to the current invention for the preparation of a medicament for use promoting bone mineralization.

[0254] In one aspect, the invention also relates to the use of a pharmaceutical composition according to the current invention for the preparation of a medicament for use promoting production of non-mineralized bone.

[0255] In one aspect, the invention also relates to the use of a pharmaceutical composition according to the current invention for the preparation of a medicament for treating a patient suffering from periodontitis, mucositis, periimplantitis.

[0256] Administration

[0257] A pharmaceutical and / or cosmetic formulation of the invention may be administered to a subject in need thereof by any suitable route depending on the tissue which the peptide is to be administered to, for example by topical (dermal), oral, buccal, nasal, aural, rectal or vaginal administration, or by administration to a body cavity such as the oral, nasal and vaginal cavity.

[0258] In particular, application within the dental / odontologic area is of great importance.

[0259] Furthermore, a composition may be adapted to administration in connection with surgery, e.g., in connection with incision within the body. The pharmaceutical and / or cosmetic formulation of the invention may also be administered via local injections, by application in a gel or via a medical device, such as a medical prosthetic device, e.g., a graft, scaffold or bioglass material.

[0260] In one aspect, a pharmaceutical and / or cosmetic formulation of the invention is administered as a minimally invasive administration by injection.

[0261] In another aspect a pharmaceutical and / or cosmetic formulation of the invention is administered by oral application.

[0262] In one embodiment, a pharmaceutical and / or cosmetic formulation of the invention is an oral gel product intended for care of the teeth and mucous membrane of the mouth. The product obtains its effect by forming a protective layer on the teeth and mucous membrane that protects against plaque and tartar formation, giving the product a prophylactic effect against caries, gingival recession, and periodontitis. It is in one embodiment provided in single use units in syringes for effective delivery in the needed region.

[0263] The current invention thus in one aspect relates to the use of a pharmaceutical and / or cosmetic composition according to the current invention for oral application: In particular, for application to the mucous membrane of the mouth and at the interface to the teeth.

[0264] Application to the mucous membrane of the mouth and at the interface to the teeth provides protection of the tissue.

[0265] Method for producing a pharmaceutical and / or a cosmetic formulation

[0266] The current invention also relates to methods for producing a pharmaceutical and / or a cosmetic formulation as disclosed herein.

[0267] In some aspects, the invention relates to a method for producing a pharmaceutical and / or a cosmetic formulation as disclosed herein comprises, g. providing an artificial peptide as defined herein, h. providing linear hyaluronic acid with a molecular weight of 0.7-4 MDa, such as between 1 .0-2.0, between 1.5-1 .8, or between 3.0-3.3 MDa, such as 1.5MDa, i. cross-linking the hyaluronic acid using a crosslinking agent, such as e.g., BDDE or PEGDE to obtain a crosslinked hyaluronic acid (HA-XL), j. optionally, dialysing the crosslinked hyaluronic acid, k. mixing 0.1-250 pg / mL of said artificial peptide and 1-40 mg / mL of said crosslinked hyaluronic acid, and adding 1-40 mg / mL of linear hyaluronic acid to obtain a mixture comprising crosslinked hyaluronic acid, linear hyaluronic acid, and artificial peptide.

[0268] The release of the peptide from the gel comprising crosslinked hyaluronic acid, linear hyaluronic acid, and the artificial peptide, in the beforementioned amounts is expected to follow a single order release profile, where the peptide is released from the gel mainly by diffusion and less by degradation of the gel. As such, a gel with a relative fast release profile as described above is expected to have positive effects on e.g., wound healing and more acute indications, where a fast onset of the action of the peptide is needed.

[0269] In other aspects, the invention relates to a method for producing a pharmaceutical and / or a cosmetic formulation as disclosed herein comprises, a. providing an artificial peptide as defined herein, b. providing linear hyaluronic acid with a molecular weight of 0.7-4 MDa, such as between 1 .0-2.0, between 1.5-1 .8, or between 3.0-3.3 MDa, such as 1.5MDa, c. mixing 0.1-250 pg / mL of said artificial peptide and 1-40 mg / mL of said hyaluronic acid, d. cross-linking the mixture of using a crosslinking agent, such as e.g., BDDE or PEGDE to obtain a mixture of intra- and / or inter-crosslinked peptide and hyaluronic acid, e. adding linear hyaluronic acid (HA) to obtain a mixture of intra- and / or intercrosslinked artificial peptide and hyaluronic acid, and linear hyaluronic acid.

[0270] In embodiments, the method further comprises f. mixing 1-40mg / mL of the intra- and / or inter-crosslinked artificial peptide and hyaluronic acid, with 0.1-250 pg / mL additional artificial peptide to obtain a mixture comprising intra- and / or inter-crosslinked artificial peptide and hyaluronic acid, linear hyaluronic acid, and artificial peptide.

[0271] The release of the peptide from the gel comprising intra- and / or inter-crosslinked hyaluronic acid and peptide, linear hyaluronic acid (HA) and non-crosslinked artificial peptide, is expected to follow a biphasic order release profile, where the peptide is released from the gel by diffusion of the non-crosslinked peptide and release of some portion of the peptide by degradation of the crosslinked gel, leading to an overall intermediate peptide release from the gel. As such, a gel with intermediate peptide release properties as described above is expected to have dual effect, both with a fast onset and a more long-lasting positive effects on both acute indications, such as acute wounds, including surgical wounds, acute inflammation and acute bone healing e.g., after surgy and traumas, and more chronic conditions, such as chronic wounds, chronic periodontitis or chronic periimplantitis.

[0272] In alternative particular embodiments, no additional artificial peptide is added, and the mixture therefore comprises crosslinked peptide and hyaluronic acid, and linear hyaluronic acid.

[0273] The release of the peptide from the gel comprising intra- and / or inter-crosslinked peptide and hyaluronic acid, and linear hyaluronic acid, is expected to follow a single order release profile, where the peptide mainly released following degradation of the crosslinked gel. As such, a gel as described above is expected to have a more long-lasting positive effects on chronic conditions, such as chronic wounds, chronic periodontitis or chronic periimplantitis etc.

[0274] Accordingly, further aspects of the invention relate to a method for producing a pharmaceutical and / or cosmetic formulation according to the current invention, said method comprises, a. providing an artificial peptide as defined herein, b. providing hyaluronic acid with a molecular weight of 0.7-4 MDa, such as between 1.0-2.0, between 1 .5-1 .8, or between 3.0-3.3 MDa, such as 1 ,5MDa, c. mixing 0.1-250 pg / mL of said artificial peptide and 1-40 mg / mL of said hyaluronic acid and d. optionally, adding a source of fluoride to said mixture, and wherein, said pharmaceutical and / or cosmetic formulation has an osmolarity of 50-400 mOsm / L, such as between 100 and 310 mOsm / L, or such as between 125 and 175 mOsm / L, such as about 150 mOsm / L, or such as between 275 and 325 mOsm / L, such as about 300 mOsm / L. In additional embodiments, the said Hyaluronic acid provided in step b. of said method comprises or consists of BDDE and / or PEGDE cross-linked hyaluronic acid.

[0275] In additional embodiments, said method may comprise a. providing the artificial peptide at a concentration of up to 100 mg / mL, b. adding hyaluronic acid fibres dissolved at a concentration of 10 wt.% in 0.3M NaOH, c. mixing in a cross-linking agent such as e.g., BDDE and / or PEGDE, d. heating the reaction vessel to 20-100 °C and incubating for between 1- 2 hours to induce gelification, e. cooling and neutralizing the solution, f. homogenizing the gel, g. dialyzing the gel for at the least 18 hours in sterile PBS, h. adding linear hyaluronic acid in an amount of about 10% of the cross-linked hyaluronic acid, i. homogenizing the formulation to reassure uniform distribution of the peptide, and j. optionally adding Phosphate Buffer Solution (PBS).

[0276] In additional embodiments, said method may comprise a. providing hyaluronic acid fibres dissolved at a concentration of 10 wt.% in 0.3M NaOH, b. mixing into the hyaluronic acid solution a cross-linking agent such as e.g., BDDE and / or PEGDE, c. heating the hyaluronic acid solution to 20-100 °C and incubating for between 1- 2 hours to induce gelification, d. cooling and neutralizing the solution, e. homogenizing the gel, f. dialyzing the gel for at the least 18 hours in sterile PBS, g. adding linear hyaluronic acid in an amount of about 10% of the cross-linked hyaluronic acid, h. adding the artificial peptide up to the desired concentration, the peptide being added either as a lyophilized powder or from an aqueous solution with a peptide concentration of up to 100 mg / ml, i. homogenizing the formulation to reassure uniform distribution of the peptide, and j. optionally adding Phosphate Buffer Solution (PBS).

[0277] In embodiments, a method for producing a pharmaceutical and / or a cosmetic formulation according to the current invention, the artificial peptide in step a. can be dissolved in 1 % acetic acid at a concentration of 10 mg / mL, followed by b. adding hyaluronic acid fibres (3.0-3.3 MDa) dissolved at a concentration of 10 wt.% in an alkaline solution, preferably NaOH, c. admixing 1 ,4-butanediol diglycidyl ether (BDDE) and / or poly(ethylene glycol) diglycidyl (PEGDE), d. heating the reaction vessel to about 40 °C and incubating for approximately 4 hours, e. cooling and neutralizing the solution with an acidic solution, preferable HCI, f. homogenizing the gel into particles of 100-400 pm.

[0278] In embodiments, the method further comprises g. dialyzing the gel in a biocompatible solution such as e.g., PBS or saline, h. optionally, adding linear hyaluronic acid, in an amount of about 10 wt.% of the cross-linked hyaluronic acid), i. homogenizing the formulation to reassure uniform distribution of the peptide, and j. optionally adding Phosphate Buffer Solution (PBS).

[0279] In embodiments, a method for producing a pharmaceutical and / or a cosmetic formulation according to the current invention comprises a. adding hyaluronic acid fibres (3.0-3.3 MDa) dissolved at a concentration of 10 wt.% in an alkaline solution, preferably NaOH, b. admixing 1 ,4-butanediol diglycidyl ether (BDDE) and / or poly(ethylene glycol) diglycidyl (PEGDE), c. heating the reaction vessel to about 40 °C and incubating for approximately 4 hours, d. cooling and neutralizing the solution with an acidic solution, preferable HOI, e. optionally dialyzing the gel in a biocompatible solution such as e.g., PBS or saline, f. homogenizing the gel into particles of 100-400 pm, g. dissolving the artificial peptide in 1 % acetic acid at a concentration of 10 mg / mL, h. mixing the dissolved peptide into the homogenized gel.

[0280] In embodiments, the method additionally comprises i. optionally, adding linear hyaluronic acid, in an amount of about 10 wt.% of the cross-linked hyaluronic acid), j. homogenizing the formulation to reassure uniform distribution of the peptide, and k. optionally adding Phosphate Buffer Solution (PBS).

[0281] In embodiments, the method additionally comprises a step of terminal sterilization of the gel.

[0282] In one embodiment, the production process comprises:

[0283] 1. Dissolving artificial peptides in distilled water to a final concentration of about 0.1-250 ug / mL, 1-10mg / mL, or of at least 0.1 ug / mL.

[0284] 2. Mixing by vertexing,

[0285] 3. Creating an aqueous solution containing water for injection, NaF, citric acid, NaCI, Na2HPC>4, NaFhPCU, and artificial peptides, such as P2 and P6.

[0286] 4. Adding NaOH for pH adjustment,

[0287] 3. Adding sodium hyaluronate,

[0288] 4. Optionally adding a crosslinking agent such as BDDE and / or PEGDE, 5. Mixing for 24h under gentle agitation,

[0289] 6. Filling into one or more syringe(s),

[0290] 7. Packing into pouch or blister pack,

[0291] 8. Autoclaving the packaged product.

[0292] In one embodiment, the production process comprises:

[0293] 1. Dissolving artificial peptides in distilled water to a final concentration of about 0.1-250 ug / mL, 1-10mg / mL, or of at least 0.1 ug / mL.

[0294] 2. Mixing by vertexing,

[0295] 3. Creating an aqueous solution containing water for injection, NaF, citric acid, NaCI, Na2HPO4, NaH2PO4, and artificial peptides, such as P2 and P6.

[0296] 4. Adding NaOH for pH adjustment,

[0297] 5. providing a mixture of crosslinked and linear hyaluronic acid fibres (HA+HA-XL) as disclosed herein, preferably the crosslinking is obtained using a crosslinking agent such as BDDE and / or PEGDE, and the mixture comprises about 90% HA-XL and about 10% HA,

[0298] 6. admixing the aqueous peptide(s) solution with the mixture of HA-XL and HA. In embodiments, the production process further comprises mixing the aqueous peptide(s) solution with the mixture of HA-XL and HA for 24h under gentle agitation. The production process may further comprise filling the mixed material into one or more syringe(s), optionally, packing the syringe into pouch or blister pack, and optionally autoclaving the packaged product. In embodiments, the syringe has a volume of about 0.5-1 .5 mL.

[0299] Accordingly, the current invention also relates to a kit for clinical use comprising a pouch, wherein said pouch comprises one or more single use syringes prefilled with a pharmaceutical formulation as disclosed herein, and instructions for use.

[0300] Due to their shorter length, compared to natural proteins, the artificial peptides comprised in the pharmaceutical and / or a cosmetic formulation according to the current invention are easier to produce, e.g., by synthetic production or biosynthesis. The artificial peptides of the invention may be produced by any known method for production of peptides, such as synthetic production by chemical synthesis. Synthetic production also allows the use of amino acid analogues which may improve the stability of the peptides produced. The skilled person knows what methods are available for the synthesis of an amino acid sequence.

[0301] Preferably, bioproduction may be used as a method for producing the peptides. Bioproduction means the production of an amino acid sequence in a biological system, such as a cell culture or in microbial cells, e.g., bacterial cells. For bioproduction, it is necessary to construct the corresponding nucleic acid sequence encoding a specific amino acid sequence. The skilled person readily knows how to construct such a nucleic acid sequence once a specific amino acid sequence to be synthesized is determined upon, and how to produce the peptide and purify it from the system used to produce it (see e.g. Svensson J, Andersson C, Reseland JE, byngstadaas SP, Bulow b. Histidine tag fusion increase expression levels of active recombinant Amelogenin in Escherichia coli. Protein Expr Purif, 48; 134-41 (2006)).

[0302] In one embodiment, the product is packed into glass syringes, such as but not limited to the BD Hylok™ pre-fillable glass syringe in which it is also autoclaved to mitigate for bacterial contamination. Prior to autoclaving, it can be packed into a E-line pouch to maintain the aseptic condition of the product. Along with the syringe with the gel, there can also be packed an application tip (19G, blunt) inside the pouch.

[0303] When delivered in a single use pre-filled syringe (e.g., at a 1 mL volume, or 0.7 mb volume), this is to allow it to be efficiently used in dental clinics. To prevent cross contamination between users, each package consists of e.g., 2, 4 or up to 8 single use units with accompanying dispensing tips. The syringe and dispensing tips are packed together in a single pouch which is packed in aseptic conditions.

[0304] The product can be a sterile or a non-sterile product.

[0305] A pharmaceutical and / or a cosmetic formulation obtained by a method according to the current invention

[0306] In one aspect, the current invention relates to a pharmaceutical and / or cosmetic formulation obtained by a method according to the current invention, as well as to its use as a medicament. The invention also relates to a pharmaceutical and / or cosmetic formulation comprising an artificial peptide as defined herein or a combination of two or more artificial peptides as defined herein. Such a pharmaceutical and / or cosmetic formulation optionally also comprises a pharmaceutically acceptable carrier, excipient and / or diluent. In addition, such a pharmaceutical and / or cosmetic formulation may also comprise a source of fluoride, such as e.g., one or more selected from the group consisting of NaF, CaF2and ZnF2.

[0307] Compositions in the present context embrace pharmaceutical and cosmetic compositions as well as compositions belonging to the so-called grey area between pharmaceuticals and cosmetics, namely cosmeceuticals.

[0308] The pharmaceutical composition is typically in the form of a gel, preferably in the form of a hydro-gel.

[0309] In one aspect, a pharmaceutical and / or cosmetic formulation of the invention is formulated for minimally invasive administration by injection.

[0310] In one aspect, a pharmaceutical and / or cosmetic formulation of the invention is formulated for oral application. In another aspect, a pharmaceutical and / or cosmetic formulation of the invention is formulated for delivery by dermal application, topical application and / or transdermal patches.

[0311] The compositions may be formulated according to conventional pharmaceutical practice, see, e.g., "Remington’s Pharmaceutical Sciences" and "Encyclopedia of Pharmaceutical Technology", edited by Swarbrick, J. & J. C. Boylan, Marcel Dekker, Inc., New York, 1988.

[0312] A pharmaceutically or cosmetically acceptable excipient, carrier and / or diluent is a substance which is substantially harmless to the individual to which the composition is to be administered. Such an excipient, carrier and / or diluent normally fulfils the requirements given by the national health authorities. Official pharmacopoeias such as e.g., the British Pharmacopoeia, the United States of America Pharmacopoeia and The European Pharmacopoeia set standards for pharmaceutically acceptable excipients. Whether a pharmaceutically acceptable excipient is suitable for use in a pharmaceutical composition is generally dependent on which kind of dosage form is chosen. In the following are given examples of suitable pharmaceutically acceptable excipients for use in different kinds of compositions for use according to the invention.

[0313] The choice of pharmaceutically acceptable excipient(s) in a composition for use according to the invention and the optimum concentration thereof cannot generally be predicted and must be determined on the basis of an experimental evaluation of the final composition. However, a person skilled in the art of pharmaceutical formulation can find guidance in e.g., "Remington’s Pharmaceutical Sciences", 18th Edition, Mack Publishing Company, Easton, 1990.

[0314] The concentration of the artificial peptide in a pharmaceutical composition according to the invention will, as the skilled person readily understands, 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 applied in vivo to a subject is typically about 10 ng / cm2to 0.1 mg / cm2, preferably about 1 ug / cm2.

[0315] In one embodiment, a pharmaceutical and / or cosmetic composition according to the current invention comprises the following ingredients as listed in table 3.

[0316] Table 3. Exemplary ingredient list in descending order after content in a final pharmaceutical and / or cosmetic composition according to the current invention, supplier of the ingredient, purity grade and weight percentage.

[0317] The pharmaceutical and / or cosmetic composition according to the current invention is based on a hyaluronic acid gel, meaning that the two main ingredients are water and hyaluronic acid. To provide an effective protection of the oral domain it also includes sodium fluoride and the artificial peptide, that both form a protective mineral layer on the teeth and protect the gum. It also includes citric acid for a fresh taste. Sodium hydroxide is added to modify the pH into the range 6.0-7.0, and sodium chloride, disodium phosphate, and monosodium phosphate are added to give osmolarity.

[0318] It is to be understood that while the present invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

[0319] The present invention is further illustrated by the following non-limiting experiments.

[0320] EXAMPLES The following Examples have been included to provide guidance to one of ordinary skill in the art for practicing representative embodiments of the presently disclosed subject matter. Considering the present invention and the general level of skill in the art, those of skill can appreciate that the following Examples are intended to be exemplary only and that numerous changes, modifications, and alterations can be employed without departing from the scope of the presently disclosed subject matter.

[0321] Example 1

[0322] The aim of this example was to compare two cross-linked hyaluronic acid gels prepared using two different crosslinking agents, BDDE and PEGDE. For both agents, the crosslinking can be induced through a similar process, thus the aim was to understand if the two agents gave different physio-chemical properties by means of rheology, FTIR, NMR and SEM. One aim was also to understand the nature of the process steps, which was done through in situ monitoring of the crosslinking kinetics with rheology, and the use of a design of experiment scheme to understand how the variables hyaluronic acid concentration, time and temperature affected the rheological properties. Materials & Method

[0323] Materials

[0324] Pharmaceutical grade high molecular weight (Mw = 1.5MDa, IV = 22.2 m3 / kg) hyaluronic acid was kindly supplied by Fidia Farmaceutici S.p.A. (Abano Terme, Italy). 1 ,4-butanediol diglycidyl ether (BDDE), poly(ethylene glycol) diglycidyl ether (PEGDE, Mn= 500 Da), anhydrous sodium hydroxide and sodium chloride was supplied from Merck KGaA, Germany. Potassium bromine (99%, IR grade) was purchased from J&K Scientific Gmbh, Germany.

[0325] Gel production

[0326] Hyaluronic acid was dissolved at 10 w / v% in 0.3M NaOH solution under manual agitation.

[0327] 1 .6 v / v% BDDE or 3.0 v / v% PEGDE was added and stirred in. The solution was incubated for 4 hours at 40 °C in a closed container. Thereafter, the gel was transferred to a cellulose membrane (MWC = 14 kDa) and dialysed in distilled water for 18 hours. The gel was granulated by extruding it through a 130pm pored mesh before the final concentration of 20 mg / mL was obtained by further addition of distilled water.

[0328] Rheology of gel samples

[0329] The viscoelastic region of the gel samples was investigated using amplitude sweeps by applying a shear strain logarithmic ramp between 0.01-100 % at a 10 rad / s frequency. Further, frequency sweep analysis was applied to understand the crosslinking properties of the gel across a frequency range of 0.1-100 rad / s at a 0.1% shear strain.

[0330] Real-time crosslinking analysis

[0331] The kinetics of the gel crosslinking was conducted using rheology. The dissolved hyaluronic acid was mixed with the cross-linked and immediately transferred to the rheology machine where the measurement was started. Over a 5-hour-time period under constant shear strain of 0.1% and 10 rad / s frequency, the crosslinking kinetics at 40 °C were observed by monitoring the change in viscoelastic properties. Material characterisation

[0332] FTIR

[0333] A FTIR spectrometer (Varian 640-IR, Agilent Technologies, Santa Clara, CA, ISA) was employed to determine the infrared spectra of pure hyaluronic acid, BDDE, PEGDE, and synthesized HA-XL(BDDE) and HA-XL(PEGDE) hydrogels. Spectra were recorded using KBr pellets in the 400-4000 cm-1range at a 4 cm-1resolution and 50 scans / spectrum. For the gel samples 300 mg KBr and 20 mg of gel was used.

[0334] Morphological characterization

[0335] After the crosslinking step, samples were dehydrated in baths of increasing concentration of ethanol up to absolute ethanol, then dried overnight at 60 °C and 10% humidity. Environmental scanning electron microscopy analysis was performed on gold sputtered samples at 15 kV with Evo 50 EP Instrumentation (Zeiss, Jena, Germany)

[0336] NMR

[0337] Liquid NMR

[0338] NMR spectroscopy was used to investigate the hyaluronic acid functionalization, and the degree of crosslinking of the gels and to confirm the removal of unreacted crosslinking agent after dialysis.

[0339] 1H-NMR spectra of the lyophilized gels diluted in deuterated water (4 mg / mL) were collected on a Bruker Advance NMR spectrometer operating at 400 MHz and 298 K. The deuterated solvent (D2O) was used as an internal deuterium lock. Chemical shifts (6) are reported in parts per million (ppm) using the residual D2O as a reference. Spectra were processed and visualized with MestReNova x64 (Mestrelab Research).

[0340] Integrations of the signals at 1.60 and 2.0 ppm were employed to measure the degree of modification (MoD), i.e., the moles of crosslinker bound per disaccharide unit (usually expressed as a percentage), of HA-XL(BDDE) gels: 100 (0-1 )

[0341] For HA-XL(PEGDE) hydrogel the peaks with a chemical shift in the range 3.20-4.0 ppm include both the hyaluronic acid protons and the PEGDE protons due to the overlap of the signals. Although PEGDE protons can be assigned to the peak at 3.68 ppm, cannot be integrated separately so the integral of the region must be subtracted by the 10 protons of hyaluronic acid in this range. The subtracted integral is then the integral of the PEGDE and 40.52 protons on average are present on the PEGDE residue. The degree of modification can consequently be calculated considering the subtracted integral and the integration of the signals at 2.20 ppm:

[0342] (0-2)

[0343] This value is an average degree of modification as cross-linked PEGDE shows a distribution of molar masses.

[0344] Leaching study

[0345] A leaching study was employed to investigate the removal of unreacted crosslinking agent. The gel was produced as described above, and before and after the dialysis, the gel was removed, the concentration of hyaluronic acid was modified to 20 mg / mb, and the sample was granulated as described above. Thereafter 2 g of each sample was moved to a cellulose membrane (MWC = 14 kDa), transferred to an enclosed bottle with 50 mb dH2O. After 7 days 1 .8 mb solution was removed from each sample, transferred to an NMR tube, before the liquid phase was removed. The residuals were then dissolved in 1 .8 mb deuterated water and analysed with NMR as described above.

[0346] Cytotoxicity testing

[0347] Cytotoxicity testing was performed to understand if any leachable could have a detrimental effect on cell proliferation. The gel was prepared similar to as described above but using sterile saline water instead of distilled water. The samples were transferred to syringes before they were sterilized by autoclaving at 121 °C for 15 minutes. Osteoblast cells (MC3T3-E1 ) were seeded in a 24-well plate at a concentration of 40 000 cells per well and 1 mb cell medium. Approximately 0.1 mb of the gels were added to inserts (0.4 pm PET-membrane, Merck) which was then added to the wells. There were used 6 wells for each group, including a positive group (cells killed with Triton X-100 1 h before finishing) and a negative control (cells only). The quantitative cytotoxicity evaluation was conducted utilizing bDH activity of the gels, and a CCK8 assay was used to calculate cell viability. The bDH assay detects the amount of bDH that leaks out through the plasma membrane of damaged cells, as a marker of cytotoxicity. A cytotoxicity level of below 30% and cell viability above 70% is counted as acceptable according to the ISO 10993-5.

[0348] Results

[0349] Employing the above-described method, cross-linked hyaluronic acid gels were produced, using both BDDE (1.6 vol.%) and PEGDE (3 vol.%) as crosslinker. The schematic of this process can be found in Figure 1. These two different gels then underwent physiochemical characterisation and cytotoxicity testing. Although a different molar equivalence of the two crosslinkers (BDDE and PEGDE) and the hyaluronic acid was used, the different gels still exhibit similar rheological data, suggesting that the crosslinker plays an essential role in the gels’ properties.

[0350] Rheology analysis

[0351] Rheology was applied to understand the mechanical properties of the gel. The gels displayed very similar properties with a wide viscoelastic region reaching up to over a 1000% shear strain (Figure 2A, left panel). Moreover, their frequency sweep behaviour was similar (Figure 2A, right panel). The concentration of PEGDE (3.0 vol.%) was chosen such that the behaviour of the gel mimics that of the 1.6 vol.% BDDE. The rheology results presented in Figure 2 do confirm this. Accordingly, although the molar equivalence between hyaluronic acid and the crosslinker is lower for the PEGDE gel than the BDDE gel the results suggests that the PEGDE cross-linked HA-XL (HA-XL(PEGDE)) gel is more elastic than BDDE cross-linked HA-XL (HA-XL(PEGDE)) gel.

[0352] Further, a real time rheology measure of the in situ crosslinking process of the gels was performed (Figure 2B). It could be observed that both gel types follow a similar kinetic behaviour, with HA-XL(PEGDE) having a quicker kinetic behaviour than HA-XL(BDDE), reaching the gelation point (G’ = G”) after approximately 30 minutes, where the HA- XL(BDDE) gel reaches gelation after roughly 1 hour. They do seem to converge towards a G’ of around 1 MPa for HA-XL(BDDE) and 2 MPa for HA-XL(PEGDE), which indicates a higher structural stability for HA-XL(PEGDE) than for HA-XL(BDDE), in line with the shear strength results presented above. Chemical characterisation

[0353] To characterize the material properties from a chemical perspective, FTIR and NMR was applied. From the FTIR analysis, multiple peaks expected from cross-linked hyaluronic acid were detected (Figure 2C). In Figure 2C the peak at 1650 cm-1is the carboxylate group (C=O) of the hyaluronic acid, meanwhile, the wide double peaks at 1050-1150 cm-1are the C-O-O & C-O stretch typical of the ether bonds that are formed during the crosslinking. This suggests that a cross-linked hyaluronic acid gel is achieved both with BDDE and with PEGDE as the crosslinker. Minimal traces of peaks at 2900-3000 cm-1, are seen, which are associated with the epoxy end-groups of the crosslinkers that binds to the hydroxyl groups of the HA, which suggests that non-reacted BDDE or PEGDE were successfully removed during the dialysis step, bastly, there is a wide peak between 3050- 3300 cm-1which is associated with intermolecular -OH groups and H-bonds. These intermolecular bonds are essential for the coherences of the gel after granulation and gives them their “sticky” properties which can be clinically favourable.

[0354] Morphological characterization

[0355] After crosslinking step samples were dehydrated in baths of increasing concentration of ethanol up to absolute ethanol, then dried overnight at 60 °C and 10% humidity.

[0356] Environmental scanning electron microscopy analysis was performed on gold sputtered samples at 15 kV with Evo 50 EP Instrumentation (Zeiss, Jena, Germany).

[0357] Leaching study

[0358] A leaching study was employed to investigate the removal of unreacted crosslinking agent. The gel was produced as described above, and before and after the dialysis gel was removed, the concentration of hyaluronic acid was modified to 20 mg / mb, and the sample was granulated as described above. Thereafter 2 g of each sample was moved to a cellulose membrane (MWC = 14 kDa), transferred to an enclosed bottle with 50 mb pureH2O. After 7 days 1 .8 mb solution was removed from each sample, transferred to an NMR tube, before the liquid phase was removed. The residuals were then dissolved in 1 .8 mb deuterated water and analysed with 1 H NMR as described above.

[0359] Discussion

[0360] The inventors have characterized two different cross-linked hyaluronic acid hydrogels, cross-linked using either BDDE or PEGDE as the crosslinker. The crosslinking can be induced using a similar methodology in both cases, and also, a granulation step was conducted, such that it is compatible with needle injection in a clinical setting. The process starts with the dissolving of hyaluronic acid. Since it typically has a solubility limit of about 4 mg / mL, dependent on salt and molecular weight, it is necessary to dissolve it in a basic / alkaline environment that was ensured using sodium hydroxide (NaOH). The basic / alkaline environment partially deprotonates the hydroxyl groups of the hyaluronic acid molecules, which increases their reactivity, thus facilitating the reaction with the epoxide groups of BDDE to enable the cross-linking. As no or very little cross linking was achieved at hyaluronic acid concentration of 5 wt.% or lower, a concentration of 10 wt.% hyaluronic acid was used. Once the hyaluronic acid was dissolved, the crosslinkers could be readily admixed. The crosslinking process is a time dependent process, that may be accelerated with elevated temperatures. This is beneficial as the high pH accelerates the degradation of hyaluronic acid, resulting in competing reactions between the BDDE or PEGDE crosslinking and hyaluronic acid degradation. In the real-time crosslinking study, a gelation point after approximately 30 minutes or 60 minutes could be observed for the PEGDE or BDDE gel, respectively, when incubated at 40 °C. Both gels seemed to follow a sigmoidal crosslinking kinetic, where the increase in crosslinking decreased with time. From an industrial perspective it can be beneficial to limit the crosslinking time out of economic considerations. In the experimental setting, crosslinking was stopped after 4 hours. This can be done by neutralizing the pH, with an acid such as e.g., HCI, but it may also be done by transferring the gel into a dialysis solution. The dialysis step is intended to allow the non-reacted crosslinking agent to diffuse out of the gel.

[0361] To understand the efficacy of the dialysis step, a leaching study was performed on the gel before and after dialysis, using NMR to characterize the leached composition. Before the dialysis, the peaks associated with the crosslinkers are found in the range from 3.3- 3.9ppm for BDDE, and 3.5-3.75 ppm for PEGDE (Figure 3), whereas after 18 hours of dialysis, the peaks in these regions have faded, indicating a that diffusion effectively removes unreacted BDDE and PEGDE from the gels. Further removal of unreacted crosslinking agent may be obtained by e.g., increasing the volume of the dialysis solution, or by exchange of the dialysis medium. The latter was implemented by changing the dialysis medium after two hours, before dialysing for another 16 hours. Diffusion is also time dependent, so increasing the time can also assist on the removal. For in vivo applications it is desired that the dialysis is conducted into a solution suitable for in vivo use in order to obtain the adequate osmolarity, such solutions are e.g., PBS buffer or saline water.

[0362] Example 2

[0363] Production of formulation comprising hyaluronic acid cross-linked with BDDE and linear hyaluronic acid and consensus peptide

[0364] 1. Hyaluronic acid (3.1 m3 / kg) was dissolved in 0.3M NaOH at a concentration of 100 mg / mL under agitation.

[0365] 2. 16 pL / mL 1 ,4-butanediol diglycidyl ether (BDDE) was admixed to the hyaluronic acid solution.

[0366] 3. The solution was incubated for 4h in a closed container at 40 °C to allow crosslinking.

[0367] 4. The gel was neutralized using 1 M HCI and shaked mildly overnight.

[0368] 5. The gel was transferred to a cellulose membrane with a cut-off molecular weight of 14 kDa, before it was dialyzed in sterile phosphate-buffered saline (PBS) for 18 hours.

[0369] 6. The gel was granulated by extruding it through a stainless-steel mesh with pore size of 200 pm.

[0370] 7. Separately a solution of phosphate-buffered saline, hyaluronic acid, sodium fluoride, and consensus peptide was created as following: a. Hyaluronic acid equivalent to 10 wt.% of the final solution was added to PBS and hydrated by stirring over 1 h. b. In parallel, the peptide (P2 or P6) solution was created by dissolving the peptide in in sterile water at a concentration of 10 mg / mL.

[0371] C. The peptide solution was added to the PBS-hyaluronic acid solution for a final mixture concentration between 1-500 pg / mL gel.

[0372] 8. The PBS-hyaluronic acid solution was added to the granulated cross-linked hyaluronic acid-BDDE and linear hyaluronic acid and mixed in. The solution was allowed to homogenize under agitation for 6 hours.

[0373] 9. The gel was transferred to an appropriate delivery system and sterilized using autoclaving. Example 3

[0374] Production of formulation consisting of hyaluronic acid cross-linked with BDDE and linear hyaluronic acid and peptide, doped with 2000 ppm NaF

[0375] The hyaluronic acid gel cross-linked with BDDE and linear hyaluronic acid was prepared in a similar manner as in example 2. Afterwards, the solution of PBS, hyaluronic acid, and peptide was produced as in example 2 with the small change that NaF is doped into a final concentration of 2000 ppm. The gel and the solutions were mixed together, transferred to a delivery system and sterilized using autoclaving. Example 4

[0376] Formulation comprising linear hyaluronic acid, a peptide, and sodium fluoride

[0377] 1 . 50 mg / mL hyaluronic acid was dissolved in 0.3M NaOH using manual stirring.

[0378] 2. The concentration is modified with sterile PBS such that the concentration of hyaluronic acid at the end of the process was 20 mg / mb.

[0379] 3. 100 ng / mb to 25 pg / mb of consensus peptide was added and mixed in.

[0380] 4. The solution is neutralized with the addition of 1 M HCI and stirred for homogenization.

[0381] 5. 2000 PPM NaF is added and mixed in. Example 5

[0382] Release of biotin-labelled peptide from hyaluronic acid solution and hyaluronic acid gel

[0383] The gels from examples 2-4 were produced with 500 pg / mb of Biotin labelled P6-peptide.

[0384] 0.2 mb of the gel was added to 24-well inserts (0.4 pm, PET), and 1 mb of distilled water was added to each well. After 3h, 7h, and 12h , 1 pb of the water was extracted and the light absorption at 205 nm was read off using a NanoDrop One (Thermo Scientific, US), and compared to a reference curve of 1 , 10, 25, and 100 pg / mb P6-Biotin in distilled water. The reference curve was used to convert to a mass release and a %-release curve was plotted demonstrating that after 12h the linear hyaluronic acid (HA) gel (example 2) has released 91% of its peptide load, meantime the cross-linked HA-XL gel (example 1 ) has released 23%(as can be seen in figure 4).This demonstrates that the different way to prepare the gel may be used to tailor the release of the peptide and shows that a combination of cross-linked and linear gel (HA+HA-XL) can give a more controlled and tuneable peptide release compared to linear gel (HA) only. Example 6

[0385] Theoretical example for treatment against gingivitis

[0386] The gel prepared in example 4 is filled into syringes. The syringes are used to apply the gel directly to the gum of patients exhibiting indications of gingival recession or who are prone to redness and other inflammatory signs in the gum. The gel is then smeared out to evenly cover the gum.

[0387] The results are expected to demonstrate that the gel consisting of the consensus peptide and fluoride will have a greater ability to reduce redness and other inflammatory indications on the mucous membrane than either ingredient would alone. Moreover, there will be a reduction in the formation of plaque at the interface between the gum and the teeth. This is of clinical importance as the prevention of inflammation and formation of plaque will have a prophylactic effect on gingival recession and periodontitis.

[0388] Example 7

[0389] Gingival tissue regeneration /

[0390] Investigation of Improved Soft Tissue Wound Healing Effect of NuPep Gel after Periodontal Surgery

[0391] The purpose of this example is to show the influence of the artificial peptide loaded gels on improved soft tissue wound healing after periodontal surgery. In six pigs, the gingival tissue of 3 premolars were detached with a scalpel. The gel from example 2 was used, with either P2 or P6 at a concentration of 50 pg / mL. They were controlled against hyaluronic acid without the peptide, Emdogain® (Straumann, Basel, CH), and sham control. The gels were filled in between the gingiva and the teeth and allowed to close by itself.

[0392] After 6 days the pigs were scarified. The gingival was able to reattach to the molars in all the gel cases, meanwhile for sham there was varying reattachment. In the case of the hyaluronic acid gel alone it was observed significant amount of puss and inflammation that was not observable for the gel loaded with the peptides nor the sham control.

[0393] Example 8

[0394] Prophylactic application The purpose of this example is to demonstrate how the described formulation can have a prophylactic effect on soft tissue using a gel as described in example 4.

[0395] Materials and method:

[0396] A solution of 5 wt.% hyaluronic acid in 0.3M NaOH was created. The solution was neutralized with a solution of 11.65M HCI and 0.9% NaCI in distilled water to yield a final concentration of hyaluronic acid at 2 wt.%. In the acidic solution, equal amounts of P2 and P6 were mixed in to yield a final peptide concentration in the range of approximately 50 ng / mL - 500 ng / mL. Remaining steps of the preparation was done as specified in Example 4.

[0397] Clinical use:

[0398] The solution can be applied to the dental mucous membrane in patients prone to periodontitis and gingivitis. When applied through a syringe or paste tube, the solution can be smeared out to uniformly cover the surface of the mucous membrane of the patients. Prior to the treatment the patient should undergo professional cleaning by trained personnel, such as e.g., a dentist or a dental hygienist to remove any plaque film from the mucous membrane or between the mucous membrane and the enamel of the teeth.

[0399] Expected results: The patients should be less likely to exhibit less redness and swelling. Moreover, patients should be less likely to experience progression of periodontitis or gingival recession.

[0400] Example 9

[0401] Soft Tissue Wound Healing Effect of the HA+HA-XL(BDDE) / consensus peptide

[0402] Aim

[0403] The purpose of this example is to show the influence of the artificial peptide loaded gels on improved soft tissue wound healing after periodontal surgery.

[0404] Methods

[0405] In six pigs, 8 rows of wounds were created on the back of the pigs down the spine, with two wounds on each side of the spine (labeled L, LM, RM, R, from left side to right side while looking in the posterior direction, with the spine running between LM and RM). There was 3 cm spacing between the wounds. The wounds were created using an 8 mm diameter histology punch, before the skin was cut off using a scalpel.

[0406] At the bottom, a 10throw was created with just two wounds in the position LM and RM, which were used as a reference for wound care.

[0407] The bleeding from the back wounds were allowed to stop by taking a 10 min pause, thereafter the treatments were applied. The following treatments were used (in brackets short code): sham (SHAM), HA+HA-XL(BDDE) , HA+HA-XL(BDDE)+NuPep P2 , HA+HA- XL(BDDE)+NuPep P6 (HA+P6), HA+HA-XL(BDDE)+mix of P2 and P6 (1 :1 mix), HA- XL(BDDE)+EMD (HA+HA-XL(BDDE)+EMD), Emdogain (EMD), NuBone Clean (with removal after; NB Rem), NuBone Clean (without removal after; NB). The gels were prepared as described in Example 2 and comprises both linear HA and cross-linked HA, referred to as HA+HA-XL(BDDE)). The treatment was applied through a syringe, with exception of HA+HA-XL(BDDE)+mix of P2 and P6 and HA+HA-XL(BDDE)+EMD for the 3 first animal where a spatula was used (application procedure was modified between due to easier application through a syringe).

[0408] After the wound treatment, a plastic cover was applied over the wounds and a dressing (with hole along the back) was applied. For the 10throw, the plastic cover was cut open to expose the wounds, and VetricynVF Plus (standard care) was applied. The 10throw functioned as a standard therapy and allowed us to determine when there was a 50% wound closure.

[0409] The wound healing was followed by histological stainings (see e.g., exemplary stainings in figure 8), and the wound healing was addressed from the following variables:

[0410] Measured wound Width (um)

[0411] Measured wound Depth (um)

[0412] Calculated wound Void Area (mm2)

[0413] Re-epithelisation (as seen by keratinisation of the skin in the wound area) score from 0-3, wherein o 0: No epithelium; o 1 : Partly re-epithelisation; o 2: Mostly complete re-epithelisation; o 3: Complete re-epithelisation

[0414] Granular Tissue (as seen by presence / absence of proliferating cells and collagen fiber deposition) score from 0-3 (see Gupta, A. and Kumar, P., 2015. Assessment of the histological state of the healing wound. Plastic and Aesthetic Research, 2, pp.239-242.), wherein o 0: Profound granulation; o 1 : Moderate granulation; o 2: Scanty granulation; o 3: Granulation absent

[0415] Inflammatory Infiltrate (as seen by presence of inflammatory cells around the wound area or necrotic tissue) score from 0-3, wherein o 0: Infection / necrotic tissue present; o 1 : Profound inflammation; o 2: Moderate inflammation; o 3: Absent

[0416] Results The hydrogel composition comprising HA+HA-XL(BDDE) gelcross-linkedand the consensus peptides P2, and / or P6 was tested for its ability to promote wound healing, as evaluated by the wound void area, re-epithelisation score, presence of granular tissue, and inflammation score (See figure 6, 7). Firstly, compositions comprising P2 (SEQ ID NO: 4), P6 (SEQ ID NO: 6) or P2 and P6 were compared to HA+HA-XL(BDDE) alone and a sham control, and the different evaluation criteria was evaluated 6 days after wound infliction. It was observed peptide P2 in BDDE cross-linked HA (P2+ HA+HA-XL(BDDE)) outcompetes sham and HA+HA- XL(BDDE)alone in relation to the wound void area, with a significant reduction in wound void area compared to HA+HA-XL(BDDE)alone or Sham (Fig 6a; in figure 6 HA+HA- XL(BDDE) is denoted as HA). Furthermore, the re-epithelisation was significantly improved for HA+HA-XL(BDDE)+P2 compared to Sham, while there was a tendency suggesting that both HA+HA-XL(BDDE)+P2 and HA+HA-XL(BDDE)+P6 improved the re- epithelisation compared to HA+HA-XL(BDDE)alone (Fig. 6b; in figure 6 HA+HA- XL(BDDE) is denoted as HA). Additionally, HA+HA-XL(BDDE)+P2 show a significant improvement in granulation (Fig. 6c; in figure 6 HA+HA-XL(BDDE) is denoted as HA), with scanty-to-absent granulation (a granulation score of 2-3), while the remaining groups showed moderate-to-scanty granulation (a granulation score of app. 1-2). Finally, when considering the wound inflammation score Sham, HA+HA-XL(BDDE)and HA+HA- XL(BDDE)+P6 / P2 show profound inflammation, while P2+ HA+HA-XL(BDDE)and P6+ HA+HA-XL(BDDE)show moderate inflammation (Fig. 6d; in figure 6 HA+HA-XL(BDDE) is denoted as HA), the data suggests that P2+HA+HA-XL(BDDE)and P6+HA+HA- XL(BDDE)provide an anti-inflammatory effect which surpasses that of HA+HA- XL(BDDE)alone.

[0417] Accordingly, the first results shows that there is a beneficial effect of formulating the consensus peptides in the combined cross-linked-linear cross-linkedHA gels (HA+HA- XL(BDDE)), and moreover, while P2 proved a beneficial effect when evaluated on the presented criteria, P6 proved beneficial in reducing the inflammation, thereby, showing that both peptides provide a beneficial effect on wound healing.

[0418] As a second validation compositions comprising P2 or P6 was compared to Emdogain® and Emdogain® (EMD) formulated in HA+HA-XL(BDDE). Emdogain® is an extract of enamel matrix and contains amelogenins of various molecular weights.

[0419] As can be seen from figure 7, both P2 and P6 containing formulations outcompeted EMD and EMD+ HA+HA-XL(BDDE)when evaluating inflammation (Fig. 7d), while the P2 comprising composition also shows an improved wound void area compared to EMD and EMD+ HA+HA-XL(BDDE), an improved re-epithelisation score compared to EMD, and an improved granulation score, compared to EMD and EMD+ HA+HA-XL(BDDE).

[0420] In summary the present results shows that the peptide P2 when embedded in the HA+HA-XL(BDDE) cross-linkedgel performs better than the HA+HA-XL(BDDE) gel cross- linkedalone on all variables with exception of re-epithelisation. There was a tendency towards the peptide P6 when embedded in the HA+HA-XL(BDDE) cross-linkedgel also leads to a reduced inflammation, seemingly to a similar extend as the P2 peptide in the HA+HA-XL(BDDE) gelcross-linked.

[0421] Additionally, the peptide P2 when embedded in the HA+HA-XL(BDDE)cross-linked gel display an improved wound healing and re-epithelization compared to EMD, and similar granulation and infiltration response. HA-XL(BDDE)+P6 provides a similar response to EMD. HA-XL(BDDE)+P2 is also better than HA-XL(BDDE)+EMD, demonstrating the efficacy of P2 vs EMD.

[0422] Taken together the present example shows that both P2 and P6 has a surprising beneficial effect on wound healing, which goes beyond the effect of the HA+HA- XL(BDDE)gel alone. Furthermore, the beneficial effects are also more profound for the consensus peptides than what was seen for EMD alone or when formulated in the HA+HA-XL(BDDE)gel.

[0423] Example 10

[0424] Aim

[0425] The aim of the present example is to illustrate the ability of the formulations disclosed herein to promote MSC homing, in order to promote tissue healing.

[0426] Methods

[0427] Animal surgeries were performed in the Department of Orthopaedic Surgery at UC Davis in compliance with the ARRIVE guidelines and were authorized by Institutional Animal Care and Use Committee (IACUC). A total of 60 C57BI6J mice were used in this study (8 animals per group per time point). A polytrauma model was created (femur osteotomy, 4 mm defect size, + chest trauma) using in a total of 60 C57BL6 / J mice and where the inflammatory response and bone formation after three weeks of healing was studied in three groups including 1) HA+HA-XL(BDDE), 2) HA+HA-XL(BDDE)+P2, and 3) HA+HA- XL(BDDE)+P2+MSCs. The hydrogels were prepared as described in Example 2. For the HA+HA-XL(BDDE)+P2+MSCs, one million MSCs was encapsulated in each hydrogel. After creating a stabilized osteotomy femur defect, hydrogels were injected at the defect site and then the chest trauma was induced using a drop weight device resulting in bilateral hemopneumothoraces. After 3 weeks of healing, fractured femur, serum, and total RNA from the fractured femur tissue were harvested. The biomineralization as well as inflammatory markers and pathways was investigated using micro CT (pCT), histology, immunohistochemistry, synchrotron SAXS / XRD, flow cytometry multiplex analysis of serum cytokines, and bulk RNA sequencing as described below or known in the art.

[0428] In brief micro CT (pCT) was conducted by using a 1172 micro-CT imaging system (Bruker microCT, Kontich, Belgium) desktop x-ray CT scanner at 5.9 pm voxel resolution, x-ray tube current 169 pA and voltage 55 kV, without any filters. Specimens were mounted vertically on a plastic support and rotated through 180° around the sample's long axis (z- axis). Four absorption images were recorded every 0. 0.5850° of rotation. These projection radiographs of the porous structure were first reconstructed to serial coronal- oriented tomograms using a 3D cone beam reconstruction algorithm (NRecon, Bruker microCT, Kontich, Belgium). The beam hardening was set to 20% and ring artifact reduction to 12. 3D reconstruction of the internal pore morphology was carried out using these axial bitmap images and analysed by CTAn (Bruker microCT, Kontich, Belgium). The grey scale threshold was set between 18 and 255; additional noise was removed by the function despeckling. All objects smaller than 300 voxels and not connected to the 3D model were thus removed prior to further analysis. Both closed and open porosity was measured.

[0429] In brief histology and IHC was conducted by After pCT imaging. Samples were cut in half to study bone formation and biomineralization in both calcified and decalcified bone tissues. Half of the defect site was embedded in methyl methacrylate (MMA) to preserve the calcified tissue. The other half was decalcified using ethylenediaminetetraacetic acid (EDTA) 10% for four months and then embedded in paraffin. The paraffin samples (eight samples per group) were cut cross-sectional to the medial plane of the animal body in 5 pm thickness. Because of the procedure difficulties, three MMA embedded samples were randomly chosen and sectioned in 5 pm thickness onto Kawamoto’s film (SECTION-LAB Co. Ltd., Hiroshima, Japan), longitudinal to the pig medial plane. Movat Pentachrome and Von Kossa / Van Gieson stains were used to evaluate the bone mineralization / non- mineralization balance over time. Alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) enzyme histochemistry were done to study the osteoblast and osteoclast balance. Collagen fibers properties (such as width, length, straightness and angle) were evaluated using Sirius Red.

[0430] Immunohistochemistry was performed using primary antibodies (Abeam Company, Cambridge, UK). The following antibodies were used: rabbit monoclonal (EPR53) to alpha-smooth muscle Actin (a-SMA), rabbit monoclonal (EPR14334) to runt-related transcription factor 2 (Runx 2), rabbit polyclonal (OAA100188) to osteopontin, mouse monoclonal (LS-C83497-100) to osteocalcin and rabbit monoclonal (EPR7785) to collagen type I.

[0431] To study the blood vessel formation, a-SMA was diluted in DAKO-Diluent (S 0809), 1 :1000. Regarding bone formation in calcified sections, osteopontin, osteocalcin and collagen type I were diluted in DAKO-Diluent, 1 :250, 1 :800 and 1 :2000, respectively. Runx 2 was diluted in DAKO-Diluent (1 :500) to study the bone formation in decalcified sections. Goat anti-rabbit (BA-1000, Vector) and methyl green staining were used as the secondary antibody and counterstain, respectively. Decalcified Movat Pentachrome and calcified a-SMA stained sections were chosen to study the general tissue formation descriptively. In the Movat Pentachrome stained sections, the tissue homogeneity and integrity as well as defect closure in all groups were studied using a 3-point scale system (poor, fair, good for 1 to 3, respectively) by two independent and calibrated examiners. Additionally, a-SMA stained sections were used to study the blood vessel phenotype and regularity over time using a 3-point scale system. The round shape vessels were defined as regular type 1 , small to moderate oval shape ones as regular type 2 and big vessels in oval or other undefined shapes as irregular type 3 vessels.

[0432] In brief flow cytometry multiplex analysis of serum cytokines using a 24-color antibody panel on the Aurora spectrum flow cytometer (Cytek Biosciences, Inc, California), ne-peak Rainbow Beads (Biolegend) informed voltage settings, and compensation controls were established using auto-compensation with single-color control ultra-comp beads (ThermoFisher Scientific). Voltages for each parameter minimized spillover between fluorophores. Data acquisition was performed on the Aurora spectrum flow cytometer (Cytek Biosciences, CA), and the SpectroFlo™ software was used for analysis. The resulting FCS files were examined using the Cytobank platform (Beckman Coulter Inc, CA)

[0433] In brief, synchrotron SAXS / XRD was conducted by using calcified bone sections with 70 pm thickness to study the collagen / hydroxyapatite (HAp) orientation and size of hydroxyapatite plates using SAXS / XRD analysis. SAXS / XRD scanning was performed at the synchrotron beamline P12, Petra III, Deutsches Elektronen-Synchrotron (DESY) Hamburg, operated by the European Molecular Biology Laboratory (EMBL). The used photon energy was 18 keV, and the beam size was determined to be 80 pm by 80 pm. The sample to detector distance was 1.5 m. A Pilatus 6 M Detector, Dectris, Switzerland, was used with a pixel size of 172 pm. The exposure time of one second was used. Due to the energy settings, the short sample to detector distance, and the large detector, it was possible to record the SAXS and XRD signal on one detector simultaneously. Data averaging and reduction were made by the EMBL analysis pipeline. A mesh scan was used with a stepping 0.15 mm perpendicular and 10 mm parallel to the sample. The total size of the scanned area was adjusted to the respective sample size. The hydroxyapatite platelets' parameter and orientation (HAp) data was evaluated using in-house written MATLAB® scripts. T parameter is the measurement of HAp platelet size and indicates the HAp platelet thickening.

[0434] Statistics

[0435] One-way ANOVA on ranks was performed when the normality test failed, using the Kruskal-Wallis test for post hoc comparison. Otherwise, regular ANOVA was performed with a Tukey test for post hoc comparison. All analyses were performed in GraphPad Prism 8 (GraphPad Software Company, San Diego, CA). Significant differences were presented as *P < 0.05 and **P < 0.01 ,* ***P < 0.001 and ****P < 0.0001.

[0436] Results

[0437] The pCT and histological staining of mineralized versus non mineralized tissues showed that HA+HA-XL(BDDE)+P2 enhanced the bone mineralization at the fracture site compared to HA+HA-XL(BDDE) alone or HA+HA-XL(BDDE)+MSC (Figure 12-15).

[0438] Additionally, the HA+HA-XL(BDDE)+P2+MSCs group stimulated significantly higher and more uniform bone formation than other groups (Figure 12-15).

[0439] In addition, the SAXS / XRD data (Figure 16 and 17) confirmed that the HA+HA- XL(BDDE)+P2 and HA+HA-XL(BDDE)+P2+MSC group had a higher degree of orientation and smaller T parameter and platelet sizes indicating a better orientation / organization of the ultrastructure (See Fig. 16 and 17) meaning more mature bone.

[0440] Proinflammatory cytokine analysis of 13 proinflammatory cytokines (IL1 alpha, IL1 beta, TNF alpha, IL6, IL23, MCP1 , IL12P70, IFN Y, IFN beta, IL27, IL10, IL17A and GM CSF) showed that both HA+HA-XL(BDDE)+P2 and HA+HA-XL(BDDE)+P2+MSCs groups attenuate the systemic inflammatory response toward the healthy baseline values after three weeks of healing (Figures 9-10).

[0441] Gene expression was performed using bulk sequencing, as described above, to obtain a deeper insight into the pathways affected by the consensus peptides and the MSG cells.

[0442] The bulk sequencing further confirmed that the HA+HA-XL(BDDE)+P2 group significantly accelerated the first stages of fracture healing by upregulating genes such as collagen biosynthesis and modifying enzymes, ECM-receptor interaction, extracellular matrix organization (see table 4). In total 7 genes showed a significant altered gene expression (see table 4), these genes are mainly involved in cartilage and bone healing process such as heparan sulfate degradation, glycosaminoglycan degradation, assembly of collagen fibrils and other multimeric structures, collagen biosynthesis and modifying enzymes, ECM-receptor interaction, extracellular matrix organization, mineral absorption. Also, its functional enrichment and gene set enrichment analysis (GSEA) showed reactive oxygen species (ROS), reactive nitrogen species (RNS) production in phagocytes, PI3K-AKT, Ras, phagosomes, lysosome, which could contribute to the low cytokine expression in the 3 weeks of fracture site and reduce inflammation.

[0443] Table 4 -HA vs. HA+HA-XL(BDDE)+P2 Table 5 - HA+HA-XL(BDDE)vs. HA+HA-XL(BDDE)+P2+MSC Table 6 - HA+HA-XL(BDDE)+P2 vs. HA+HA-XL(BDDE)+P2+MSC

[0444] Comparison of the HA+HA-XL(BDDE) +P2+MSC group to the HA group showed that in total 14 genes had a significantly changed expression (See table 5). It was surprising that compared to HA+HA-XL(BDDE)+P2, HA+HA-XL(BDDE)+P2+MSC significantly changed the pathway involving in more epigenetic changes, cell cycles, protein expressions of the cells at the fracture site. For example, E2F mediated regulation of the DNA replication, TP53 regulated metabolic and DNA repair genes, snRNP assembly. IL-2, IL-5, IL- 3 and B cell receptor signalling pathways were significantly down regulated in the HA+HA- XL(BDDE)+P2+MSC group. Similarly, as for the HA+HA-XL(BDDE)+P2 group, bone mineralization and BMP pathway were activated in the HA+HA-XL(BDDE)+P2+MSC group.

[0445] Accordingly, it was seen that the HA+HA-XL(BDDE)+P2+MSC formulation induced the epigenetic changes, local inflammation downregulation and fracture healing.

[0446] These results indicated the synergistic advantage of using both HA+HA-XL(BDDE)+P2 and HA+HA-XL(BDDE)+P2+MSCs for reducing inflammation and improving fracture healing in polytrauma. The results presented herein represent the HA+HA-XL(BDDE)+P2 both with and without MSCs as efficient formulations for attenuating the inflammatory responses and directing bone formation in polytrauma. Additionally, the present examples show that the hydrogel-embedded consensus peptide promotes MSC homing, thereby maintaining, e.g., invading mesenchymal stem cells into the gel after implantation, enable proliferation and differentiation into bone, as well as keep the metabolic activity for the MSC adequate. Example 11

[0447] Release of P2 peptide from hyaluronic acid solution and HA+HA-XL(BDDE).

[0448] Aim

[0449] The aim of the present example is to show the effect of the amount of cross-linked hyaluronic acid in the gels on the swelling of the gel and the release of the consensus peptide from the gels.

[0450] Methods

[0451] A hyaluronic acid gel crosslinked with BDDE, HA-XL(BDDE), a hyaluronic acid solution (linear), HA, and a gel obtained by mixing 90% of HA-XL(BDDE) and 10% of HA were produced with 500 pg / mL of P2-peptide (HA-XL(BDDE)+P2, HA+P2, HA+HA- XL(BDDE)+P2). 0.1ml of each gel was added to 24-well inserts (0.4 pm, PET), and 1 mb of distilled water was added to each well and kept for 12h or 24h. After the time point, the remaining distilled water in the well was extracted, the swollen hydrogel in the insert was weighted, and a Micro BCATMProtein Assay Kit (Thermo Scientific, US) and a BioTek ELxSOO Absorbance Microplate Reader (BioTek Instruments, Inc., Winooski, VT, USA) were used to measure the absorbance at 562nm compared to a reference curve of 0.5, 1 , 2.5, 5, 10, 20, 40, 200 pg / mL of albumin standard in distilled water. The reference curve was used to convert the absorbance to protein concentration, and a bubble plot representing the %-release curve and %-relative weight change over time was plotted.

[0452] Results

[0453] Figure 18 shows that the peptide release is higher from the cross-linked HA-XL(BDDE) gel (12h : 27%; 24h: 28%) and the combination of cross-linked / linear gel (HA+HA- XL(BDDE)+P2, 12h: 25%; 24h: 24%) compared to the linear gel (12h: 14%; 24h: 18%) However, the swelling is reduced in the cross-linked HA-XL(BDDE) gel (12h: 234%; 24h: 306%) and the HA+HA-XL(BDDE) gel (12h: 249%; 24h: 342%) compared to the linear, non-cross-linked gel (HA) (12h: 468%; 24h: 484%). This indicates that the method of preparing the gel can be adjusted to control swelling and peptide release.

[0454] Table 7: Relative weight change [%], percentage peptide release [pg / ml] and remaining peptide concentration in the gel [pg / ml] in P2 peptide-enforced cross-linked HA-XL(BDDE) [HA-XL], linear HA [HA] and combination of cross-linked HA-XL(BDDE) and linear HA [HA+HA-XL(BDDE)] gels over time.

[0455] Example 12

[0456] Aim The aim of this study is to understand the early immunological response to a novel hyaluronic acid gel loaded with proline-rich peptides for periodontal regeneration in comparison with conventional biomaterials.

[0457] Methods The gingiva of three premolars in each quartile of six conventional pigs were detached using a scalpel. Using a blunt application tip, the defects were treated with either nothing (sham; 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). The hyaluronic acid was cross-linked with BDDE and mixed with 10% sodium hyaluronate. P2 and P6 were two different varieties of the proline-rich peptide mimicking the sequence of amelogenin. The animals were euthanised after 6 days, and the local immunological response was investigated using histology stain (Masson Goldner Trichrome, scored by experts) and RT-PCR. The in vitro biocompatibility was evaluated using a cell viability assay.

[0458] Results The histology score shows that the HA+HA-XL(BDDE)+P2, HA+HA-XL(BDDE)+P6 improves the immunological response of the cross-linked hyaluronic acid, also compared to Emdogain® (See figure 19).

[0459] In vitro viability testing demonstrated improved viability for both peptide groups (Median: HA+HA-XL(BDDE): 98%, HA+HA-XL(BDDE)+P2: 104%; HA+HA-XL(BDDE)+P6: 115%) compared to the control (see figure 20).

[0460] The RT-PCR results are anticipated to show alterations in the gene expression profile of the different treatment regimens.

[0461] Example 13

[0462] Production of formulation comprising hyaluronic acid cross-linked with BDDE and linear hyaluronic acid and consensus peptide

[0463] 1 . Hyaluronic acid (3.1 m3 / kg) was dissolved in 0.3M NaOH at a concentration of 100 mg / mL under agitation.

[0464] 2. In parallel, the peptide (P2 or P6) solution was created by dissolving the peptide in in sterile water at a concentration of 10-20 mg / mL.

[0465] 3. Optionally, the peptide solution was added to the hyaluronic acid solution for a final concentration between 1-2500 pg / mL gel.

[0466] 4. 16 pL / mL 1 ,4-butanediol diglycidyl ether (BDDE) was admixed to the hyaluronic acid-peptide solution.

[0467] 5. The solution was incubated for 4-20h in a closed container at 40 °C to allow crosslinking.

[0468] 6. The gel was neutralized using 1M HCI and shaked mildly overnight. 7. The gel was transferred to a cellulose membrane with a cut-off molecular weight of 14 kDa, before it was dialyzed in sterile phosphate-buffered saline (PBS) for 18 hours.

[0469] 8. Optionally, the peptide concentration in the crosslinked gel was quantified using Micro BCA as described in Example 11 .

[0470] 9. The gel was granulated by extruding it through a stainless-steel mesh with pore size of 200 pm.

[0471] 10. Separately the hyaluronic acid equivalent to 10-90 wt.% of the final solution was added to PBS and hydrated by stirring over 1 h.

[0472] 11 .The PBS-hyaluronic acid solution was added to the granulated cross-linked hyaluronic acid-BDDE and linear hyaluronic acid and mixed in to reach the desired HA / HA-XL ratio, optionally with additional addition of the peptide solution for a final peptide concentration between 1-500 pg / mL gel.

[0473] 12. The solution was allowed to homogenize under agitation for 6 hours.

[0474] 13. The gel was transferred to an appropriate delivery system and sterilized.

Claims

CLAIMS1. A pharmaceutical and / or cosmetic formulation in the form of a gel comprising a. an artificial peptide comprising, or consisting of, the amino acid sequence:Pro-X-X-Pro-Y-Y-Y-Pro-X-X-Pro-Y-Y-Pro-X-X-Pro-X- Pro-Y-Y-Y-Y-Y-Y-Pro-Y-Y-Y-Y-Y-Y-Pro-X-X-Pro-X-Pro-Y-Y-Y-Pro-Y-Y-Pro-Y-Pro-X-X-Pro-Y-Pro-Y-Y-Pro-X-X- Pro-Y-Y-Pro-X-X-Pro-Y-Y-Pro-X-X-Pro-Y-Pro-Pro-X-Pro-Pro-X-X-X-X-X-X-X-X-Pro-X-X-Pro-X-X-X-X (SEQ ID NO 1), wherein i) Pro is proline; ii) X is an amino acid selected from the group consisting of Ala, lie, Leu, Met, Phe, Trp and Vai; iii) Y is an amino acid selected from the group consisting of Asn, Cys, Gin, Ser, Thr and Tyr, and b. 1-40mg / mL linear hyaluronic acid fibres (HA) and 1-40 mg / mL cross-linked hyaluronic acid fibres (HA-XL).

2. A pharmaceutical and / or cosmetic formulation in the form of a gel comprising a. an artificial peptide comprising, or consisting of, the amino acid sequence:Pro-X-X-Pro-Y-Y-Pro-X-X-Pro-Y-Y- Pro-X-X-Pro-Y-Y- Pro-Y-Pro-Pro-X-Pro-Pro (SEQ ID NO 2), wherein i) Pro is proline; ii) X is an amino acid selected from the group consisting of Ala, lie, Leu, Met, Phe, Trp and Vai; and iii) Y is an amino acid selected from the group consisting of Asn, Cys, Gin, Ser, Thr and Tyr, and b. 1-40mg / mL linear hyaluronic acid fibres (HA) and 1-40 mg / mL cross-linked hyaluronic acid fibres (HA-XL).

3. A pharmaceutical and / or cosmetic formulation according to claim 1 or 2, wherein the artificial peptide is at least 90% identical to an artificial peptide selected from the group consisting of 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, such as wherein the artificial peptide is one or more artificial peptide(s) selected from the group consisting ofartificial peptides comprising the amino acid sequence of SEQ ID NO 4 and SEQ ID NO 5.

4. A pharmaceutical and / or cosmetic formulation according to any of the preceding claims, comprising a. 0.1-250 pg / mL, such as 0.1 , 1.0, 5.0, 10, 50, 100, 200 or 250 pg / mL of said artificial peptide, b. 1-40 mg / mL, such as1.0, 2.5, 4, 10, 20, 25, 30 or 40 mg / mL cross-linked hyaluronic acid fibres (HA-XL), and c. 1-40 mg / mL, such as 1.0, 2.5, 4, 10, 20, 25, 30 or 40 mg / mL linear hyaluronic acid fibres (HA).

5. A pharmaceutical and / or cosmetic formulation according to any one of the preceding claims, wherein the cross-linked hyaluronic fibres (HA-XL) comprise or consist of 1 ,4-butanediol diglycidyl ether cross-linked hyaluronic acid fibres (HA- XL(BDDE)) and / or poly(ethylene glycol) diglycidyl ether (PEGDE) cross-linked hyaluronic acid fibres (HA-XL(PEGDE)).

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

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

8. A pharmaceutical and / or cosmetic formulation according to any of the preceding claims, wherein the hyaluronic acid fibres are between 0.7-4.0 MDa, such as between 1 .0-2.0, between 1.5-1 .8, or between 3.0-3.3 MDa, such as 1.5MDa.

9. A pharmaceutical and / or cosmetic formulation according to claim 6, 7 or 8, further comprising d. one or more buffering agent(s) e. a source of fluoride, f. one or more salt(s), and g. water.

10. A 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 Phosphate and j. Sodium Phosphate.11 . A pharmaceutical and / or cosmetic formulation according to any of the preceding claims, wherein the artificial peptide is released at a controlled rate of 0.01-10 ug pr hour, such as about 0.01 , 0.1 , 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 ug per hour.

12. A pharmaceutical and / or cosmetic formulation according to any of the preceding claims, wherein the composition further comprises Mesenchymal Stromal Cells (MSCs).

13. A pharmaceutical and / or cosmetic formulation according to claim 12, wherein the composition comprises between 100.000-10.000.000 MSCs pr. ml, preferably about 1.000.000 MSCs pr. ml.

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

15. A pharmaceutical formulation according to any of the preceding claims for use as a medicament.

16. A pharmaceutical formulation according to any of the preceding claims for use in soft tissue healing, for inducing neovascularization, for inducing reepithelization, for stimulating collagen production, for promoting oriented collagen formation, for graft taking, post-grafting, for chronic wound healing, for use in an antiinflammatory and / or antimicrobial treatment, and / or for use in treating periodontitis, periimplantitis, peri-mucositis, gingivitis, aphthous stomatitis and / or infections and / or inflammations in the soft tissue of the craniomaxillofacial complex.

17. A pharmaceutical formulation according to any of claims 1-13, for use as an antiinflammatory composition.

18. A pharmaceutical formulation for use according to claim 17, wherein the antiinflammatory effect is measured as a reduction in proinflammatory cytokines.

19. A pharmaceutical formulation for use according to claim 18, wherein the proinflammatory cytokines comprise one or more cytokines selected from the group consisting of IL-23, IL-1 alpha, IL-1 beta, TNF-alpha, MCP-1 , IL-12P70, IFN-Y, IFN-beta, IL-6, IL-10, IL-27, IL-17A and GM-CSF.

20. A method for producing a pharmaceutical and / or cosmetic formulation according to any of the preceding claims, said method comprises, a. providing an artificial peptide as defined in any one of claims 1-3, b. providing hyaluronic acid with a molecular weight of 0.7-4 MDa, such as between 1 .0-2.0, between 1.5-1 .8, or between 3.0-3.3 MDa, such as 1.5MDa, c. mixing 0.1-250 pg / mL of said artificial peptide and 1-40 mg / mL of said HA and d. optionally, adding a source of fluoride to said mixture,wherein, said pharmaceutical and / or cosmetic formulation has an osmolarity of 50-400 mOsm / L, such as between 100 and 310 mOsm / L, or such as between 125 and 175 mOsm / L, such as about 150 mOsm / L, or such as between 275 and 325 mOsm / L, such as about 300 mOsm / L.21 . A method according to claim 20, wherein said hyaluronic acid comprises or consists of BDDE and / or PEGDE cross-linked hyaluronic acid and linear hyaluronic acid.

22. A method for producing a pharmaceutical and / or cosmetic formulation according to any of claims 1-19 the method comprises, a. providing an artificial peptide as defined in any one of claims 1-3, b. providing linear hyaluronic acid with a molecular weight of 0.7-4 MDa, such as between 1 .0-2.0, between 1.5-1.8, or between 3.0-3.3 MDa, such as 1.5MDa, c. cross-linking the hyaluronic acid using a crosslinking agent, such as e.g., BDDE or PEGDE to obtain a crosslinked hyaluronic acid (HA-XL), d. optionally, dialysing the crosslinked hyaluronic acid, e. mixing 0.1-250 pg / mL of said artificial peptide and 1-40 mg / mL of said crosslinked hyaluronic acid, and adding 1-40 mg / mL of linear hyaluronic acid to obtain a mixture comprising crosslinked hyaluronic acid, linear hyaluronic acid, and artificial peptide.

23. A method for producing a pharmaceutical and / or cosmetic formulation according to any of claims 1-19 the method comprises, a. providing an artificial peptide as defined in any one of claims 1-3, b. providing linear hyaluronic acid with a molecular weight of 0.7-4 MDa, such as between 1 .0-2.0, between 1.5-1.8, or between 3.0-3.3 MDa, such as 1.5MDa,C. mixing 0.1-250 pg / mL of said artificial peptide and 1-40 mg / mL of said hyaluronic acid,d. cross-linking the mixture of using a crosslinking agent, such as e.g., BDDE or PEGDE to obtain a mixture of intra- and / or inter-crosslinked peptide and hyaluronic acid, e. adding linear hyaluronic acid (HA) to obtain a mixture of intra- and / or intercrosslinked artificial peptide and hyaluronic acid, and linear hyaluronic acid.

24. A method according to claim 23, wherein the method further comprises g. mixing 1-40mg / mL of the intra- and / or inter-crosslinked artificial peptide and hyaluronic acid, with 0.1-250 pg / mL additional artificial peptide to obtain a mixture comprising intra- and / or inter-crosslinked artificial peptide and hyaluronic acid, linear hyaluronic acid, and artificial peptide.

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

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