Biocompatible materials

Abstract

The present invention provides compositions comprising a polymer and a natural or synthetic peptide or protein (NSPP). The compositions form hydrogels with water. The compositions are useful as fillers for cosmetic and therapeutic applications. Embodiments of the present invention provide methods for treating certain conditions using the compositions or hydrogels, as well as surgical kits for the simultaneous or sequential administration of each component of the composition, allowing for in situ formation of the hydrogel.

Description

[Technical Field]

[0001] Related Applications This application claims convention priority to Australian Provisional Patent Applications Nos. 2019904817 (filed December 19, 2019) and 2020903462 (filed September 25, 2020), the entire disclosures of which are each incorporated herein by reference.

[0002] The present invention relates to biocompatible materials that are useful in tissue regeneration and repair.

[0003] The present invention relates to a filler that supports the natural healing of damaged tissue without inducing any specific tissue formation. The present invention can be used to partially or completely fill or cover tissue cavities or defects, providing the necessary space filling with minimal foreign body reaction.

[0004] In one embodiment, the present invention relates to a tissue-conductive medical filler. In one embodiment, the polymer of the present invention can be formulated as a hydrogel. In another embodiment, the hydrogel is thermoresponsive. In yet another embodiment, the compositions disclosed herein have been developed for delivery in a flowable form that can be injected, poured, or sprayed. In one embodiment, the composition forms a hydrogel after administration into, on, or near the body.

[0005] The present invention is useful in tissue engineering applications, including both cosmetic and therapeutic applications. The present invention is useful in dermatological applications for the treatment of chronic, acute, or surgically created defects, as well as in dental and orthopedic applications. However, it will be understood that the present invention is not limited to these particular fields of use. [Background technology]

[0006] The following discussion of the prior art is provided to place the present invention in its proper technical context so that its advantages may be more fully understood. It should be understood, however, that any discussion of prior art throughout this specification should not be construed as an explicit or implicit admission that such prior art is widely known or forms part of the common general knowledge in the art, or that this prior art would be reasonably expected to be recognized, understood, or considered relevant by those skilled in the art.

[0007] All patents and publications referenced herein are incorporated by reference in their entirety.

[0008] Patent Document 1 relates to polymers, particularly polymers useful as hydrogels, and the use of hydrogels for tissue repair or reconstruction. In particular, the polymers and hydrogels of Patent Document 1 can be used for the repair or reconstruction of cartilage, particularly articular cartilage. The polymers contain at least monomers for binding water, monomers for imparting mechanical properties, and monomers for binding to extracellular proteins. Hydrogels include polymers containing at least monomers for binding water and monomers for binding to extracellular proteins. Crosslinking of the polymers by binding of the extracellular matrix proteins forms the hydrogel.

[0009] Patent Document 2 discloses a biocompatible material useful for tissue regeneration and repair, in which the bioactive polymer can be in the form of a hydrogel, such as a thermoresponsive hydrogel. The bioactive polymer of Patent Document 2 and the resulting hydrogel can be used for regenerating bone tissue. Accordingly, Patent Document 2 discloses a method for treating a bone defect in a mammal, comprising administering to the mammal a therapeutically effective amount of a hydrogel formed by the bioactive polymer, thereby treating the bone defect.

[0010] Patent Document 3 discloses a polymer comprising at least one bactericidal / analgesic / anti-inflammatory monomer unit together with at least three additional monomer units, wherein the three additional monomer units provide properties selected from the group consisting of temperature activation, water solubility, mechanical strength, protein / polysaccharide binding ability, and combinations thereof. In particular, Patent Document 3 discloses a polymer in which the water-soluble monomer unit is a hydrophilic ethylene glycol (OEGMA) moiety, the mechanical strength-providing monomer unit is polylactide-co-2-hydroxyethylmethylacrylate (PLA / HEMA), the protein-reactive monomer unit is an N-acryloxysuccinimide (NAS) moiety, and the thermosetting monomer unit is an N-isopropylacrylamide (NIPAAm) moiety. The bactericidal / analgesic / anti-inflammatory monomer unit comprises a methacrylic acid ester derivative of salicylic acid (5-HMA or 4-HMA, or a combination thereof).

[0011] All three of the above referenced applications are assigned to the present applicant.It is against this background that the present invention has been developed. [Prior art documents] [Patent documents]

[0012] [Patent Document 1] International Publication No. 2013 / 091001 (PCT / AU2012 / 001566) [Patent Document 2] International Publication No. 2017 / 035587 (PCT / AU2016 / 050817) [Patent Document 3] International Publication No. 2017 / 015703 (PCT / AU2016 / 050653) Summary of the Invention [Problem to be solved by the invention]

[0013] In one form, the present invention is embodied as a flowable filler that, upon administration into the body (e.g., by injection) or onto the body surface (i.e., at 30-37°C), forms an adhesive hydrogel. The hydrogel is well tolerated within the body with minimal inflammatory response. The hydrogel only exhibits regenerative properties in the presence of active bleeding or other fluids containing regenerative biological components, making it host tissue conductive but not inductive. The filler can be injected with a fine-gauge needle (e.g., 21G). The hydrogel adheres to the injection site. The filler can be formulated into an aerosol for administration by nebulization. The hydrogel can be administered in a minimally invasive manner, either to create a 3D structure layered within the body or locally. Key innovative aspects of the filler of the present invention include minimal foreign body reaction, host tissue conductivity, blood mixing, injectability, adhesive properties, layered packing, and an optimal degradation profile.

[0014] The present invention is useful in soft tissue applications, such as dermatological applications, and hard tissue applications, such as dental and orthopedic applications. For example, the present invention is useful in cosmetic applications, such as wrinkle reduction. The present invention is useful for promoting scar healing, including burn scars and post-surgical scars. The present invention may also be used in the management of chronic wounds, such as diabetic ulcers.

[0015] The present invention is also useful in dental applications. Tooth extraction is an inherently traumatic procedure that damages soft tissues, underlying bone, and ultimately leads to significant mandibular or alveolar bone loss. Clinically, alveolar bone loss results in aesthetic and functional complications associated with future prosthetic replacement of the missing tooth. When the missing tooth is replaced with an implant-supported reconstruction, complex bone grafting procedures are routinely required. In an attempt to reduce or potentially eliminate complex bone grafting procedures, extraction socket or alveolar ridge preservation techniques have been proposed.

[0016] Several techniques have been described in the literature, most of which involve placing graft filling material into the extraction socket immediately after tooth extraction. There are several commercially available products that have been used to fill the extraction site. In fact, none of the currently available products are able to restore bone volume to pre-extraction levels or provide improved healing outcomes.

[0017] Clinical studies using demineralized bone allografts, synthetic bioactive glass, and deproteinized bovine bone mineral (xenografts) have shown that implant particles are surrounded by connective tissue or a fibrous bone environment even 6 to 9 months after administration. These findings indicate that healing is physically hindered by the implant particles. The resulting surgical site presents an unpredictable surgical environment for implant placement, even after prolonged waiting periods. Given the range of problems associated with bone loss after tooth extraction, there is a significant unmet need for scaffolds to improve bone healing outcomes in patients undergoing tooth extraction.

[0018] There is a general need for compositions for tissue repair that are injectable at room temperature and form hydrogels at body temperature.

[0019] The composition of the present invention is intended to be used as a filler to support the natural healing of damaged tissue without inducing any specific tissue formation.The present invention is intended to be used to partially or completely fill tissue cavities to provide the necessary space filling with minimal foreign body reaction.The present invention can adhere to the cavity, mix with blood, and facilitate the repair of soft and hard tissues in the host.To induce the growth of any specific tissue, such as soft or hard tissue, the present invention can be used in combination with other active ingredients.

[0020] It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.

[0021] A particularly preferred embodiment of the present invention is to use PNPHO-co-TB4 as a filler material at a specific concentration and in a specific formulation. The filler material does not have any tissue-inducing properties. The filler may be used in combination with other materials, such as inert materials that provide 3D structures or active materials that induce the formation of specific tissues. The present invention can be injected, poured, or sprayed. The concentrations of the polymer and TB4 can be adjusted to form different forms of the present invention.

[0022] The present invention is intended for use in soft and hard tissues. For soft tissues, the present invention can be added to bioactive ingredients (such as cells or fat grafts) for skin-type applications, and the product can be injected or sprayed. For hard tissue or hard / soft tissue applications, the present invention can be applied with 3D fillers (e.g., inert bone particles) or with active compounds (e.g., growth factors) to promote both soft and hard tissue growth. Contemplated commercial forms are injectables or putties.

[0023] Although the present invention is described below with respect to preferred embodiments thereof, those skilled in the art will understand that the spirit and scope of the invention can be embodied in many other forms. [Means for solving the problem]

[0024] In the present invention, applicants have optimized their proprietary smart polymer, PNPHO, to be conjugated with thymosin beta-4 to form a cytotropic medical packing material.

[0025] According to a first aspect of the present invention, there is provided a composition comprising a polymer and a natural or synthetic peptide or protein (NSPP), wherein said polymer is a first monomer for binding water; a second monomer to impart mechanical properties to the hydrogel; a third monomer for attachment to a natural or synthetic peptide or protein (NSPP), and a fourth monomer to impart phase transition behavior; Compositions are provided wherein the natural or synthetic peptide or protein (NSPP) is thymosin beta-4 or a functional homolog thereof.

[0026] In one embodiment, the first monomer is selected from polyethers, polyvinyl alcohol (PVA), poly(vinylpyrrolidone) (PVP), poly(amino acids), and dextran.

[0027] In one embodiment, the polyether is selected from polyethylene glycol (PEG), oligo(ethylene glycol) (OEG) or macromonomers thereof, polyethylene oxide (PEO), polyethylene oxide-co-propylene oxide (PPO), co-polyethylene oxide block or random copolymers thereof.

[0028] In one embodiment, the first monomer is oligo(ethylene) glycol monomethyl ether methacrylate (OEGMA).

[0029] In one embodiment, the second monomer is a methacrylate or a random copolymer that includes a methacrylate.

[0030] In one embodiment, the second monomer is selected from hydroxyethyl methacrylate (HEMA), hydroxyethyl methacrylate poly(lactic acid) copolymer (HEMA-PLA), poly(lactic acid), poly(caprolactone), poly(glycolide), poly(glycolide-co-lactide), or poly(glycolide-co-caprolactone).

[0031] In one embodiment, the second monomer is hydroxyethyl methacrylate poly(lactic acid) (HEMA-PLA).

[0032] In one embodiment, the third monomer has an electrophilic functional group for attachment to the NSPP.

[0033] In one embodiment, the third monomer is selected from N-hydroxysulfosuccinimide (SNHS), N-hydroxyethoxylated succinimide (ENHS), and N-acryloxysuccinimide (NAS).

[0034] In one embodiment, the third monomer is N-acryloxysuccinimide (NAS). In one embodiment, the fourth monomer is selected from poly(ethylene oxide) / poly(propylene oxide) and poly(N-isopropylacrylamide) (PNIPAAm) homopolymers and copolymers.

[0035] In one embodiment, the fourth monomer is (N-isopropylacrylamide).In one embodiment, the polymer comprises the first monomer in an amount of about 3 to about 8 mol %.

[0036] In one embodiment, the polymer comprises a second monomer in an amount of about 5 to about 9 mol %.

[0037] In one embodiment, the polymer comprises a third monomer in an amount of at least about 7 mol %, such as 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 mol %.

[0038] In one embodiment, the polymer comprises a first monomer in an amount of about 3 to about 8 mol %, a second monomer in an amount of about 5 to about 9 mol %, a third monomer in an amount of at least about 7 mol %, and a fourth monomer in an amount that comprises the remainder up to 100% of the composition of the polymer.

[0039] In one embodiment, the polymer comprises a fourth monomer in an amount of about 60 to about 85 mol %, preferably about 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84 or 85 mol %.

[0040] In one embodiment, the first monomer is OEGMA, the second monomer is HEMA-PLA, the third monomer is NAS, and the fourth monomer is NIPAAm, and the polymer comprises OEGMA in an amount of about 3 to about 8 mol%, HEMA-PLA in an amount of about 5 to about 9 mol%, NAS in an amount greater than about 7 mol%, and NIPAAm in an amount up to about 85 mol%.

[0041] In one embodiment, the polymer comprises OEGMA in an amount of about 5 mol %, HEMA-PLA in an amount of about 7 mol %, NAS in an amount greater than about 7 mol %, and NIPAAm in an amount of about 81 mol %.

[0042] In one embodiment, the natural or synthetic peptide or protein (NSPP) is thymosin beta-4.

[0043] In one embodiment, the composition comprises essentially equimolar amounts of the polymer and thymosin beta-4.

[0044] In one embodiment for partially or completely filling the cavity, the concentration of the polymer is from about 100 mg / mL to about 300 mg / mL of the composition.

[0045] According to a second aspect of the present invention, there is provided a hydrogel comprising a composition according to the first aspect of the present invention and water, wherein binding of the NSPP to a third monomer crosslinks the polymer, thereby forming a hydrogel having water contained therein.

[0046] According to a third aspect of the present invention there is provided a method of making a hydrogel comprising adding water to a composition of the first aspect of the present invention.

[0047] According to a fourth aspect of the present invention there is provided a method of making a hydrogel comprising mixing an aqueous solution of the composition of the first aspect of the present invention with an aqueous solution of a natural or synthetic peptide or protein (NSPP) as defined above.

[0048] In one embodiment, the hydrogel forms at body temperature. In one embodiment, the hydrogel forms after administration of the composition and NSPP to a mammal by injection or by administering an aerosol.

[0049] According to a fifth aspect of the present invention, there is provided a use of a composition according to the first aspect of the present invention in the manufacture of a hydrogel for the repair and / or reconstruction of both hard and soft tissues. Hard tissues (also called mineralized tissues) are tissues that are mineralized and have a hard intercellular matrix. Human hard tissues include bone, tooth enamel, dentin, and cementum. Soft tissues include tissues that connect, support, or surround other structures and organs of the body that are not hard tissues, such as bone. Soft tissues include tendons, ligaments, fascia, skin, fibrous tissue, fat, and synovial membranes (which are connective tissues), as well as muscles, nerves, and blood vessels (which are not connective tissues).

[0050] According to a sixth aspect of the present invention there is provided the use of a composition according to the first aspect of the present invention in the manufacture of a hydrogel for wound healing.

[0051] According to a seventh aspect of the present invention there is provided the use of a composition according to the first aspect of the present invention in the manufacture of a hydrogel for temporary wrinkle reduction.

[0052] According to an eighth aspect of the present invention there is provided the use of a composition according to the first aspect of the present invention in the manufacture of a hydrogel for temporarily lifting the base of a scar and promoting healing.

[0053] According to a ninth aspect of the present invention there is provided the use of a composition according to the first aspect of the present invention in the manufacture of a hydrogel for supporting dermal connective tissue formation and promoting healing in scar tissue following surgical intervention.

[0054] According to a tenth aspect of the present invention there is provided the use of a composition according to the first aspect of the present invention in the manufacture of a hydrogel for supporting dermal connective tissue formation in the management of post-burn scars.

[0055] According to an eleventh aspect of the present invention there is provided the use of a composition according to the first aspect of the present invention in the manufacture of a hydrogel for supporting vascular ingrowth and promoting healing in acute skin defects involving bleeding.

[0056] According to a twelfth aspect of the present invention there is provided the use of a composition according to the first aspect of the present invention in the manufacture of a hydrogel for filling a surgically created skin cavity.

[0057] According to a thirteenth aspect of the present invention there is provided the use of a composition according to the first aspect of the present invention in the manufacture of a hydrogel for supporting skin graft surgery.

[0058] According to a fourteenth aspect of the present invention there is provided the use of a composition according to the first aspect of the present invention in the manufacture of a hydrogel for the physical delivery of a bone graft substitute.

[0059] According to a fifteenth aspect of the present invention there is provided the use of a composition according to the first aspect of the present invention in the manufacture of a hydrogel for filling a prosthesis.

[0060] According to a sixteenth aspect of the present invention there is provided the use of a composition according to the first aspect of the present invention in the manufacture of a hydrogel for use as a filler without tissue inductive properties.

[0061] According to a seventeenth aspect of the present invention there is provided the use of a composition according to the first aspect of the present invention in the manufacture of a hydrogel for supporting and repairing periodontal tissue following tooth extraction.

[0062] According to an eighteenth aspect of the present invention there is provided the use of a composition according to the first aspect of the present invention in the manufacture of a hydrogel for temporarily lifting periodontal ligament tissue and / or supporting a periodontal ligament tissue transplant.

[0063] According to a nineteenth aspect of the present invention there is provided the use of a hydrogel according to the second aspect of the present invention in the manufacture of a medicament for tissue repair and / or reconstruction.

[0064] According to a twentieth aspect of the present invention there is provided a method of tissue repair and / or reconstruction comprising administering to a mammal a composition according to the first aspect of the present invention, in one embodiment the administering step is carried out by injection or by administering an aerosol, thereby forming a hydrogel at body temperature of the mammal.

[0065] According to a twenty-first aspect of the present invention there is provided a method of wound healing comprising administering to a mammal a composition according to the first aspect of the present invention, in one embodiment the administering step is carried out by injection or by administering an aerosol, thereby forming a hydrogel at body temperature of the mammal.

[0066] According to a twenty-second aspect of the present invention there is provided a method for temporary wrinkle reduction comprising administering to a mammal a composition according to the first aspect of the present invention, in one embodiment the administering step is carried out by injection or by administering an aerosol, which forms a hydrogel at body temperature of the mammal.

[0067] According to a twenty-third aspect of the present invention there is provided a method of temporarily lifting the base of a scar and promoting healing, comprising administering to a mammal a composition according to the first aspect of the present invention. In one embodiment the administering step is carried out by injection or by administering an aerosol, which forms a hydrogel at body temperature of the mammal.

[0068] According to a 24th aspect of the present invention there is provided a method of supporting dermal connective tissue formation and promoting healing in scar tissue following surgical intervention, comprising administering to a mammal a composition according to the first aspect of the present invention, in one embodiment the administering step is carried out by injection or by administering an aerosol, thereby forming a hydrogel at body temperature of the mammal.

[0069] According to a 25th aspect of the present invention there is provided a method of supporting dermal connective tissue formation in the management of post-burn scars, comprising administering to a mammal a composition according to the first aspect of the present invention, in one embodiment the administering step is carried out by injection or by administering an aerosol, which forms a hydrogel at body temperature of the mammal.

[0070] According to a 26th aspect of the present invention there is provided a method of supporting vascular ingrowth and promoting healing in acute skin defects involving bleeding, comprising administering to a mammal a composition according to the first aspect of the present invention, in one embodiment the administering step is carried out by injection or by administering an aerosol, thereby forming a hydrogel at body temperature of the mammal.

[0071] According to a 27th aspect of the present invention there is provided a method of filling a surgically created skin cavity comprising administering to a mammal a composition according to the first aspect of the present invention, in one embodiment the administering step is carried out by injection or by administering an aerosol, thereby forming a hydrogel at body temperature of said mammal.

[0072] According to a 28th aspect of the present invention there is provided a method of supporting skin graft surgery comprising administering to a mammal a composition according to the first aspect of the present invention, in one embodiment the administering step is carried out by injection or by administering an aerosol, thereby forming a hydrogel at body temperature of the mammal.

[0073] According to a 29th aspect of the present invention there is provided a method of physically delivering a bone graft substitute comprising administering to a mammal a composition according to the first aspect of the present invention, in one embodiment the administering step is carried out by injection or by administering an aerosol, thereby forming a hydrogel at body temperature of the mammal.

[0074] According to a 30th aspect of the present invention, there is provided a method of filling a prosthesis, comprising administering to a mammal a composition according to the first aspect of the present invention. In one embodiment, the filler does not have tissue inductive properties. In one embodiment, the administering step is carried out by injection or by administering an aerosol, thereby forming a hydrogel at the body temperature of the mammal.

[0075] According to a thirty-first aspect of the present invention there is provided a method of supporting and repairing periodontal tissue following tooth extraction, comprising administering to a mammal a composition according to the first aspect of the present invention, in one embodiment the administering step is carried out by injection or by administering an aerosol, which forms a hydrogel at body temperature of the mammal.

[0076] According to a 32nd aspect of the present invention there is provided a method for temporarily lifting periodontal ligament tissue and / or supporting a periodontal ligament tissue graft, comprising administering to a mammal a composition according to the first aspect of the present invention. In one embodiment the administering step is carried out by injection or by administering an aerosol, which forms a hydrogel at body temperature of the mammal.

[0077] According to a thirty-third aspect of the present invention there is provided a method of tissue repair and / or reconstruction comprising administering to a mammal a hydrogel according to the second aspect of the present invention, in one embodiment the administering step is carried out by injection or by administering an aerosol, thereby forming a hydrogel at body temperature of the mammal.

[0078] According to a thirty-fourth aspect of the present invention there is provided a composition according to the first aspect of the present invention for use in tissue repair and / or reconstruction, in one embodiment the administering step is carried out by injection or by administering an aerosol, thereby forming a hydrogel at body temperature of the mammal.

[0079] According to a thirty-fifth aspect of the present invention there is provided a composition according to the first aspect of the present invention for use in wound healing, in one embodiment the administering step is carried out by injection or by administering an aerosol, which forms a hydrogel at body temperature of the mammal.

[0080] According to a 36th aspect of the present invention there is provided a composition according to the first aspect of the present invention for use in temporary wrinkle reduction, in one embodiment the administering step is carried out by injection or by administering an aerosol, which forms a hydrogel at body temperature of the mammal.

[0081] According to a 37th aspect of the present invention there is provided a composition according to the first aspect of the present invention for use in temporarily lifting the base of a scar and promoting healing. In one embodiment the administering step is carried out by injection or by administering an aerosol which forms a hydrogel at body temperature of the mammal.

[0082] According to a thirty-eighth aspect of the present invention there is provided a composition according to the first aspect of the present invention for use in supporting dermal connective tissue formation and promoting healing in scar tissue following surgical intervention, in one embodiment the administering step is carried out by injection or by administering an aerosol, which forms a hydrogel at body temperature of the mammal.

[0083] According to a thirty-ninth aspect of the present invention there is provided a composition according to the first aspect of the present invention for use in supporting dermal connective tissue formation in the management of post-burn scars, in one embodiment the administering step is carried out by injection or by administering an aerosol, which forms a hydrogel at body temperature of the mammal.

[0084] According to a fortieth aspect of the present invention there is provided a composition according to the first aspect of the present invention for use in supporting vascular ingrowth and promoting healing in acute skin defects involving bleeding, in one embodiment the administering step is carried out by injection or by administering an aerosol, thereby forming a hydrogel at body temperature of the mammal.

[0085] According to a forty-first aspect of the present invention there is provided a composition according to the first aspect of the present invention for use in filling a surgically created skin cavity, in one embodiment the administering step is carried out by injection or by administering an aerosol, thereby forming a hydrogel at body temperature of the mammal.

[0086] According to a forty-second aspect of the present invention there is provided a composition according to the first aspect of the present invention for use in supporting skin graft surgery, in one embodiment the administering step is carried out by injection or by administering an aerosol, thereby forming a hydrogel at body temperature of the mammal.

[0087] According to a forty-third aspect of the present invention there is provided a composition according to the first aspect of the present invention for use in physically delivering a bone graft substitute, in one embodiment the administering step is carried out by injection or by administering an aerosol, thereby forming a hydrogel at body temperature of the mammal.

[0088] According to a forty-fourth aspect of the present invention, there is provided a composition according to the first aspect of the present invention for use in filling a prosthesis. In one embodiment, the filler does not have tissue inductive properties. In one embodiment, the administering step is carried out by injection or by administering an aerosol, which forms a hydrogel at mammalian body temperature.

[0089] According to a forty-fifth aspect of the present invention there is provided a composition according to the first aspect of the present invention for use in supporting and repairing periodontal tissue following tooth extraction, in one embodiment the administering step is carried out by injection or by administering an aerosol, which forms a hydrogel at mammalian body temperature.

[0090] According to a 46th aspect of the present invention there is provided a composition according to the first aspect of the present invention for use in temporarily lifting periodontal ligament tissue and / or supporting a periodontal ligament tissue transplant, in one embodiment the administering step is carried out by injection or by administering an aerosol, which forms a hydrogel at body temperature of the mammal.

[0091] According to a 47th aspect of the present invention there is provided a hydrogel according to the second aspect of the present invention for use in tissue repair and / or reconstruction, in one embodiment the administering step is carried out by injection or by administering an aerosol, thereby forming the hydrogel at body temperature of the mammal.

[0092] In all aspects and embodiments in which the administering step is performed by administering an aerosol, the aerosol may be applied to any orifice of the body, including, but not limited to, the nasal cavity, mouth, or open wound.

[0093] According to a 48th aspect of the present invention, there is provided a kit for forming a hydrogel, comprising a polymer and a natural or synthetic peptide or protein (NSPP), wherein the polymer comprises a first monomer for binding water, a second monomer for imparting mechanical properties to the hydrogel, a third monomer for binding to the natural or synthetic peptide or protein (NSPP), and a fourth monomer for imparting phase transition behavior, and wherein the natural or synthetic peptide or protein (NSPP) is thymosin beta-4 or a functional homolog thereof. In one embodiment, the kit further comprises water in a separate container.

[0094] According to a forty-ninth aspect of the present invention, there is provided a kit for forming a hydrogel, comprising, in separate containers, a natural or synthetic peptide or protein (NSPP), and a composition comprising a first monomer for binding water, a second monomer for imparting mechanical properties to the hydrogel, a third monomer for binding to the natural or synthetic peptide or protein (NSPP), and a fourth monomer for imparting phase transition behavior, wherein the natural or synthetic peptide or protein (NSPP) is thymosin beta-4 or a functional homolog thereof, and the NSPP and the second monomer are crosslinked, thereby enabling the formation of the hydrogel when the composition is contacted with water.

[0095] In one embodiment, one or both of the NSPP and the composition are in solid form. In one embodiment, the kit further comprises water in a separate container. In one embodiment, the kit further comprises instructions for sequential or simultaneous administration of the components of the kit. In one embodiment, the kit is configured so that the composition, NSPP, and water are mixed together when dispensed.

[0096] In one embodiment, the NSPP and the composition are used as a filler for in situ delivery of a bone graft substitute in a patient in need of such treatment. The filler maintains the BGS in place (degrades) for at least 6 weeks (remains attached) and provides a scaffold for cell ingrowth (osteoblasts in the bone).

[0097] In another embodiment, the polymer is present in the composition at a concentration of about 5 mg / mL to about 70 mg / mL to form an aerosol for topical administration, e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 mg / mL. Thus, the polymer can be delivered by aerosol in very low concentrations and adhered to the surgical site for on-the-spot application.

[0098] Definitions and Nomenclature In describing and claiming the present invention, the following terminology will be used in accordance with the definitions provided below. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments of the present invention only, and is not intended to be limiting. Unless otherwise defined, 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.

[0099] Unless the context clearly requires otherwise, throughout this specification and the claims, the terms "comprise," "comprising," and the like are to be construed in an inclusive sense, i.e., "including but not limited to," rather than an exclusive or exhaustive sense.

[0100] The terms "preferred" and "preferably" refer to embodiments of the invention that may provide certain benefits, under certain circumstances. However, other embodiments may be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the invention.

[0101] As used herein, expressions defining range or length limits, such as, for example, "1 to 5," refer to any integer between 1 and 5, i.e., 1, 2, 3, 4, and 5. In other words, any range defined by two explicitly stated integers is intended to include and disclose any integers defining said limits and any integers included in said range.

[0102] Other than in the examples, or unless otherwise indicated, all numbers expressing amounts of ingredients or reaction conditions used herein should be understood to be modified in all instances by the term "about." These examples are not intended to limit the scope of the invention. Hereinafter, or unless otherwise indicated, "%" means "% by weight," "ratio" means "weight ratio," and "parts" means "parts by weight."

[0103] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible. However, any numerical value inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0104] The term "room temperature" is intended to mean a temperature of about 20 to about 25°C.

[0105] The term "animal" includes humans and non-humans, eg, mammals, such as horses, cows, pigs, sheep, cats, dogs, and the like.

[0106] As used herein, "implant" refers to an article or device that is completely or partially placed in an animal, for example, by a surgical procedure.

[0107] As used herein, the term "natural or synthetic peptide or protein" (or NSPP) refers to a protein or peptide that is naturally present in the extracellular portion of animal tissues, which provides structural support to animal cells (in addition to performing a variety of other important functions). The term also refers to synthetically prepared proteins or peptides that have functions similar to those of naturally occurring proteins and peptides. By way of example, naturally occurring proteins and peptides are those commonly found in the extracellular matrix (or ECM), a defining feature of animal connective tissue. Naturally occurring proteins commonly found in ECM include collagen, fibrin, fibronectin, and laminin (and their isoforms).

[0108] The NSPP employed in the present invention is thymosin beta-4 or a functional homologue thereof.

[0109] The following abbreviations are used herein:

[0110] [Table 0]

[0111] In an exemplary composition according to the present invention, 140 mg / mL of PNPHO (ratio 5:8 (5:7:81) is used with 30 mg / mL of thymosin beta-4, and this composition is labeled PNPHO-co-TB4 (or alternatively PNPHO-co-NSPP or "TR001") for applicant's clinical trials and will be referred to as such throughout the body of this specification.

[0112] Preferred embodiments of the present invention will now be described, by way of example only, with reference to the accompanying drawings, in which: [Brief explanation of the drawings]

[0113] [Figure 1a] 1 is a macroscopic image of a solution of the present invention (polymer and NSPP) that forms a hydrogel in simulated physiological conditions (PBS at 37° C.) and retains its structure upon gelation. [Figure 1b] 10 is a macroscopic image of PNPHO-co-TB4 injection into a site with active bleeding showing immediate hydrogel formation despite the presence of active bleeding at the defect site. [Figure 1c] 10 is a macroscopic image sequence showing PNPHO-co-TB4 solution in contact with a wound at body temperature and hydrogel formation. [Figure 1d] Macroscopic image of PNPHO-co-TB4 mixed with blood forming an adhesive hydrogel used to fill 3D areas via layer-by-layer filling. [Figure 2a] 1 shows the synthetic preparation of PNPHO in DMF at 70° C. [Figure 2b] The H NMR spectrum of PNPHO in CDCl is shown; the resonances at approximately 2.9–3.0 ppm for the residual trace of DMF solvent overlap with the NAS proton resonance (e), making initial calculations based on the same data set (integral / area under each peak based on the relative mole % of each monomer within the overall PNPHO polymer) erroneous. Subsequent correction confirms that a third monomer (NAS) is present in an amount greater than approximately 7 mole %. [Figure 3] Solubility of copolymers with lactate numbers 3 (a) and 6 (b) synthesized with different mole fractions of HEMA-PLA in aqueous solution at 4 °C (*, **, and *** indicate p<0.05, <0.01, and <0.001, respectively). [Figure 4] LCST measurements and a comparison of PNPHO-co-TB4 (a) and PNPHO (b) are shown. The difference between the two LCST values ​​confirms the presence of a chemical interaction between the two components, as well as the physical role of TB4 in accelerating the gelation kinetics. [Figure 5] 1A-C show the investigation of the scaffolding effect of PNPHO-co-TB4 on integrating with host tissue. (a) Formation of full-thickness skin wounds; (b) Use of PNPHO-co-TB4 and Integra for skin grafting; and (c) Engraftment of grafts at different time points treated with Integra or PNPHO-co-TB4. [Figure 6] Figure 1 shows the assessment of the inflammatory response to PNPHO-co-TB4 and direct comparison with Integra (the gold standard for dermal matrix); H&E staining of Integra-treated sites 2 weeks (a) and 4 weeks (b) after surgery; and H&E staining of PNPHO-co-TB4-treated sites 2 weeks (c) and 4 weeks (d) after implantation. White arrows indicate PNPHO-co-TB4 structures, and black arrows indicate peri-implant fibrous tissue formation. [Figure 7] Figure 1 shows the angiogenic response in mice at different time points for PNPHO-co-TB4 and Integra treatment sites. Angiogenesis and ingrowth at the implantation site were determined by fluorescence emission efficiency using an IVIS Lumina XR live imager. Two and four weeks after implantation, the angiogenic response was determined using the AngioSense750 EX in vivo blood pool fluorescence imaging probe. This near-infrared fluorescent macromolecular probe persists in the vasculature, allowing imaging of blood vessels and angiogenesis. At each post-surgery time point, each mouse was injected with 2 nmol of AngioSense750 EX in 100 μL of PBS. Twenty-four hours later, each mouse was scanned for fluorescence emission efficiency (n=8). Emission efficiency was used to indicate the density of new blood vessels in the wound area. Results showed that two weeks after surgery, the fluorescence emission efficiency for PNPHO-co-TB4 treatment sites was significantly higher than that for Integra treatment sites (p<0.01). In contrast, 4 weeks after surgery, angiogenic signals were very low for both treatment groups, indicating that angiogenesis was controlled and healing of the site was complete. [Figure 8] Histological evaluation of the PNPHO-co-TB4-treated site two weeks after skin grafting surgery is shown. The white arrows indicate vascular ingrowth within the PNPHO-co-TB4 hydrogel structure, along with fibroblast infiltration into the injectable scaffold. The results in Figures 8(c) and 8(d) demonstrate the formation of blood vessels within the PNPHO-co-TB4 structure. In addition, staining of the skin biopsy sample showed clear infiltration of host fibroblasts within the PNPHO-co-TB4 hydrogel structure; see Figure 8(d). [Figure 9]Figure 9 shows Masson's trichrome staining of the PNPHO-co-TB4-treated skin graft site 4 weeks after surgery. The black arrow indicates collagen fibers deposited from fibroblast ingrowth within the PNPHO-co-TB4 structure. To further confirm fibroblast infiltration and dermal extracellular matrix formation within the PNPHO-co-TB4 structure, the skin graft site was stained with Masson's trichrome 4 weeks after grafting. The results in Figure 9 show collagen fiber formation within the PNPHO-co-TB4 structure 4 weeks after grafting surgery. These results confirm fibroblast infiltration within the PNPHO-co-TB4 structure and its potential to integrate with host tissue and promote neodermis formation. [Figure 10] Masson's trichrome staining of skin grafts treated with Integra four weeks after surgery. Collagen fiber formation within the PNPHO-co-TB4 structure was significantly higher than that detected within the Integra structure. Results showed significantly less collagen formation within the Integra structure compared to PNPHO-co-TB4. [Figure 11] Use of PNPHO-co-TB4 after tooth extraction, showing (a) the tooth extraction site with active bleeding, (b) injection of PNPHO-co-TB4 via a 21G needle into the tooth extraction socket site, (c) immediate gelation of PNPHO-co-TB4 at the site, and (d) mixing of PNPHO-co-TB4 with blood at the site. [Figure 12] The application of PNPHO-co-TB4 after tooth extraction in 10 patients is shown. Clinical use of this device in a PET trial showed that injection of PNPHO-co-TB4 into the tooth extraction socket site was successful in all 10 patients, with no device malfunctions reported by the investigator. [Figure 13]Soft tissue regeneration and wound healing are shown 7 days after surgery and treatment with PNPHO-co-TB4. All 10 patients treated with PNPHO-co-TB4 returned for the first follow-up visit 1 week after surgery. No pain or discomfort was reported by the patients. During oral examination (1 week after administration), there were no signs of infection or inflammation at the site. In addition, wound closure and soft tissue formation were examined by the investigator. Wound closure was observed and rapid soft tissue formation was detected in all 10 patients. [Figure 14] H&E and Masson's Trichrome staining of PNPHO-co-TB4 treated samples is shown. [Figure 15] Representative images of plume patterns for formulations A) 17.5 mg / mL, B) 35 mg / mL, and C) 70 mg / mL are shown. [Figure 16] The maximum and minimum plume envelope diameter and derived ellipticity (mean±SD, n=3) for the three formulations are shown. [Figure 17] 1 shows the deposition pattern of the formulations using a human nasal model. [Figure 18] 1 shows the in vitro release of ciprofloxacin HCl from PNPHO and PNPHO-co-TB4 hydrogels at 37° C., shown as cumulative mass (results shown are n=1±SD). DETAILED DESCRIPTION OF THE INVENTION

[0114] The present invention will now be more fully described with reference to the accompanying examples and drawings, but it should be understood that the following description is for illustrative purposes only and should not be construed in any way as a restriction on the generality of the invention described above.

[0115] Reference will now be made in detail to certain specific embodiments of the invention. While the invention will be described in conjunction with the embodiments, it will be understood that the intention is not to limit the invention to these embodiments. On the contrary, the invention is intended to encompass all alternatives, modifications, and equivalents that may be included within the scope of the invention as defined by the claims. Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described.

[0116] It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or apparent from the text or drawings, all of these different combinations constituting various alternative aspects of the invention.

[0117] The use of tissue compatible polymers is disclosed herein.The compositions of the present invention are preferably injectable.

[0118] 1. Polymers The term "polymer," as used herein, refers to a large molecule (macromolecule) made up of repeating structural units (monomers). These subunits are typically joined by covalent chemical bonds. Polymers can be linear or branched polymers. Preferably, the polymers of the present invention are copolymers comprising three or more different monomers.

[0119] Thus, in one embodiment, a preferred polymer for use herein comprises a first water-binding monomer, a second monomer capable of imparting mechanical properties to the hydrogel, and a third monomer having a functional group for binding to an NSPP.

[0120] The term "monomer," as used herein, refers to a structural unit that can be combined to form a polymer, but which may itself be a polymer, or a derivative of a monomer or polymer. This latter type of monomer is also referred to herein as a "macromonomer."

[0121] As used herein, a "macromonomer" is a polymer or oligomer whose molecules each have one end group that acts as a monomer molecule, such that each polymer or oligomer molecule contributes only a single monomer unit to the product polymer chain.

[0122] The polymer of the present composition comprises a first monomer for binding water, a second monomer for imparting mechanical properties to the hydrogel, a third monomer for binding to a natural or synthetic peptide or protein (NSPP), and a fourth monomer for imparting phase transition behavior.

[0123] 1.1. First Monomer: Water-Binding Monomer As mentioned above, the advantageous properties of the preferred hydrogels used herein can be attributed to the combination of the specific components of the NSPP and the preferred polymer. One particularly advantageous property of these preferred polymers is their water-binding ability. The presence of water in the hydrogel provides the hydrogel with both an environment similar to the natural environment of the damaged tissue (which aids in tissue regeneration) and the necessary compression resistance.

[0124] Therefore, preferred polymers for use herein must contain monomers or units capable of binding water so that a hydrogel can form when the polymer is in contact with an NSPP and water, and the hydrogel thus formed must have the necessary compression resistance and recovery properties.

[0125] Those skilled in the art will understand that the water-binding monomer must be present in the preferred polymers used in the present invention in a proportion sufficient to produce a polymer that meets these requirements. Generally, the proportion of water-binding monomer in the polymer can be about 10:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, or about 1:5 in terms of molar ratio of water-binding monomer to mechanical strength monomer. In fact, the water-binding monomer must not only render the polymer hydrophilic, but also impart significant water-binding ability to the polymer.

[0126] Therefore, preferred polymers used in the present invention have a water binding capacity of about 70% to about 500%, about 80% to about 400%, about 90% to about 300%, or about 100% to about 200%. For example, the water binding capacity of preferred polymers used herein is about 70%, about 80%, about 90%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, about 200%, about 210%, about 220%, about 230%, about 240%, about 250%, about 260%, about 270%, about 280%, about 290%, about 300%, about 310%, about 320%, about 330%, about 340%, about 350%, about 360%, about 370%, about 380%, about 390%, about 400%, about 410%, about 420%, about 430%, about 440%, about 450%, about 460%, about 470%, about 480%, about 490%, about 500%, about 510%, about 520%, about 530%, about 540%, about 550%, about 560%, about 570%, about 580%, about 590%, about 600%, about 610%, about 620%, about 630%, about 640%, about 650%, about 660%, about 670%, about 680%, about 690%, about 700%, about 710%, about 720%, about 730%, about 740%, about 750%, about 760%, about 770%, about 780%, about 70%, about 280%, about 290%, about 300%, about 310%, about 320%, about 330%, about 340%, about 350%, about 360%, about 370%, about 380%, about 390%, about 400%, about 410%, about 420%, about 430%, about 440%, about 450%, about 460%, about 470%, about 480%, about 490%, or about 500%.

[0127] Suitable examples of water-binding monomers include monomers that can be synthesized into polymers such as polyethers (e.g., alkaline polyimides such as polyethylene glycol (PEG), oligo(ethylene glycol) (OEG), polyethylene oxide (PEO), polyethylene oxide-co-propylene oxide (PPO), co-polyethylene oxide block or random copolymers, polyvinyl alcohol (PVA), poly(vinylpyrrolidone) (PVP), poly(amino acids), and dextran. Polyethers, especially oligo(oxyalkylenes) (e.g., OEG), are particularly preferred because they have the requisite water-binding capacity, are simple to synthesize and / or purchase, and are inert, meaning that they provoke minimal or no immune response from tissues into which they are placed.

[0128] Additionally, any of a variety of hydrophilic functional groups may be used to render the monomers (and thus the polymers formed from such monomers) water soluble. For example, functional groups such as phosphate, sulfate, quaternary amine, hydroxyl, amine, sulfonic acid, and carboxylic acid, which are water soluble, may be incorporated into the monomer to render it water soluble.

[0129] Monomers can also react with other compounds to form "macromonomers." Thus, the first monomer can optionally be a macromonomer.

[0130] A preferred first monomer that is a macromonomer is oligo(ethylene glycol) monomethyl ether methacrylate (OEGMA), which is a hydrophilic monomer that is composed of two hydrophilic monomers: ethylene glycol and methacrylate.

[0131] Preferably, the polymer comprises the first monomer in an amount of about 3 to about 8 mol %, preferably about 3, 4, 5, 6, 7 or 8 mol %.

[0132] 1.2. Second Monomer: Monomer Imparting Mechanical Properties As noted above, the advantageous properties of preferred hydrogels used with the present invention can be attributed, in part, to the particular components that make up the polymer. In some embodiments, preferred polymers used in the present invention can provide additional mechanical properties to the hydrogel.

[0133] Those skilled in the art will understand that monomers capable of imparting mechanical properties to hydrogels must be present in the preferred polymer in a proportion sufficient to produce a hydrogel with the desired mechanical properties. Generally, the proportion of "mechanical" monomers in the polymer can be about 10:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, or about 1:5 in terms of molar ratio of water-binding monomers to mechanical strength monomers. Suitable examples of monomers capable of imparting mechanical properties (e.g., compression resistance) to hydrogels include methacrylates, such as hydroxyethyl methacrylate (HEMA), hydroxyethyl methacrylate poly(lactic acid) copolymer (HEMA-PLA), polyesters, such as poly(lactic acid), poly(caprolactone), poly(glycolide), and random copolymers thereof (e.g., poly(glycolide-co-lactide) and poly(glycolide-co-caprolactone)).

[0134] Monomers can also react with other compounds to form “macromonomers.” A preferred second monomer that is a macromonomer is hydroxyethyl methacrylate poly(lactic acid) (HEMA-PLA).

[0135] Preferably, the polymer comprises the second monomer in an amount of about 5 to about 9 mol %, preferably about 5, 6, 7, 8 or 9 mol %.

[0136] 1.3. Third Monomer: NSPP-Linked Monomer As mentioned above, preferred hydrogels for use in the present invention are formed by combining a polymer with an NSPP in the presence of water. To effectively combine the polymer with the NSPP, the polymer preferably contains a monomer or unit capable of crosslinking.

[0137] This crosslinkability means that the polymer can bind to the NSPP (discussed further below) and, in doing so, crosslink the NSPP to form a hydrogel containing the NSPP. Alternatively, by a similar mechanism, the NSPP can act as a crosslinker, thereby crosslinking the polymer to form a hydrogel.

[0138] The inventors have recognized that by utilizing a polymer design in which the polymer is equipped with monomers having functional groups for binding to thymosin beta-4, etc., it is not necessary to further crosslink the polymer, for example by chemical or UV crosslinking, to form a hydrogel.

[0139] Additionally, by covalently bonding the NSPP to the polymer, the NSPP is more effectively retained within the hydrogel network, meaning that once the hydrogel is administered to the repair site, the NSPP cannot easily migrate away from the site, which means that the structural integrity of the gel at the repair site is maintained (due to the mechanical properties of the NSPP, as mentioned above), helping to provide an environment at the repair site that closely mimics the tissue's native environment.

[0140] Those skilled in the art will understand that to produce a polymer capable of binding to an NSPP, the monomer capable of binding to the NSPP must be present in the polymer of the invention in a proportion sufficient to crosslink with the NSPP so that a hydrogel can be formed in the presence of water. Generally, the proportion of "crosslinking" monomers in the polymer is at least about a 1:1 molar ratio of crosslinking monomer to water-binding monomer. This ratio can be increased to, for example, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, or about 10:1.

[0141] Monomers capable of linking to an NSPP generally have electrophilic or nucleophilic functional groups, such that, for example, a nucleophilic functional group on the NSPP can react with an electrophilic functional group on the monomer to form a covalent bond. Preferably, the polymer contains more than two NSPP-linking monomers, whereby the polymer links to the NSPP as a result of an electrophilic-nucleophilic reaction to form a crosslinked polymer product. Such a reaction is referred to as a "crosslinking reaction."

[0142] Thus, for example, if the NSPP has a nucleophilic functional group such as an amine, the polymer may have an electrophilic functional group such as N-hydroxysuccinimide (NHS). Other electrophilic functional groups suitable for use in the present invention are N-hydroxysulfosuccinimide (SNHS) and N-hydroxyethoxylated succinimide (ENHS). An example of this type of monomer is N-acryloxysuccinimide (NAS). On the other hand, if the NSPP has an electrophilic functional group, the polymer may have a nucleophilic functional group such as an amine or a thiol.

[0143] Preferably, the polymer comprises a third monomer in an amount of at least about 7 mol %, more preferably at least about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 mol %.

[0144] 1.4. Fourth Monomer: Phase Transfer Monomer In another embodiment, preferred polymers may further comprise a fourth monomer capable of imparting phase transition properties to the hydrogel, thereby enabling the composition to be injectable at room temperature and capable of gel formation (i.e., hydrogel formation) at body temperature. Furthermore, these phase transition properties enable preferred polymers used with the present invention to form hydrogels whose various properties (e.g., viscosity) can be changed by altering factors such as pH and temperature.

[0145] Thermoresponsive injectable hydrogels are designed to have a lower critical solution temperature (LCST) below body temperature. Therefore, gelation can be achieved simply by increasing the temperature of the hydrogel, for example, by warming the hydrogel to body temperature (which occurs when the hydrogel is administered into the body). Various thermoresponsive injectable polymers are suitable for use in the present invention, including poly(ethylene oxide) / poly(propylene oxide) and poly(N-isopropylacrylamide) (PNIPAAm) homopolymers and copolymers. NIPAAm (as a monomer component or polyNIPAAm) is particularly suitable because it has an LCST of 32°C, allowing it to form a gel at body temperature.

[0146] Those skilled in the art will understand that to produce a thermoresponsive polymer, the phase transition monomer must be present in the polymer used with the present invention in a proportion sufficient to allow the viscosity of the hydrogel containing the polymer to be changed by exposing the hydrogel to different conditions of temperature and pH. Generally, the proportion of "phase transition" monomer in the polymer is at least about a 9:1 molar ratio of phase transition monomer to water-binding monomer. This ratio can be increased, for example, to a molar ratio of phase transition monomer to water-binding monomer of about 10:1, about 11:1, about 12:1, about 13:1, about 14:1, about 15:1, about 16:1, about 17:1, about 18:1, about 19:1, about 20:1, about 21:1, about 22:1, about 23:1, about 24:1, about 25:1, about 26:1, about 27:1, about 28:1, about 29:1, or about 30:1.

[0147] The viscosity of preferred hydrogels for use with the present invention at lower temperatures (e.g., 4°C) is such that the hydrogel is injectable. As the temperature is then increased, the hydrogel becomes more viscous, forming a gel with the desired viscosity at a temperature of about 37°C. This means that preferred hydrogels for use with the present invention can be easily administered to the repair site at cold temperatures, for example, by injection or aerosol administration. The hydrogel is then converted to a more viscous gel with the desired strength and elastic properties by warming within the body to the body's natural temperature.

[0148] Preferably, the polymer includes a fourth monomer in an amount that constitutes the remainder up to 100% of the polymer's composition. In one embodiment, the mol% of the fourth monomer can be up to about 85%, preferably about 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, or 85 mol%.

[0149] 1.5. Other Polymer Properties Those skilled in the art will understand that combining different types of monomers can produce polymers with a variety of different properties. Additionally, the properties of a polymer can be modified by incorporating specific monomers or functional groups into an existing polymer. For example, copolymerization of HEMA monomers with other monomers (e.g., methyl methacrylate) can be used to modify properties such as swelling and mechanical properties. Monomers can also be reacted with other compounds to form "macromonomers" (mentioned above), which are then included in preferred polymers used in the present invention. For example, HEMA can be reacted with lactide to form HEMA-polylactic acid polymer (HEMA-PLA), and HEMA-PLA itself can be used as a monomer in the polymers of the present invention. Additionally, the monomer itself can be a combination of monomer units, which are then incorporated into the polymer. An example of this type of monomer is oligo(ethylene glycol) monomethyl ether methacrylate (OEGMA), a hydrophilic monomer composed of two hydrophilic monomers: ethylene glycol and methacrylate.

[0150] The preferred polymers used in the present invention can be further modified with one or more moieties and / or functional groups. Any moiety or functional group can be used as needed. In some embodiments, the polymer can be modified with acyclic polyacetals derived from polyethylene glycol (PEG), carbohydrates, and / or polysaccharides. In addition, as mentioned above, hydrophilic groups can be incorporated into the monomers (and thus the polymer) to enhance the water-binding ability of the polymer.

[0151] In terms of arrangement, the copolymer can be a block copolymer, a graft copolymer, a random copolymer, a blend, a mixture, and / or an adduct of any of these and other polymers. Typically, the polymer used in accordance with the present invention is an organic polymer. Preferably, the polymer used in the present invention is biocompatible. In some embodiments, the polymer is biodegradable. In other embodiments, the polymer is both biocompatible and biodegradable.

[0152] Preferred polymers for use in the present invention may also include other monomers in their structure, for example, the monomers may be polymers such as poly(vinyl alcohol) (PVA), polyesters, acrylic polymers, and ionic polymers, or monomers thereof.

[0153] If it is desired that the polymer be biodegradable or absorbable, one or more monomers having biodegradable linkages can be used. Alternatively or additionally, the monomers can be selected so that the product of the reaction between the monomers is a biodegradable linkage. For each approach, the monomers and / or linkages can be selected so that the resulting biodegradable polymer will degrade or be absorbed in a desired period of time, e.g., from about 6 hours to about 6 months. Preferably, the monomers and / or linkages can be selected so that the resulting product is non-toxic when the polymer degrades under physiological conditions.

[0154] Biodegradable linkages can be chemically or enzymatically hydrolyzable or absorbable. Exemplary, non-limiting, chemically hydrolyzable biodegradable linkages that are useful for understanding include polymers, copolymers, and oligomers of glycolide, lactide, caprolactone, dioxanone, and trimethylene carbonate. Enzymatically hydrolyzable biodegradable linkages that are useful for understanding include peptide bonds cleavable by metalloproteinases and collagenases. Additional biodegradable linkages that are useful for understanding include polymers and copolymers of poly(hydroxyl acids), poly(orthocarbonates), poly(anhydrides), poly(lactones), poly(amino acids), poly(carbonates), and poly(phosphonates).

[0155] The chemical hydrolysis of lactide in the present invention results in an increase in the lower critical solution temperature (LCST) of the polymer (by decreasing the overall hydrophobicity of the polymer) and therefore its bioresorbability.

[0156] 1.6. Preferred Polymers The polymer preferably comprises the first monomer in an amount of about 3 to about 8 mol %, such as about 4 to about 6 mol %, or about 4, 5, 6 mol %.

[0157] The polymer preferably comprises the second monomer in an amount of about 5 to about 9 mol %, such as about 6 to about 8 mol %, or about 6, 7, or 8 mol %.

[0158] The polymer preferably comprises a third monomer in an amount of at least about 7 mol %, such as about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 mol %.

[0159] The polymer preferably includes a fourth monomer in an amount that constitutes the remainder up to 100% of the polymer's composition, for example, about 60 to about 81 mol %, for example, about 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, or 85 mol %.

[0160] The percentages given herein relate to the composition of the final polymer, not the amount of feed utilized in forming the polymer.

[0161] In one embodiment, the polymer preferably comprises: i. a first monomer in an amount of about 3 to about 8 mol % (e.g., about 4 to about 6 mol %); ii. a second monomer in an amount of about 5 to about 9 mol % (e.g., about 6 to about 8 mol %); iii. a third monomer in an amount of at least about 7 mol %, and iv. A fourth monomer in an amount of up to about 85 mol % (e.g., up to about 81 mol %).

[0162] In another embodiment, the polymer is preferably i. a first monomer in an amount of about 5 mol %; ii. a second monomer in an amount of about 7 mol %; iii. a third monomer in an amount of about 7 mol %, and iv. containing a fourth monomer in an amount of about 81 mol %.

[0163] In one embodiment, the preferred polymers used in the present invention are polymers of formula (I):

[0164] [ka] During the ceremony, A is the first monomer (water-binding monomer), B is a second monomer (a monomer capable of imparting mechanical properties to the hydrogel); C is a third monomer (a monomer having a functional group for binding to the NSPP), D is a fourth monomer (a monomer capable of imparting phase transition properties to the hydrogel); m is an integer from 1 to 20, for example, 1 to 19, 1 to 18, 1 to 17, 1 to 16, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2; n is an integer from 1 to 20, for example, 1 to 19, 1 to 18, 1 to 17, 1 to 16, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2; p is an integer from 1 to 20, for example, 1 to 19, 1 to 18, 1 to 17, 1 to 16, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2; q is an integer of 1 to 20, for example, 1 to 19, 1 to 18, 1 to 17, 1 to 16, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2.

[0165] Preferably, the ratio of m:n:p:q is about 5:8(5):7:81. Those skilled in the art will recognize that monomers A, B, C and D can be present in the polymer in any order, provided the required water binding, reinforcing and / or crosslinking capacity is achieved.

[0166] A, B, C and D may preferably be present in the mol % ranges provided above in the context of the first, second, third and fourth monomers, respectively.

[0167] An example of a polymer of formula (I) is a polymer of formula (Ia):

[0168] [ka] where A is the water-binding monomer OEGMA, B is the reinforcing monomer HEMA-PLA, C is the crosslinker NAS, D is the phase-transfer monomer NIPAAm, and m, n, p, q, x, and y are as defined above.

[0169] Those skilled in the art will know that monomers A, B, C and D can be present in the polymer in any order, provided the required water binding, strengthening and / or crosslinking capacity is achieved.

[0170] It has also been discovered that some monomers, such as HEMA-PLA, polyesters such as poly(lactic acid), poly(caprolactone), poly(glycolide), and their random copolymers (e.g., poly(glycolide-co-lactide) and poly(glycolide-co-caprolactone)), as well as other biodegradable and biocompatible polymers, can increase the LCST of preferred polymers used in the present invention during degradation of the biodegradable segment (e.g., PLA) in vivo, leading to bioabsorption of the polymer. This provides the additional advantage that the polymers used in the present invention can be designed to be biodegradable in vivo.

[0171] The overall size of preferred polymers for use in the present invention can vary depending on factors such as the type of monomer incorporated into the polymer, the type of NSPP to be used to form the hydrogel, and the conditions under which the protein binds to the polymer, but generally, preferred polymers for use in the present invention can be molecules of about 1 to about 100 kDa, about 5 to about 60 kDa, or about 30 kDa.

[0172] 1.7.PNPHO A preferred polymer is PHPHO. The polymer PHPHO preferably contains OEGMA in an amount of about 3 to about 8 mol %, for example about 4 to about 7 mol %, or about 3, 4, 5, 6, 7 or 8 mol %.

[0173] The polymer preferably contains HEMA-PLA in an amount of about 5 to about 9 mol%, e.g., about 6 to about 8 mol%, or about 3, 4, 5, 6, 7, or 8 mol%. The polymer preferably contains NAS in an amount of at least about 7 mol%, e.g., about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 mol%.

[0174] The polymer preferably contains NIPAAm in an amount that makes up the remainder up to 100% of the polymer's composition, for example, about 64 to about 85 mol %, for example, about 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, or 85 mol %.

[0175] The percentages given herein relate to the composition of the final polymer, not the amount of feed utilized in forming the polymer.

[0176] In one embodiment, preferably the polymer is i. OEGMA in an amount of about 3 to about 8 mol % (e.g., about 4 to about 6 mol %); ii. HEMA-PLA in an amount of about 5 to about 9 mol % (e.g., about 6 to about 8 mol %); iii. NAS in an amount of at least about 7 mol %, and iv. Contains NIPAAm in an amount of up to about 85 mol % (e.g., up to about 81 mol %).

[0177] In another embodiment, the polymer is i. OEGMA in an amount of about 5 mol %, ii. HEMA-PLA in an amount of about 7 mol %; iii. NAS in an amount of about 7 mol %, and iv. Containing NIPAAm in an amount of about 81 mol %.

[0178] A preferred form of polymer PNPHO for use in this application is the polymer of formula (Ia) depicted above.

[0179] Based on formula I defined previously, iA is oligo(ethylene) glycol monomethyl ether methacrylate OEGMA; ii.B is hydroxyethyl methacrylate poly(lactic acid) (HEMA-PLA); iii.C is N-acryloxysuccinimide (NAS), iv.D is N-isopropylacrylamide (NIPAAm).

[0180] Additionally, x is in the range of 1-1000, y is in the range of 1-1000, and m, n, p, and q are in the range of 1-20.

[0181] Those skilled in the art will recognize that monomers A, B, C and D can be present in the polymer in any order, provided the required water binding, strengthening and / or crosslinking capacity is achieved.

[0182] 1.8. Polymer Synthesis Those skilled in the art will be aware of suitable methods for synthesizing the preferred polymers used in the present invention, including methods such as ring-opening polymerization, addition polymerization (including free radical polymerization), and condensation polymerization.

[0183] The formation of the preferred polymer PNPHO is described in the Examples below.

[0184] 2. Composition for forming hydrogels The present invention also relates to preferred compositions useful in forming hydrogels for use in the present invention.

[0185] The composition of the present invention comprises a polymer and an NSPP, wherein the polymer is i. a first water-binding monomer, and ii. a second monomer that imparts mechanical properties; iii. a third monomer that is an NSPP-binding monomer, comprising a functional group capable of binding to an NSPP; iv. a fourth monomer capable of imparting phase transition properties to the hydrogel; the natural or synthetic peptide or protein (NSPP) is thymosin beta-4 or a functional homolog thereof; The attachment of the NSPP to the second monomer crosslinks the polymer, which allows for the formation of a hydrogel when the composition is contacted with water.

[0186] The term "composition," as used herein, refers to a solid or liquid composition containing the above-mentioned components. In some embodiments, other components, such as pharmaceutically acceptable excipients and bioactive agents (e.g., drugs, vitamins, and minerals), for assisting in the repair and / or regeneration of target bone tissue and / or for providing a method for achieving targeted delivery of bioactive compounds, may also be included in the preferred compositions used in the present invention.

[0187] Generally, the amount of polymer in the compositions used in the present invention is that amount that allows for the formation of a hydrogel.

[0188] In some embodiments, the amount of polymer in the composition ranges from about 1% w / w to about 90% w / w, from about 2% w / w to about 80% w / w, from about 4% w / w to about 70% w / w, from about 5% w / w to about 60% w / w, from about 5% w / w to about 50% w / w, from about 6% w / w to about 40% w / w, from about 7% w / w to about 30% w / w, or from about 8% w / w to about 20% w / w.

[0189] In some embodiments, the amount of polymer is about 1% w / w, about 2% w / w, about 3% w / w, about 4% w / w, about 5% w / w, about 6% w / w, about 7% w / w, about 8% w / w, about 9% w / w, about 10% w / w, about 15% w / w, about 20% w / w, about 25% w / w, about 30% w / w, about 35% w / w, about 40% w / w, about 45% w / w, about 50% w / w, about 55% w / w, about 60% w / w, about 65% w / w, about 70% w / w, about 75% w / w, about 80% w / w or more. In some embodiments, the amount of polymer is approximately 85% w / w.

[0190] Generally, the stiffness of the hydrogel increases with increasing polymer concentration in the composition.

[0191] Generally, the amount of NSPP in the compositions of the present invention is that amount that allows for the formation of a hydrogel.

[0192] In some embodiments, the amount of NSPP in the composition ranges from about 0.01% w / w to about 60% w / w, from about 1% w / w to about 50% w / w, from about 1% w / w to about 40% w / w, from about 5% w / w to about 30% w / w, from about 5% w / w to about 20% w / w, or from about 5% w / w to about 10% w / w.

[0193] In some embodiments, the percentage of NSPP is about 1% w / w, about 2% w / w, about 3% w / w, about 4% w / w, about 5% w / w, about 6% w / w, about 7% w / w, about 8% w / w, about 9% w / w, about 10% w / w, about 20% w / w, about 30% w / w, about 40% w / w, about 50% w / w, or more.

[0194] The % w / w is based on the total weight of the composition before it is contacted with water.

[0195] In one embodiment, the composition comprises equimolar amounts of the polymer and thymosin beta-4 or a functional homologue thereof.

[0196] 2.1. Excipients and Bioactive Agents Pharmaceutically acceptable excipients may be included in the preferred compositions and / or hydrogels used in the present invention and include any and all solvents, dispersion media, inert diluents, or other liquid vehicles, dispersing or suspending aids, granulating agents, surfactants, disintegrants, isotonicity agents, thickening or emulsifying agents, preservatives, binders, lubricants, buffers, oils, etc., appropriate for the particular dosage form desired. Remington (Gennaro, AR, Remington: The Science and Practice of Pharmacy, 21st Edition (2006) Lippincott Williams & Wilkins) discloses various excipients used in formulating pharmaceutical compositions and known techniques for their preparation. Unless any conventional excipient is incompatible with the substance or its derivatives, such as by producing any undesirable biological effects or otherwise interacting in a deleterious manner with any other component of the pharmaceutical composition, its use is contemplated within the scope of this invention.

[0197] Excipients such as coloring agents, coating agents, sweetening, flavoring, and perfuming agents can be present in the composition, according to the discretion of the formulator.

[0198] Bioactive agents or drug compounds that may be added to preferred compositions and / or hydrogels used in the present invention include proteins, glycosaminoglycans, carbohydrates, nucleic acids, and inorganic and organic bioactive compounds, such as enzymes, antibiotics, antitumor agents, local anesthetics, hormones, angiogenic agents, angiogenesis inhibitors, growth factors (e.g., insulin-like growth factor-1 (IGF-1), basic fibroblast growth factor (bFGF), and transforming growth factor-b (TGFb), antibodies, neurotransmitters, psychotropic drugs, anticancer drugs, chemotherapeutic drugs, drugs affecting the reproductive organs, genes, and oligonucleotides.

[0199] Compositions containing ingredients such as excipients and / or bioactive agents can be produced by combining a preferred polymer as disclosed herein with an NSPP, drying the resulting composition, and then combining it with one or more other ingredients. The resulting composition may be in the form of a powder or other particulate form, to which water is then added in accordance with the present invention to form a hydrogel. Thus, hydrogels containing these ingredients can be produced simply by adding the desired aqueous solvent to the composition.

[0200] The amounts of polymer, NSPP, and bioactive agent present in preferred compositions used in the present invention will necessarily depend on the particular drug and condition being treated. One of ordinary skill in the art will know the appropriate agents and amounts to use to treat a condition.

[0201] Exemplary embodiments include fat grafts, demineralized bone matrix (DBM), and autografts (i.e., bioactive grafts). The role of this type of additive is to impart tissue-inductive properties to the composite. For example, low concentrations of growth factors are present in DBM / autografts, etc.

[0202] Further exemplary embodiments include purely inert space-filling agents such as bone particles (human or animal derived) and glass beads. The role of this type of additive is to provide the necessary 3D structure to the composite.

[0203] Those skilled in the art will recognize that bone graft substitute refers to a wide range of particles, including but not limited to synthetic calcium / phosphate particles, animal-derived bone particles, and unprocessed human bone.

[0204] 2.2. Natural or Synthetic Peptides or Proteins (NSPPs) In the context of the present invention, NSPP is appropriate because it crosslinks the polymer, as described above, thereby allowing the polymer to form a hydrogel. A preferred hydrogel for use in the present invention can be formed, for example, by exposing thymosin beta-4 to a polymer of formula (I). NSPP is also important because it provides additional mechanical properties (such as strength and resilience) to the hydrogel and also provides an environment at the repair site that mimics the natural environment, thereby aiding in tissue repair and regeneration.

[0205] It is important that the NSPP contain a side chain or other functional group that is exposed to allow reaction with a functional group on the NSPP-binding monomer, thereby attaching the NSPP to the polymer via the NSPP-binding monomer. Examples of suitable side chains include glutamic acid or lysyl side chains.

[0206] The present invention also contemplates the use of variants of NSPP, such as species variants or polymorphic variants. The present invention is intended to encompass all functionally active variants of NSPP that exhibit the same activity. This includes apo and halo forms of NSPP, post-translationally modified forms, and glycosylated or deglycosylated derivatives. Such functionally active fragments and variants include, for example, those with conservative amino acid substitutions.

[0207] Preferably, the NSPPs used in the present invention are obtained from recombinant sources, but can also be extracted from natural sources or synthesized.

[0208] 2.3 Thymosin beta-4 A preferred NSPP is thymosin beta-4. Thymosin beta-4 is a highly conserved, naturally occurring, water-soluble regenerative peptide found in all tissues and all cell types except red blood cells. It is also found in blood and other body fluids, including tears, saliva, cerebrospinal fluid, and wound fluid.

[0209] Human thymosin beta-4 has the following sequence: SDKPDMAEIE KFDKSKLKKT ETQEKNPLPS KETIEQEKQA GES.

[0210] Thymosin beta-4 is alternatively written Ser-Asp-Lys-Pro-Asp-Met-Ala-Glu-Ile-Glu-Lys-Phe-Asp-Lys-Ser-Lys-Leu-Lys-Lys-Thr-Glu-Thr-Gln-Glu-Lys-Asn-Pro-Leu-Pro-Ser-Lys-Glu-Thr-Ile-Glu-Gln-Glu-Lys-Gln-Ala-Gly-Glu-Ser.

[0211] For the purposes of further discussion, the terms thymosin beta-4 and TB4 will be used interchangeably, with TB4 being shorthand for thymosin beta-4.

[0212] According to an exemplary embodiment of the present invention, thymosin beta-4 is most preferably used with PHPHO in a molar ratio of approximately 1:1. However, this may be varied depending on the intended use. In an exemplary composition, 140 mg / mL of PNPHO (ratio 5:8 (5:7:81)) was used with 30 mg / mL of thymosin beta-4, and this composition was labeled "PNPHO-co-TB4" for the applicant's clinical trials.

[0213] 2.4 Functional homologs (isoforms) of thymosin beta-4 Another preferred NSPP is the functional homolog of thymosin beta-4.The functional homolog of the above polypeptide is also suitable for use in the compositions and methods described herein.Functional homolog is a polypeptide that has sequence similarity with reference polypeptide and performs one or more of the biochemical or physiological functions of reference polypeptide.

[0214] Functional homologue and reference polypeptide can be naturally occurring polypeptides, and sequence similarity can be due to convergent or divergent evolution events.Therefore, functional homologue can be referred to in literature as homologue, or orthologue, or paralogue.Variants of naturally occurring functional homologues, such as the polypeptides encoded by mutants of wild-type coding sequences, can themselves be functional homologues.

[0215] Functional homologs can also be created by site-directed mutagenesis of the coding sequence of a polypeptide or by combining domains from coding sequences of different naturally occurring polypeptides ("domain swapping"). Techniques for modifying genes encoding the functional polypeptides described herein are known and include, among others, directed evolution, site-directed mutagenesis, and random mutagenesis techniques, which may be useful to increase a specific activity of a polypeptide, alter substrate specificity, alter expression levels, alter subcellular location, or modify polypeptide:polypeptide interactions in a desired manner. Such modified polypeptides are considered functional homologs. The term "functional homolog" is sometimes applied to nucleic acids encoding functionally homologous polypeptides.

[0216] Functional homologs can be identified by analyzing nucleotide and polypeptide sequence alignments. For example, polypeptide homologs can be identified by querying a database of nucleotide or polypeptide sequences. Sequence analysis can include BLAST, Reciprocal BLAST, or PSI-BLAST analysis of non-redundant databases using an amino acid sequence as a reference sequence. The amino acid sequence is inferred from the nucleotide sequence in some cases. Those polypeptides in the database with sequence identity greater than 40% are candidates for further evaluation of their suitability as polypeptides. Amino acid sequence similarity allows for conservative amino acid substitutions, such as substituting one hydrophobic residue for another or one polar residue for another. If necessary, manual inspection of such candidates can be performed to narrow the number of candidates to be further evaluated. Manual inspection can be performed by selecting those candidates that appear to have domains present in the polypeptide, such as conserved functional domains.

[0217] Conserved regions can be identified by locating regions within the primary amino acid sequence of a polypeptide that are repetitive, form some secondary structure (e.g., helices and beta sheets), establish positively or negatively charged domains, or represent protein motifs or domains; see, for example, the Pfam website, which lists consensus sequences for various protein motifs and domains, at www.sanger.ac.uk / Software / Pfam / and pfam.janelia.org / . Information contained in the Pfam database is described in Sonnhammer et al., Nucl. Acids Res., 26:320-322 (1998); Sonnhammer et al., Proteins, 28:405-420 (1997); and Bateman et al., Nucl. Acids Res., 27:260-262 (1999). Conserved regions can also be determined by aligning sequences of the same or related polypeptides from closely related species. The closely related species are preferably from the same family. In some embodiments, alignment of sequences from two different species is appropriate.

[0218] Typically, polypeptides that exhibit at least about 40% amino acid sequence identity are useful for identifying conserved regions. Conserved regions of related polypeptides exhibit at least 45% amino acid sequence identity (e.g., at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% amino acid sequence identity). In some embodiments, conserved regions exhibit at least 92%, 94%, 96%, 98%, or 99% amino acid sequence identity.

[0219] 3. Hydrogels The present invention also provides a hydrogel comprising a polymer according to the present invention, an NSPP and water, wherein the polymer is i. a first water-binding monomer; ii. a second monomer that imparts mechanical properties; iii. a third monomer that is an NSPP-binding monomer, comprising a functional group capable of binding to an NSPP; iv. a fourth monomer capable of imparting phase transition properties to the hydrogel; the natural or synthetic peptide or protein (NSPP) is thymosin beta-4 or a functional homolog thereof; The attachment of the NSPP to a third monomer crosslinks the polymer, thereby forming a hydrogel in which water is contained.

[0220] In one embodiment, the hydrogel comprises a polymer having the above-mentioned monomers to enable the hydrogel to undergo a phase transition from a liquid state at low temperatures to a gel state at body temperature. One example of a useful monomer for this purpose is NIPAAm. By using this monomer, thymosin beta-4 can transition from a liquid state to a gel state according to a temperature profile. Therefore, an advantage of the preferred hydrogel used in the present invention is that it can be easily administered at a cold temperature, for example, by injection or aerosol. After the hydrogel warms up to natural body temperature in the body, it is transformed into a more viscous gel with the desired strength and elastic properties.

[0221] Given the above polymer compositions, hydrogels can be formed by adding water to the composition by any method known to those skilled in the art. Indeed, one advantage of the present invention is that the polymer does not need to be crosslinked in any way prior to contact with the NSPP in order for the hydrogel to form.

[0222] 3.1.Cells Preferred hydrogels for use in the present invention may also include cells that assist in the repair and / or regeneration of target tissue.

[0223] Generally, the cells used in accordance with the present invention can be any type of cell. These cells must be viable when encapsulated or immobilized within the preferred hydrogels used in the present invention. The products of the present invention are effective for immobilizing cells. For example, a layer of hydrogel can be injected / sprayed into the site, after which cells can be added to the adhesive hydrogel and then covered with another layer of hydrogel to achieve the desired results. Thus, encapsulation occurs within the body, not outside the body.

[0224] In some embodiments, cells that may be encapsulated within the hydrogel include, but are not limited to, mammalian cells (e.g., human cells, primate cells, mammalian cells, rodent cells, etc.), avian cells, fish cells, insect cells, plant cells, fungal cells, bacterial cells, and hybrid cells. In some embodiments, exemplary cells that may be encapsulated within the hydrogel include stem cells, totipotent cells, pluripotent cells, and / or embryonic stem cells.

[0225] In some embodiments, exemplary cells that may be encapsulated / immobilized within the hydrogel include, but are not limited to, primary cells and / or cell lines derived from any tissue, such as cardiomyocytes, myocytes, hepatocytes, keratinocytes, melanocytes, neurons, astrocytes, embryonic stem cells, adult stem cells, hematopoietic stem cells, hematopoietic cells (e.g., monocytes, neutrophils, macrophages, etc.), ameloblasts, fibroblasts, chondrocytes, osteoblasts, osteoclasts, nerve cells, sperm cells, egg cells, liver cells, lung epithelial cells, gastrointestinal epithelial cells, intestinal epithelial cells, liver, skin epithelial cells, and / or hybrids thereof, may be encapsulated within preferred hydrogels used in accordance with the present invention.

[0226] Exemplary mammalian cells that may be encapsulated within preferred hydrogels used in accordance with the present invention include, but are not limited to, Chinese hamster ovary (CHO) cells, HeLa cells, Madin-Darby canine kidney (MOCK) cells, baby hamster kidney (BHK) cells, NSO cells, MCF-7 cells, MDA-MB-438 cells, U87 cells, A172 cells, HL60 cells, A549 cells, SP10 cells, DOX cells, DG44 cells, HEK293 cells, SHSY5Y, Jurkat cells, BCP-1 cells, COS cells, Vero cells, GH3 cells, 9L cells, 3T3 cells, MC3T3 cells, C3H-10T1 / 2 cells, NIH-3T3 cells, and C6 / 36 cells.

[0227] In some embodiments, it is desirable for the cells to be uniformly distributed throughout the hydrogel. A uniform distribution can help provide a more uniform tissue-like hydrogel that provides a more uniform environment for the encapsulated cells. In some embodiments, the cells are disposed on the surface of the hydrogel. In some embodiments, the cells are disposed within the hydrogel. In some embodiments, the cells are layered within the hydrogel. In some embodiments, the hydrogel contains different cell types.

[0228] In some embodiments, the conditions under which cells are encapsulated within the hydrogel are altered to maximize cell viability. For example, lower polymer concentrations increase cell viability in some embodiments. In some embodiments, cells positioned at the periphery of the hydrogel tend to have reduced viability relative to cells fully encapsulated within the hydrogel. In some embodiments, the conditions of the surrounding environment (e.g., pH, ionic strength, nutrient availability, temperature, oxygen availability, osmolarity, etc.) may need to be adjusted and / or altered to maximize cell viability.

[0229] In some embodiments, cell viability can be measured by monitoring one of many indicators of cell viability. In some embodiments, indicators of cell viability include, but are not limited to, intracellular esterase activity, cell membrane integrity, metabolic activity, gene expression, and protein expression. By way of example only, when cells are exposed to a fluorogenic esterase substrate (e.g., calcein AM), live cells fluoresce green as a result of intracellular esterase activity that hydrolyzes the esterase substrate to a green fluorescent product. In another example, when cells are exposed to a fluorescent nucleic acid dye (e.g., ethidium homodimer-1), dead cells fluoresce red because their cell membranes are compromised and permeable to the high-affinity nucleic acid dye.

[0230] Generally, the number / amount of cells in the composition is an amount that allows for the formation of a preferred hydrogel for use in accordance with the present invention. In some embodiments, the amount of cells suitable for forming a hydrogel ranges from about 0.1% w / w to about 80% w / w, about 1.0% w / w to about 50% w / w, about 1.0% w / w to about 40% w / w, about 1.0% w / w to about 30% w / w, about 1.0% w / w to about 20% w / w, about 1.0% w / w to about 10% w / w, about 5.0% w / w to about 20% w / w, or about 5.0% w / w to about 10% w / w. In some embodiments, the number / amount of cells in a composition suitable for forming a hydrogel is approximately 5% w / w.

[0231] In some embodiments, the concentration of cells in the precursor solution suitable for forming a hydrogel is about 10 to about 1 x 10 8 cells / mL, approximately 100 to approximately 1×10 7 cells / mL, approximately 1×10 3 ~Approx. 1×10 6 cells / mL, or approximately 1 x 10 4 ~Approx. 1×10 5In some embodiments, a single hydrogel comprises a population of identical cells and / or cell types. In some embodiments, a single hydrogel comprises a population of non-identical cells and / or cell types. In some embodiments, a single hydrogel may comprise at least two different types of cells.

[0232] In some embodiments, a single hydrogel may contain three, four, five, ten, or more types of cells. By way of example only, in some embodiments, a single hydrogel may contain only embryonic stem cells. In some embodiments, a single hydrogel may contain both embryonic stem cells and hematopoietic stem cells.

[0233] 3.2. Culture medium Cells can be grown and / or maintained using any of a variety of cell culture media, including complex media and / or serum-free culture media, capable of supporting the growth of one or more cell types or cell lines. Typically, cell culture media contain buffers, salts, energy sources, amino acids (e.g., natural amino acids, unnatural amino acids, etc.), vitamins, and / or trace elements. Cell culture media may optionally contain a variety of other components, including, but not limited to, carbon sources (e.g., natural sugars, unnatural sugars, etc.), cofactors, lipids, sugars, nucleosides, animal-derived components, hydrolysates, hormones, growth factors, surfactants, indicators, minerals, activators of specific enzymes, inhibitors of activators of specific enzymes, enzymes, organic substances, and / or small molecule metabolites.

[0234] Cell culture media suitable for use in accordance with the present invention are commercially available from a variety of sources, such as ATCC, Manassas, Va. In certain embodiments, cells are grown using one or more of the following media: RPMI-1640 medium, Dulbecco's Modified Eagle's Medium, Eagle's Minimum Essential Medium, F-12K medium, Iscove's Modified Dulbecco's Medium.

[0235] Those skilled in the art will recognize that the cells listed herein represent an exemplary, non-exhaustive list of cells that can be encapsulated within precursor solutions (and thus ultimately within hydrogels) in accordance with the present invention.

[0236] 4.Applications The present invention aims to provide a filler that supports the natural healing of damaged tissue without inducing any specific tissue formation. The present invention can be used to partially or completely fill tissue cavities or cover tissue defects to provide the necessary space filling with minimal foreign body reaction.

[0237] Preferably, the compositions of the invention are administered to a subject (eg, a mammal) by injection or by spraying particles embodying the invention, such as by aerosol.

[0238] Uniquely, the compositions of the present invention can fill defects partially or completely, regardless of the shape and / or depth / size of the defect, and can be added in layers to build up the required volume. The compositions of the present invention adhere to the site without the need for physical confinement, mix with blood in situ, and can be injected into deep tissues with a fine gauge needle. Tissue gaps can be refilled with the compositions of the present invention at different time intervals as needed.

[0239] The present invention provides an injectable filler. After injection into the body, the composition forms an adhesive hydrogel. Due to the unique properties of the present invention, the composition of the present invention is well tolerated within the body with minimal inflammatory response. The product is host tissue conductive, but not inductive, as it only exhibits regenerative properties in the presence of active bleeding or other fluids containing regenerative biological components.

[0240] The compositions of the present invention can be injected through a fine gauge needle, and can be injected in a manner that adheres to the injection site and creates a 3D structure layered within the body in a minimally invasive manner.

[0241] Tissue types for which this application is useful include skin tissue and periodontal tissue.

[0242] Uses of the compositions of the present invention include: a) an injectable dermal filler for filling skin cavities for temporary reduction of skin wrinkles (cosmetic use); b) an injectable dermal filler for temporary lifting of scar bases and promoting healing (therapeutic use); c) an injectable dermal filler for supporting dermal connective tissue formation in scar tissue after surgical intervention and promoting healing (therapeutic use); d) an injectable dermal filler for supporting dermal connective tissue formation in post-burn scar management (cosmetic / therapeutic use); e) a ready-to-use dermal matrix for supporting vascular ingrowth in acute skin defects with bleeding and promoting healing (therapeutic use); f) a ready-to-use dermal matrix for filling surgically created skin cavities (therapeutic use); g) a ready-to-use dermal matrix for supporting skin graft surgery (cosmetic and therapeutic use); h) a carrier system for the physical delivery of bone graft substitutes (therapeutic use; see, e.g., Expert Rev Med Devices., January 2006;3(1):49-57); i) a prosthesis (e.g., a cage) ex vivo and / or in vivo j) a filler without tissue inductive properties; k) a ready-to-use matrix for supporting and repairing periodontal tissue after tooth extraction (therapeutic use); l) a ready-to-use matrix for supporting periodontal ligament tissue grafts (therapeutic use); and m) an injectable matrix for temporary lifting of periodontal ligament tissue (therapeutic use).

[0243] Advantages of preferred embodiments of the present invention include: a) cell affinity, b) injectability, c) host tissue adhesion, d) lack of tissue-specific induction properties, and e) being well tolerated in the body (minimal immune response).

[0244] The hydrogels of the present invention preferably result in a minimal foreign body reaction upon injection into the body of an animal.

[0245] The hydrogels of the present invention are preferably host tissue conductive, meaning that they exhibit regenerative properties in the presence of active bleeding or other fluids containing regenerative biological components. The hydrogels of the present invention are preferably miscible with blood.

[0246] The compositions of the present invention are preferably injectable, preferably capable of multiple injections at the same site, and preferably form a hydrogel in situ after administration to a mammal by injection.

[0247] The hydrogels of the present invention preferably exhibit good adhesion to host tissue. The adhesive properties allow the underlying tissue bed to be gradually laid down to support healing. The adhesive properties also allow the formation of 3D structures in a minimally invasive manner.

[0248] The hydrogels of the present invention can preferably be used for layered filling, which allows filling of 3D cavities with different heights.

[0249] The hydrogels of the present invention preferably degrade in situ over a period of days, weeks or months, leaving healthy tissue behind.

[0250] The present invention has been developed as a safe and easy-to-use biomaterial for use as an injectable scaffold. The present invention is based on a single, homogeneous molecule consisting of a synthetic smart polymer (PNPHO) conjugated with thymosin beta-4. The composition of the present invention is liquid at room temperature, allowing for direct injection into the desired clinical location. The composition of the present invention forms an elastic gel after exposure to body temperature, mixes with blood, and stabilizes the clot at the site. Based on in vitro and in vivo studies, the absorption rate of the composition of the present invention is expected to be absorbed by the body in less than three months. Furthermore, sheep osteotomy models and cellular studies of osteoblasts and preosteoblasts have confirmed that the device supports vascular ingrowth and bone formation.

[0251] It was hypothesized that the composition of the present invention could be injected into the base of the tooth extraction socket after tooth extraction, mix with blood, stabilize the blood clot, and provide a uniform scaffold for vascular ingrowth and bone regeneration. Direct administration of the composition of the present invention into the tooth extraction socket was intended to enhance wound healing at the site and preserve alveolar bone after tooth extraction. To investigate this hypothesis, the applicant conducted a clinical trial involving 10 participants scheduled for tooth extraction. Soft tissue closure (wound healing), the appearance of the tooth extraction site, the participants' well-being, and the quality of the underlying bone were studied in this clinical trial.

[0252] The uses of the compositions of the present invention and the existence of supporting data for these potential uses of this technology are summarized in Table 1.

[0253] [Table 1-1] [Table 1-2]

[0254] 5. Kit Various kits containing one or more of the preferred hydrogels for use in the present invention are disclosed herein. For example, the present invention provides a kit containing a hydrogel and instructions for use in repairing or regenerating bone defects. The kit can include multiple different hydrogels.

[0255] The kit may optionally include polymers, cells, NSPPs, bioactive compounds, water, etc. The kit may include any of several additional components or reagents in any combination. Although not all of the various combinations are explicitly set forth, each combination is within the scope of the present invention. Some exemplary kits provided in accordance with the present invention are described in the following paragraphs.

[0256] According to certain embodiments of the present invention, the kit comprises, for example: i. a solution containing a polymer, a solution containing an NSPP, and ii. Instructions for forming a hydrogel from the solution may include:

[0257] According to another embodiment, the kit comprises, e.g. i. a composition comprising a polymer and an NSPP, and ii. Instructions for forming a hydrogel from the composition. may include:

[0258] According to another embodiment, the kit comprises, e.g. i. a composition comprising a polymer and an NSPP, one or both of which are in solid form, optionally with a solvent such as water; and ii. Instructions for forming a hydrogel from the composition. may include:

[0259] Kits may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes. Exemplary kits can be in the form of an aerosol or a form having components that can be combined to form an aerosol or similar means for applying the compositions of the invention.

[0260] The kits typically include instructions for use of the preferred hydrogels for use in the present invention. The instructions may include, for example, protocols and / or may describe conditions for generating the hydrogel, administering the hydrogel to a subject in need thereof, generating a hydrogel assembly, etc. The kits generally include one or more vessels or containers such that some or all of the individual components and reagents may be housed separately. The kits may also include a means for enclosing the individual containers in relatively tight confinements for commercial sale, such as a plastic box, into which the instructions, packaging material such as Styrofoam, etc. may be placed.

[0261] The kit or "article of manufacture" may include a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, blister packs, etc. The container may be formed from a variety of materials, such as glass or plastic. The container holds a hydrogel or composition that is effective in treating a condition and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle). The label or package insert indicates that the hydrogel or composition is used to treat the condition of choice. In one embodiment, the label or package insert includes instructions for use and indicates that the therapeutic composition can be used to repair or regenerate tissue.

[0262] Preferred Embodiments of the Invention The preferred polymer used in the present invention is PNPHO. The concentration of PNPHO in the composition of the present invention is preferably about 100 to about 300 mg / mL of the composition. When embodied in aerosol form, the concentration is about 5 mg / mL to about 70 mg / mL of the composition.

[0263] A preferred NSPP for use in the present invention is thymosin beta-4. Alternatively, a functional homologue of thymosin beta-4 may be used.

[0264] Preferably, PNPHO is conjugated to thymosin beta-4 (or a functional homologue thereof), and both the protein / peptide segment and PNPHO have defined roles.

[0265] Thymosin beta-4 (or its functional homologue) i. Serve as a source of bioactive signaling for tissue regeneration; ii. Promotes the formation of blood vessels in and around the hydrogel filler.

[0266] PNPHO polymers are chemically conjugated to proteins / peptides, i. tailoring the physicochemical properties of this biopolymer for tissue applications; ii. imparting rapid thermal cure to the hydrogel filler and securing it locally; iii. Imparting bioresorbable properties to injectable hydrogels.

[0267] The combination of these two main segments results in the formation of a new type of superior tissue filler with a variety of favorable properties for tissue regeneration and repair. An advantage of PNPHO polymers is that all of their components are approved by the US FDA for use in biomedical applications.

[0268] PNPHO polymers contain lactide, ethylene glycol, and N-acryloxysuccinimide segments, which impart high mechanical strength, water solubility, and amine group reactivity to the product, along with a thermoresponsive segment (N-isopropylacrylamide) that induces hydrogel formation at body temperature. The molecular structure of PNPHO polymers and the role of each segment are illustrated in the scheme below in the Examples.

[0269] Preferably, equimolar amounts of PNPHO and thymosin beta-4 are used, although those skilled in the art will appreciate that the molar ratio may be varied according to each scenario encountered in practice. [Example]

[0270] material Unless otherwise stated, chemicals were purchased from Sigma-Aldrich. Stannous 2-ethylhexanoate (Sn(Oct)), N-isopropylacrylamide (NIPAAm), 2-hydroxyethyl methacrylate (HEMA), 4,4'-azobis(4-cyanovaleric acid) (ACVA), and N-acryloxysuccinimide (NAS) were used as received. Oligo(ethylene glycol) monomethyl ether methacrylate (OEGMA, M nThe HCl (H O ) was purified by passing its solution in dichloromethane (1:1 volume ratio) through a neutral alumina column to remove inhibitors before use. D,L-Lactide (LA) monomer was dried under vacuum at 40 °C for 24 h before use. Azobisisobutyronitrile (AIBN) was a kind gift from the Department of Chemistry, University of Sydney, Australia.

[0271] Synthesis of HEMA-poly(lactide) (HEMA-PLA) macromonomer HEMA-PLA macromonomer was synthesized by ring-opening polymerization of LA using the hydroxyl group of HEMA as an initiator and Sn(Oct)2 as a catalyst (see Scheme 1).

[0272] [ka] Scheme 1. Synthesis of HEMA-poly(lactide) (HEMA-PLA) macromonomer

[0273] LA and HEMA were mixed in a three-neck flask under a nitrogen atmosphere at 110°C for 15 minutes. Then, a mixture of 1 mol% Sn(Oct)2 (relative to the HEMA feed amount) in 1 ml of anhydrous toluene was added to the LA / HEMA solution. The resulting mixture was stirred at 300 rpm at 110°C for 1 hour under a nitrogen atmosphere. After the reaction, the mixture was dissolved in tetrahydrofuran and precipitated in cold distilled water at 1°C. The formed precipitate was separated by centrifugation at 3000 rpm for 5 minutes.

[0274] The centrifugation cycle was repeated three times to remove all unreacted monomers and by-products (mainly salts). The precipitate was then dissolved in ethyl acetate. The suspended solid particles were removed from this solution using centrifugation at 6000 rpm for 5 minutes, and the supernatant was dried over MgSO4 for 12 hours. The dried supernatant was filtered to remove the MgSO4 particles. The polymer solution was then dried under reduced pressure at 60°C, and the solvent residue was further removed under vacuum at 40°C for 24 hours. The resulting viscous oil was stored in a refrigerator for further use.

[0275] The HEMA:LA feed ratio was varied from 1:1.5 to 1:2.5 to obtain PLA / HEMA macromonomers with different lactate lengths. Two PLA / HEMA macromonomers with lactate lengths of 3 and 6 were synthesized by using a molar ratio of HEMA to LA monomer of 1:1.5 and 1:2.5, respectively.

[0276] 1 1 H NMR spectra were used to confirm the synthesis of PLA / HEMA macromonomers based on the proton peaks from both HEMA and LA. 1 From the 1 H NMR spectrum, the molar ratio of LA to HEMA in the PLA / HEMA macromonomer was calculated using the total integral of the peak at 5.2 ppm for methine in lactate and the peaks at 5.7 ppm and 6.0 ppm for HEMA.

[0277] Synthesis of poly(NIPAAm-co-NAS-co-(HEMA-PLA)-co-OEGMA) (PNPHO) PNPHO was synthesized using either method (1) or (2) described below (see Scheme 2).

[0278] [ka] Scheme 2. Synthesis of poly(NIPAAm-co-NAS-co-(HEMA-PLA)-co-OEGMA) (PNPHO)

[0279] Method 1 PNPHO was synthesized by free-radical polymerization using AIBN as the initiator. A Schlenk flask equipped with a magnetic stir bar and a rubber septum was charged with NIPAAm (12 mmol), NAS (1.0 mmol), HEMA-PLA (0.57 mmol), OEGMA (0.56 mmol), AIBN (0.07 mmol), and anhydrous N,N'-dimethylformamide (DMF). The flask was deoxygenated by three freeze-pump-thaw cycles, then sealed and immersed in a preheated oil bath at 70 °C to initiate polymerization. After 24 h, the reaction mixture was cooled to room temperature, precipitated in diethyl ether, filtered, and then dried under vacuum. The polymer was purified twice by redissolution / reprecipitation with THF / ethyl ether and finally dried under vacuum for 2 days.

[0280] Method 2 PNPHO was synthesized by free radical polymerization using ACVA as an initiator. The copolymer composition was varied by varying the lactate length (3 and 6 in HEMA-PLA) and the molar ratios of HEMA-PLA (6, 8, and 11 mol%) and OEGMA (3, 5, and 8 mol%). In a single-neck round-bottom flask, known amounts of NIPAAm, NAS, HEMA-PLA, OEGMA, and ACVA (7.0 × 10 -5 (mol) was dissolved in 13 ml of anhydrous N,N'-dimethylformamide. The system was then deoxygenated by purging with nitrogen for 15 minutes. The results also showed that it was feasible to deoxygenate the monomer solution by purging with nitrogen gas for 10 minutes under vacuum. This technique provides a more efficient method for removing oxygen from solutions on a large scale.

[0281] The reactor was then sealed and immersed in an oil bath at 70°C for 24 hours. The resulting polymer solution was then cooled to room temperature for 1 hour and precipitated in 250 ml of diethyl ether. The precipitate was then collected by filtering the suspension and dried under vacuum for 6 hours. The dried powder was dissolved in tetrahydrofuran and precipitated in diethyl ether to further remove macromonomer residues. The final powder was dried under vacuum for at least 48 hours.

[0282] [Table 2]

[0283] PNPHO composition PNPHO was synthesized based on the scheme shown in Figure 2a. As shown in Figure 2b, 1The synthesis of PNPHO copolymers was confirmed based on the proton peaks for each monomer in the H NMR spectrum. Characteristic proton peaks for NIPAAm (a and b), NAS (e), HEMA-PLA (f, h, k), and OEGMA (m and n) were detected. Regarding the NAS proton resonances (e), these overlap with resonances at approximately 2.9–3.0 ppm for the DMF solvent residue trace, leading to erroneous initial calculations based on the same data set (integral / area under each peak based on the relative mole percent of each monomer within the overall PNPHO polymer). Subsequent corrections confirmed the presence of a third monomer (NAS) in an amount greater than approximately 7 mole percent. The final composition of the copolymer was calculated based on the integrals of these peaks from each monomer for NIPAAm (a), NAS (e / 2-f), HEMA-PLA (h), and OEGMA (n / 2). In this study, copolymers are designated as PNPHO, with an additional subscript corresponding to the molar ratio of HEMA-PLA (lactate length) to OEGMA. For example, PNPHO5:8(5):7:81 represents a copolymer synthesized with 8 mol% HEMA-PLA with a lactate length of 5, and 5 mol% OEGMA. A variety of copolymers were produced; these are listed in Table 2, which also provides data on their gelation times and temperatures.

[0284] Solubility of PNPHO in PBS For the development of injectable formulations, the monomer ratio of PNPHO was modified to obtain a range of compositions that would be soluble in aqueous media such as PBS. NIPAAm-based copolymers are soluble in aqueous solutions below their LCST due to the formation of hydrogen bonds between the copolymer polar groups and water molecules. In this study, the effects of lactate length, HEMA-PLA, and OEGMA content on the solubility of PNPHO were investigated by measuring the saturation concentration of PNPHO with different compositions in PBS.

[0285] The results in Figure 3 demonstrate that increasing the lactate length in the HEMA-PLA backbone within the range of 3 to 6 did not significantly affect the solubility of PNPHO in PBS (p > 0.05). Thus, the hydrophobic nature of the side chains in the PNPHO backbone had minimal impact on the overall solubility of PNPHO in aqueous media within the range examined. Therefore, by varying the lactate length, it is possible to tune other characteristics of PNPHO, such as gelation behavior and mechanical properties, without affecting its solubility in aqueous media.

[0286] The solubility of PNPHO in PBS can be tuned by varying both the hydrophobic and hydrophilic content. The PLA segment is the predominant hydrophobic backbone, while both NAS and HEMA monomers exhibit relatively limited hydrophilic properties. Therefore, OEGMA was included in the synthesis of PNPHO to promote the hydrophilic properties of the copolymer.

[0287] Increasing the HEMA-PLA (i.e., hydrophobic content) in the copolymer from 6 mol% to 8 mol% and 11 mol% decreased the solubility of PNPHO in PBS by 30% and 50%, respectively. This decrease in solubility was also due to a decrease in the concentration of the relatively hydrophilic segment NIPAAm in the copolymer (p<0.05). Thus, decreasing the NIPAAm content in PNPHO substantially affected the hydration of the copolymer.

[0288] The solubility of PNPHO in water increased dramatically when more than 3 mol% (e.g., 1.5 mol%) OEGMA was used as the hydrophilic segment. This result indicated that copolymers with an OEGMA content less than 3 mol% were insoluble in aqueous media. The results in Figure 3 show that the solubility of PNPHO copolymers with 6 mol% PLA-HEMA significantly increased by two and three times when the OEGMA concentration was increased from 3 mol% to 5 mol% and 8 mol%, respectively. However, in copolymers containing higher molar ratios of the hydrophobic segment HEMA-PLA (i.e., 8 mol% and 11 mol%), the OEGMA concentration had little effect on the solubility of PNPHO. This behavior is due to the longer chains and higher M W This was due to the formation of a copolymer with ##STR1## which hindered the hydration and solubility of the copolymer in aqueous solution.

[0289] The effect of the concentration of water-soluble PNPHO copolymer on the injectability of these solutions through an 18G needle was determined. A 150 mg / mL PNPHO solution in PBS was found to be injectable through an 18G needle, and this concentration of copolymer was used for further analysis. Higher concentrations of the polymer can be used for other biomedical applications, such as scaffold fabrication for in vitro tissue growth.

[0290] Thymosin beta-4 Because the succinimide linker exhibits high reactivity and optimized accessibility toward compounds containing amino groups, it is reasonable to infer that this polymer can be applied to TB4, which has amino groups, for the preparation of flowable hydrogels. To confirm this assumption, the feasibility of the reaction between the polymer and TB4 was investigated. A TB4 solution was formed in phosphate-buffered saline. 1000 μL of the TB4 solution was thoroughly mixed with 140 mg of the polymer at 4°C for at least 24 hours, and then the mixture was allowed to gel at 37°C.

[0291] Gelation behavior (time and temperature) The results in Table 2 showed that the gelation times of hydrogels prepared using the PNPHO composition ranged from 2.5 to 5 minutes. It is important to note that these measurements were performed under simulated physiological conditions (a 37°C incubator), and the gelation rate of hydrogels in contact with body fluids and / or body surfaces is significantly faster at this temperature. This gelation time is desirable for clinical use because it allows for the administration of the product while simultaneously preventing migration of the administered product from the site. Additionally, the gelation temperatures of these PNPHO formulations are below 37°C, making their clinical use possible. The high gelation temperature of this formulation makes it highly convenient for clinicians to deliver the solution in vivo.

[0292] Conjugation efficiency of PNPHO To test the conjugation efficiency of PNPHO, the TB4 synthetic peptide was used as a model system. Results showed that TB4 was efficiently conjugated with different PNPHO formulations. This demonstrated the high efficacy of PNPHO for conjugation with synthetic or natural peptides or proteins. The two components (TB4 and PNPHO) interact at the molecular level through the succinimide ester group of PNPHO and the amino group of TB4. To determine this, the lower critical solution temperatures (LCSTs) of PNPHO and PNPHO-co-TB4 were compared.

[0293] The LCST of the PNPHO polymer and conjugate system (PNPHO-co-TB4) is driven by the chemical composition of PNPHO and TB4 and the ratio between hydrophilic and hydrophobic groups within the molecular structure. PNPHO and PNPHO-co-TB4 exhibit inverse solubility upon heating. This thermoresponsive behavior arises from the ability of the NIPAAm group and related side chains (e.g., NAS, PLA / HEMA, OEGMA, and TB4) to undergo a transition from a molten coil to a collapsed globule (i.e., a transition from a hydrophilic to a hydrophobic state) when the temperature is elevated above the LCST (Figure 1). 1 H-NMR provides an accurate profile of the LCST by monitoring the transition of the polymer during the temperature change of the NMR data acquisition.1 H-NMR spectra are recorded at temperatures ranging from 10°C to 30°C at 1°C intervals. As the temperature increases, the NMR signal decreases for various peaks as the conjugate system transitions from coil to globule (inverse solubility). A plot of the NMR peak area against temperature reveals the LCST.

[0294] PNPHO polymer and PNPHO-co-TB4 1 Based on the H-NMR spectra, the LCST of the conjugate system is found to be 2–4°C lower than that of the PNPHO polymer (Figure 4). DO / m (a characteristic peak for OEGMA) was used to quantify the gelation temperature. The shift in gelation temperature of the conjugate system compared to PNPHO confirmed the presence of a chemical interaction between the two components. In addition, the decrease in gelation temperature increases the dynamics of gelation in vivo, thus promoting adhesion of the product at the administration site.

[0295] Bioabsorption behavior of protein-PNPHO hydrogels Clinical studies of PNPHO-co-TB4 have shown that the product is fully resorbed by the body within three months. Histological evaluation of the implant site showed no trace of the product 3-4 months after implantation.

[0296] Feasibility Study

[0297] [Table 3]

[0298] Samples from the batch were incubated on 90 mm Trypticase soy agar plates at 37° C. The absence of bacterial growth was verified before batch release was performed.

[0299] The sample size(s) used in the execution of this protocol are shown in Table 4.

[0300] [Table 4]

[0301] The sample size(s) selected for all studies performed will be chosen with consideration of statistical validity, with a significant number of animals / test article and control groups (e.g., six replicates per group per time point, far exceeding the three replicates typically used for preclinical, proof-of-concept studies).

[0302] [Table 5]

[0303] The PNPHO-co-TB4 test article was manufactured by the applicant in accordance with documented procedures and processes. All equipment, instruments, and materials used during product manufacturing were approved within the applicant's QMS. The PNPHO-co-TB4 device was supplied to the testing facility in a ready-to-use syringe. Each test article was for single use.

[0304] The following equipment / instruments / materials were required for the performance of this study protocol: Histochemical analysis, staining and visualization were performed according to industry standards.

[0305] [Table 6]

[0306] result The PNPHO-co-TB4 solution transitions from a liquid at room temperature (20–25°C) to a gel at body temperature (37°C) and retains its structural stability after injection under simulated physiological conditions (Figure 1a). A live ovine osteotomy model was used to investigate the injectability and adhesive properties of PNPHO-co-TB4 under active bleeding conditions. The results in Figure 1b demonstrate that this product can be injected into the defect site and immediately forms a hydrogel that fills the cavity, mixes with blood, and stabilizes the clot. Hydrogel formation was achieved despite active bleeding at the site and subsequent dilution of the hydrogel with blood. This is an important attribute of the hydrogel system of the present invention, making it useful for clinical applications of injectable systems and preventing device failure. Due to the presence of body fluids during in vivo injection, better heat transfer facilitates the dynamic kinetics of hydrogel formation. For example, Figure 1c shows that hydrogel formation on the skin is slower. This unique behavior of the present invention can be used to form 3D structures through the administration of layer after layer of this product into exposed wounds, which typically have a lower temperature compared to the core / deep tissue (Figure 1d). The present invention is adhesive and mixes with blood and host cells to support healing.

[0307] The ability of PNPHO-co-TB4 to support tissue regeneration is based on the physical scaffold it forms upon administration. This matrix is ​​suggested to support cell infiltration and vascular ingrowth throughout its structure. To test this hypothesis, a mouse animal model was used to investigate the in vivo potential of PNPHO-co-TB4 to integrate with the host environment and support vascular ingrowth. Additionally, PNPHO-co-TB4 was directly compared with Integra Regenerative Dermal Matrix (Integra) as a positive control.

[0308] Animal models and surgical procedures A full-thickness mouse animal model (n=20) was used; two full-thickness skin defects were surgically created in each animal; see Figure 5(a). The wounds were treated with PNPHO-co-TB4 or Integra (positive control), and the skin grafts were sutured into place as shown in Figure 5(b). Conventional dressings, specifically Atrauman and IV 3000, were then applied to the treatment sites. Angiogenesis and ingrowth at the graft sites were determined by fluorescence emission efficiency using an IVIS Lumina XR live imager. The defects were periodically checked for graft survival up to 8 weeks after treatment. In addition, animals were sacrificed at different time points; biopsy samples were collected to quantify host tissue integration, vascular ingrowth, and inflammatory responses.

[0309] skin graft survival The survival of the grafted skin was examined at different time points throughout the study. Successful skin grafts remained intact at the host skin site, while failed sites folded and peeled over time; see Figure 5(c). Results from this study showed that within one week after surgery, four skin grafts treated with Integra failed, representing an 80% survival rate. This finding is consistent with previous studies reporting an approximately 85% survival rate for Integra ("Design of an artificial skin. I. Basic design principles," n.d.; Vyas & Vasconez, 2014). All graft sites treated with PNPHO-co-TB4 survived throughout the study; a 100% survival rate for PNPHO-co-TB4-treated skin grafts. The effectiveness of PNPHO-co-TB4 in supporting full-thickness skin grafts suggests that PNPHO-co-TB4 is well tolerated with minimal inflammatory responses. Successful skin graft engraftment suggests that PNPHO-co-TB4 acts as a regenerative scaffold for neovascular ingrowth, host tissue infiltration, and extracellular matrix formation. To investigate these characteristics of PNPHO-co-TB4, we performed intravital fluorescence imaging of animals at different time points and histological evaluation of the treatment sites.

[0310] Inflammatory response to the compositions of the present invention Skin biopsy samples were collected 2 and 4 weeks after implantation to histochemically assess the biological behavior of PNPHO-co-TB4. H&E staining of the Integra-treated sites showed a mild inflammatory response to the Integra construct at both 2 and 4 weeks; Figures 6(a) and 6(b), respectively, show multilayered fibrous tissue visible around the Integra site. Previous studies have shown similar inflammatory responses to the construct and the formation of fibrous tissue around Integra (Hori, Osada, Isago, & Sakurai, 2017; Y. Wang et al., 2015).

[0311] In contrast, H&E staining of skin biopsies from PNPHO-co-TB4-treated sites in Figures 6(c) and 6(d) shows that a very narrow layer of fibrous tissue had formed around the PNPHO-co-TB4 gel two weeks after implantation. The white arrows in Figure 6(c) indicate the structure of the PNPHO-co-TB4 gel, while the black arrows identify minimal fibrous tissue formation around the PNPHO-co-TB4 hydrogel. Recognizing that increased inflammation correlates with impaired wound remodeling, the reduction in fibrous tissue formation around PNPHO-co-TB4 suggests that this material contributes to effective soft tissue repair.

[0312] Neovascularization Angiogenesis and ingrowth at the implantation site were determined by fluorescence emission efficiency using an IVIS Lumina XR live imager. Two and four weeks after implantation, the angiogenic response was determined using the AngioSense750 EX in vivo blood pool fluorescence imaging probe. This near-infrared fluorescent macromolecular probe persists in the vasculature, allowing imaging of blood vessels and angiogenesis. At each post-surgery time point, each mouse was injected with 2 nmol of AngioSense750 EX in 100 μL of PBS. After 24 hours, each mouse was scanned for fluorescence emission efficiency (n=8).

[0313] Radiation efficiency was used to indicate the density of new blood vessels in the wound area. The results in Figure 7 show that at two weeks after surgery, the fluorescence emission efficiency for the PNPHO-co-TB4-treated site was significantly higher (p<0.01) than that for the Integra-treated site. In contrast, at four weeks after surgery, the angiogenic signal was very low for both treatment groups, indicating that angiogenesis was controlled and healing of the site had ceased. Similar findings showing that treatment sites after four weeks did not exhibit angiogenic signals have been reported in a previous study (Yiwei Wang et al., 2015).

[0314] Vascular ingrowth and cell infiltration within the compositions of the present invention Four weeks after skin grafting, histological evaluation of the PNPHO-co-TB4-treated site confirmed the potential of PNPHO-co-TB4 to support vascular ingrowth and host cell infiltration. H&E staining of the skin biopsy sample is shown in Figure 8. As previously outlined, a thin layer of fibrous tissue formed around the PNPHO-co-TB4 construct, indicating that the injectable hydrogel is well tolerated within the body; Figures 8(a) and 8(b). More importantly, this thin layer of fibrous tissue can be used as an indicator of the PNPHO-co-TB4 boundary in the dermis.

[0315] The results in Figures 8(c) and 8(d) show the formation of blood vessels within the PNPHO-co-TB4 structure. In addition, staining of skin biopsy samples showed clear infiltration of host fibroblasts within the PNPHO-co-TB4 hydrogel structure; Figure 8(d). To further confirm fibroblast infiltration and dermal extracellular matrix formation within the structure of the composition of the present invention, the skin graft site was stained with Masson's trichrome four weeks after grafting. The results in Figure 9 show collagen fiber formation within the composition of the present invention four weeks after grafting surgery. These results confirm the infiltration of fibroblasts within the structure of the composition of the present invention and its potential to integrate with host tissue and promote neodermis formation.

[0316] The formation of collagen fibers within the PNPHO-co-TB4 structure was significantly higher than that detected within the Integra structure. The CCC results showed that the formation of collagen within the Integra structure was significantly less than that of the composition of the present invention. This result is also consistent with previous findings reported in the literature (Yiwei Wang et al., 2015).

[0317] Findings from this study confirmed the scaffolding effect of PNPHO-co-TB4, which supported host tissue integration, vascular ingrowth, cell infiltration, and neodermis formation.

[0318] Technical Feasibility: Conclusion PNPHO-co-TB4 was successfully tested in an expanded animal study. The expanded animal study included 40 full-thickness skin grafts with a direct comparison of PNPHO-co-TB4 with a gold standard skin template (Integra skin matrix). During the study, all skin grafts treated with the composition of the present invention survived (100% survival), compared with an 82% success rate in the Integra group. In addition, the composition of the present invention rapidly vascularized the structure, minimized the inflammatory response, and promoted skin cell infiltration to form neodermis and collagen fibers at the site. These results demonstrate the potential of this technology in post-burn scar management.

[0319] Clinical Research - Investigational Device Description The composition of the present invention, PNPHO-co-TB4, is supplied in a ready-to-use sterile syringe. The product is single-use and double-sachet packaged. The composition of the present invention is liquid at room temperature, allowing for direct injection into the desired clinical location. At body temperature, the composition of the present invention forms a white, stretchable scaffold.

[0320] Device composition The composition of the present invention is an injectable scaffold. The formulation of the composition of the present invention has two main components: (1) a synthetic polymer (PNPHO) and (2) a synthetic non-human or animal-derived peptide, i.e., thymosin beta-4. These two components are chemically bonded to form a single, homogeneous molecule (e.g., PNPHO-co-TB4).

[0321] This smart polymer is poly(N-isopropylacrylamide-co-(N-acryloxysuccinimide)-co-(polylactide / 2-hydroxymethacrylate)-co-(oligo(ethylene glycol)), designated PNPHO. The specific formulation of PNPHO used in the compositions of the present invention is PNPHO 5:8(5):7:81. Equimolar amounts of PNPHO and thymosin beta-4 are used in the formulation of PNPHO-co-TB4.

[0322] Intended use of the device PNPHO-co-TB4 is intended to promote bone regeneration. Specifically, PNPHO-co-TB4 has been shown to reduce bone resorption after tooth extraction, promoting healthy patient outcomes.

[0323] Clinical research plan - Research objectives The primary objective was to identify qualitative measurements and analytical methodologies to further explore the use of the compositions of the present invention. Specifically, the healing process and the adequacy of histologically and CT-assessed bone regeneration in the presence of the compositions of the present invention after tooth extraction compared to a historical (literature) control population. A secondary objective was to investigate the in vivo characteristics of the compositions of the present invention in humans.

[0324] Clinical Study Design - Clinical Trial Endpoints The clinical trial endpoints for this study were to identify qualitative measurements and analytical methodologies to further investigate the use of the compositions of the present invention up to 3-4 months after surgery. Specifically, the healing process and adequacy of histologically and CT-assessed bone regeneration in the presence of the compositions of the present invention after tooth extraction compared to a historical (literature) control population. In addition, clinical trial endpoints were designed to examine the in vivo characteristics of the compositions of the present invention in humans 3-4 months after surgery.

[0325] Data Quality Assurance This clinical study was designed, configured, monitored, and data generated, documented, recorded and reported under the applicant's certified ISO 13485 quality management system in accordance with ISO 14155 and in accordance with SSR's internal procedural requirements.

[0326] Management of the clinical study was outsourced to an independent CRO, Southern Start Research (SSR). All data points were monitored 100% by SSR throughout the trial. Data quality assurance and quality control monitoring visits were conducted at intervals specified in Table 7.

[0327] [Table 7]

[0328] Participant group Ten patients (10) were enrolled in this study. A summary of patient demographics is shown in Table 8.

[0329] [Table 8]

[0330] Treatment Schedule The study procedures and schedule for visits and follow-up are outlined in Table 9. The protocol was carried out as planned in the Clinical Investigation Plan (CIP). All participants except Participant #7 followed the plan and follow-up visits occurred as scheduled. Due to personal circumstances, Participant #7 changed histology from his planned treatment; therefore, histology was not collected at Visit 4 for Participant #7.

[0331] Follow-up period Study participants were followed for three months (3) after tooth extraction and treatment with the composition of the present invention.

[0332] [Table 9]

[0333] Table 10 outlines the intended follow-up visit schedule, along with the individual visit windows. Time point zero (0) is the time of tooth extraction (and implantation of the composition of the present invention for the treatment population).

[0334] [Table 10]

[0335] Results - Disposal of Objects and Investigation Devices Ten patients were treated with the composition of the present invention. A device was implanted in all participants. No study device was left in place after completion of the clinical study. All study devices were regulated under device accountability procedures by the Southern Start Research (SSR) Clinical Research Organization.

[0336] All findings were qualitative; these included, but were not limited to, wound appearance at different time points after tooth extraction, patient reports of pain and discomfort, radiological appearance of the underlying bone using a CT scan 3 months after surgery, visual appearance of the site during implant placement surgery, and histological examination of the treated site 3-4 months after surgery.

[0337] Device Usability No device malfunctions were reported throughout the clinical study. The composition of the present invention is provided in a ready-to-use format; it eliminates the need for premixing or any other preparation steps by the clinician prior to surgery. Figure 11b in particular shows that the composition of the present invention can be easily injected into the tooth extraction site via a 21G needle. Thereafter, due to the hydrophilic nature of the product, the composition of the present invention mixes with blood at the site, forming a hydrogel and stabilizing the clot.

[0338] Clinical use of this device in a PET trial demonstrated that injection of the composition of the present invention into the tooth extraction socket site was successful in all 10 patients, with no device malfunctions reported by the investigator. The results in Figure 12 showed a 100% success rate in application and gelation of the composition of the present invention for all 10 patients.

[0339] [Table 11]

[0340] A summary of findings from the utility of the compositions of the present invention is outlined in Table 11. According to the investigators, use of the compositions of the present invention can reduce time on-site by up to 45 minutes. This can be achieved because use of the compositions of the present invention after tooth extraction eliminates the need for preliminary closure, such as the use of a membrane and micro-suturing of the site.

[0341] [Table 12]

[0342] Device Safety and Wound Healing All 10 patients treated with the composition of the present invention returned for the first follow-up visit one week after surgery. No pain or discomfort was reported by the patients. During oral examination (one week after administration), there were no signs of infection or inflammation at the site. In addition, the investigator examined wound closure and soft tissue formation. In all 10 patients, wound closure was observed and rapid soft tissue formation was detected (results in Figure 12).

[0343] Three months after tooth extraction and treatment with the composition of the present invention, the patient underwent an implant placement procedure. At this time, biopsy samples were collected from the injection site of the composition of the present invention. Histochemical analysis of the samples was performed by an independent laboratory (Sonic Clinical Trials Pty Ltd). The findings from these histochemical analyses are summarized in Table 12. There was no evidence of necrosis, foreign body giant cells, or foreign body reaction in all analyzed samples. These findings suggest that the composition of the present invention is well tolerated by the body and is biocompatible in vivo.

[0344] Indicators of effectiveness for bone regeneration Three months after surgery, CT scan imaging of the sites was used to examine healing progress and the extent of bone resorption at the sites. An independent CT scan reported by Dr. Tom Huang at Envision Medical Imaging confirmed minimal bone resorption. In addition to CT scan imaging of the sites, biopsy samples were collected from the injection sites of the compositions of the present invention during the implant placement procedure. These samples were fixed and sent for independent histochemical analysis. Hematoxylin and eosin (H&E) and Masson's trichrome staining of the sites were used to analyze the pathological behavior and bone regeneration process at the sites. In all analyzed samples, histochemical analysis demonstrated the formation of viable, interconnected trabecular bone, fibrous and lamellar bone, as well as a mixture of active osteoblasts and osteoclasts. Active periodontal bone remodeling was observed in all patients.

[0345] All reports from independent laboratories for H&E and Masson's Trichrome stained histochemical specimens are shown in Table 13.

[0346] [Table 13-1] [Table 13-2] [Table 13-3]

[0347] Aerosol formation performance of PNPHO-co-TB4 Particle size analysis as a volume-diameter distribution and derived parameters (d10, d50, d90, and %V>10) was performed for each formulation with different solids content (PNPHO concentrations ranging from 17.5 mg / mL to 140 mg / mL). Regardless of the formulation concentration and temperature tested, aerosol droplets from all formulations exhibited a bimodal distribution. In general, the first peak (approximately 50 μm) tended to increase with decreasing formulation concentration, while the second peak (approximately 500–600 μm) increased with increasing concentration. This suggests that the majority of aerosol droplets were in the 10–100 μm range.

[0348] The percentile sizes for all diluted formulations were 21.4 ± 3.9 μm, 45.6 ± 9.4 μm, and 238.6 ± 181.5 μm (mean) for d10, d50, and d90, respectively. The prevalent median size for nasal delivery is 30–120 μm (1), and therefore all diluted formulations were within the specifications for nasal delivery. Importantly, all formulations had less than 3% by volume of droplets with diameters <10 μm, suggesting their suitability for nasal delivery and avoidance of lower respiratory tract deposition. The 90th percentile size showed the greatest variability, suggesting potential issues with poor droplet ejection, especially for the more concentrated formulations.

[0349] Plume Cover Characterization of spray patterns for nasal formulations is recommended by the FDA. The plume patterns for the tested formulations (operated at room temperature) are shown in Figure 15. All formulations showed a wide coverage area, but the most obvious feature was the lack of deposition in the center of the plume with increasing formulation concentration.

[0350] Despite these differences, D max and D min The values ​​(Figure 16) were consistent across samples, and the resulting ellipticity (approximately 1) is an indicator of spray symmetry during aerosolization. The ellipticity for all formulations was within FDA specifications (1.00-1.30). Indeed, the best option for nasal delivery should be a trade-off between coverage area and improved residence time. Larger coverage areas did not provide the best outcome if runoff occurred.

[0351] Nasal deposition pattern The deposition patterns of the formulations in the human nasal model are shown in Figure 17. Both formulations tested (17.5 mg / mL and 35 mg / mL) demonstrated rapid adhesion, as the deposition pattern remained stable and relatively unchanged up to 15 minutes after actuation. Furthermore, no throat dripping was observed. The short-term adhesion of the formulations may indicate a higher residence time and bioavailability of the delivered cargo. Both formulations were able to reach the olfactory region (upper nasal region), which is important for nose-to-brain delivery.

[0352] In vitro drug release from PNPHO-based hydrogels Using ciprofloxacin HCl as a model drug, we investigated the potential of PNPHO-based hydrogels to control the release of small hydrophilic drugs and prevent sudden drug release after administration. The assembly of hydrophobic domains in the polymer chains during the gelation process expels water from the matrix. To determine the amount of ciprofloxacin HCl leaching from PNPHO-co-TB4, the hydrogels were assayed using a snapwell device. Briefly, ciprofloxacin HCl powder was dissolved in a PNPHO-co-TB4 solution (20 mg / mL). Then, 200 μL of the PNPHO-co-TB4 / ciprofloxacin HCl formulation was placed in the apical chamber of the snapwell, and the hydrogel was allowed to form at 37 °C for 30 minutes while the snapwell was placed flat to avoid polymer loss through the membrane. The liquid supernatant layer on top of the hydrogel was then collected.

[0353] To further analyze drug release from the gels over time, 2 ml of PBS was added to the basolateral compartment of each snapwell, and the plates were then incubated at 37°C with constant orbital shaking (60 rpm). At predetermined time points over 24 h, samples (200 μL) were removed from the basolateral medium and replaced with an equal volume of fresh pre-warmed PBS each time. All samples were quantified for ciprofloxacin HCl using a validated high-performance liquid chromatography method (HPLC, Shimadzu, Sydney NSW, Australia).

[0354] Quantification of Ciprofloxacin HCl via HPLC Ciprofloxacin HCl quantification was determined using an HPLC system consisting of an LC20AT pump, a SIL20AHT autosampler, and an SPD-20A UV-VIS detector (Shimadzu, Sydney, NSW, Australia). Sample quantification was performed using a reversed-phase Luna C-18 column (Phenomenex, Torrance, USA) 150 x 4.6 mm and 3 μm particle size. Measurements were performed using a mobile phase consisting of phosphate buffer (pH 7.2):acetonitrile (75:25 v / v), a flow rate of 0.7 mL / min, a detection wavelength of 275 nm, and an injection volume of 100 μL. Standard solutions were prepared fresh daily with a needle wash of acetonitrile:water (50:50 v / v). Linearity was confirmed within the concentration range of 0.05 to 100 μg / mL, with a regression value >0.999.

[0355] During the gelation process (30 min), the initial release of ciprofloxacin HCl from PNPHO hydrogels was evaluated under physiological conditions. An average of 2.7 ± 1.4 μg and 28.9 ± 17.9 μg (n = 3) (Figure 18; t = 0) of ciprofloxacin HCl was found to be released into the liquid apical layer of PNPHO and PNPHO-co-TB4, respectively. It is important to note that the initial release of the test drug from the hydrogels was negligible compared to the loading amount, i.e., at a loading amount of 20 mg / mL, the release amounts from PNPHO and PNPHO-co-TB4 were 2.7 ± 1.4 μg and 28.9 ± 17.9 μg, respectively.

[0356] The relatively low percentage of ciprofloxacin released from the hydrogels upon administration demonstrates the high potential of the present invention for drug delivery applications. The time-dependent release of ciprofloxacin HCl from the hydrogels at 37°C is shown in Figure 18 as cumulative mass for each formulation. After 24 hours, 137.4 ± 32.4 μg (89.5 ± 15.8%) and 133.7 ± 42 μg (65.3 ± 10.5%) were released from PNPHO and PNPHO-co-TB4, respectively, with no significant difference in the amount of ciprofloxacin HCl released between the two hydrogels. These results confirmed the sustained and controlled drug release profile from PNPHO-based hydrogels.

[0357] Summary of Adverse Events No medical device adverse events were reported throughout the clinical trial. The only adverse event reported in this trial was related to the treatment plan for participant #7; participant #7 elected to modify their treatment plan to exclude bone histology, so histology was not collected for this participant at Visit 4.

[0358] Conclusions regarding device usability The composition of the present invention was administered to all 10 patients without difficulty. No device (syringe) malfunctions were reported in this study. No preparation and / or mixing of the device was required prior to use. The composition of the present invention immediately forms a white hydrogel upon injection into the extraction socket site. The product mixes with the blood and adheres to the extraction site. The product adheres to the site and does not require pre-closure at the site to contain the device.

[0359] Device Safety No device-related or serious adverse events were reported in this study. All 10 (10) patients returned for their first follow-up visit 7 days after tooth extraction, and no patients reported inflammation, infection, pain, or discomfort. Nine (9) patients underwent implant placement surgery, during which the original tissue was collected for histochemical analysis. H&E staining of the sites indicated that the product was well tolerated by the body and there was no evidence of abnormality or foreign body giant cells at the sites. No necrosis or necrotic tissue was observed at the sites. Additionally, CT scans of the tooth extraction sites and the sites treated with the composition of the present invention showed no bone-like abnormalities at the sites.

[0360] Device Availability Seven days after tooth extraction and treatment with the composition of the present invention, wound closure was observed in all patients. Cellular levels, osteoblast and osteoclast activity, and active bone remodeling were observed. Viable bone and bone remodeling were observed at the treatment site, demonstrating the osteoinductive properties of the device. The product did not induce bone formation and, as hypothesized, had no other phenotypic effects on the host tissue.

[0361] Risk and benefit assessment Given careful consideration of the potential benefits of the device, the potential benefits and identified residual risks of the device were assessed on an individual and collective basis to determine whether the device was acceptable.

[0362] Emerging evidence suggests that bone-anchored prostheses improve quality-of-life measures, including mastication, speech, and overall health outcomes. However, financial barriers and access to oral surgeons are recognized limitations to the widespread use of implant-retained prostheses. From a clinical perspective, minimizing bone loss after tooth extraction is essential to simplify and therefore improve the use of bone-anchored prostheses.

[0363] Bone volume loss after tooth extraction is difficult for clinicians to manage and places a financial burden on patients and health systems, especially in rural, socioeconomically disadvantaged areas. The extent of this problem and difficulty in dentistry is highlighted by the fact that one in two patients requires a secondary grafting procedure to augment bone volume for successful implant placement. The compositions of the present invention are easy to use materials that can preserve ridge bone volume after tooth extraction. Dentists appreciate the ease of use and predictability of the implant placement procedure. Patients benefit from the use of the compositions of the present invention by promoting wound healing and accelerating bone healing after tooth extraction, potentially preventing the need for secondary augmentation procedures.

[0364] Application of the composition of the present invention in a fresh extraction socket was simple and easy to use in clinical practice. Unlike all other bone substitutes, the composition of the present invention is delivered as a liquid to the extraction socket and forms a stretchable matrix at the site. The primary objective of the pilot PET trial was to investigate the safety, efficacy, and osteoinductivity of the composition of the present invention. Measures of efficacy were collected, including radiographic imaging and histochemical analysis. In summary, while the trial was not in operation, PET confirmed the safety and efficacy of the device; no device malfunctions were observed, and no device-related adverse events were detected.

[0365] Considering the intended use, the intended users, reasonably foreseeable misuses, and potential effective uses of the device family of compositions of the present invention, and taking into account the generally recognized state of the art, it can be concluded that the risk management process has determined that the potential device benefits outweigh the residual risks.

[0366] Clinical Relevance of the Compositions of the Invention The composition of the present invention has been developed as a safe and easy-to-use biomaterial for filling tissue defects / cavities. The composition of the present invention is a single, homogeneous molecule composed of a synthetic "smart" polymer (PNPHO) crosslinked with thymosin beta-4. The composition of the present invention is liquid at room temperature, allowing for direct injection into the desired clinical location. The composition of the present invention forms a stretchable gel upon exposure to body temperature, mixes with blood, and stabilizes the clot at the site. Ten patients (10) were administered the composition of the present invention. No device malfunctions or device-related adverse events were reported or observed during use of the composition of the present invention. Wound healing was observed one week after administration.

[0367] Three months after surgery, no pathological abnormalities, such as inflammation, infection or giant cells, were detected at the administration site.In addition, active bone remodeling, as well as osteoblast and osteoclast activity, were detected at the site treated with the composition of the present invention, confirming the osteoinductive properties of the device.These findings indicate the high potential of the composition of the present invention for wound healing in both soft and hard tissues, since this product does not induce phenotypic effects, such as soft and hard tissue repair at the site.However, it is believed that further health benefits can be achieved by increasing the osteoinductive properties of the composition of the present invention.

[0368] Clinical Study Summary A pilot trial involved the use of the PNPHO-co-TB4 scaffold of the present invention for post-extraction socket preservation in 10 patients. In all patients, the applicant's device was successfully administered; there was no need for membranes or microsutures at the extraction site, allowing the investigator to spend less time on-site. At a follow-up visit, wound closure was observed 7 days after extraction, with no signs of infection or inflammation in any patient. Three months after use of the composition of the present invention, tissue biopsies were collected from the sites for histochemical analysis; the results showed that the product was fully resorbed and the sites showed no signs of pathological abnormalities. Additionally, active bone remodeling was observed at the sites.

[0369] Although the present invention has been described with reference to specific embodiments, it will be apparent to those skilled in the art that the present invention may be embodied in many other forms.

[0370] Applicants acknowledge the contributions of Dr. Hui Ong and Dr. Dina Silva in conducting the in vitro aerosol formation studies and benchtop drug release tests.

Claims

1. 1. A composition for use in soft tissue as a tissue-conductive medical filler, comprising: The composition comprises a polymer and a natural or synthetic peptide or protein (NSPP), wherein the polymer is a first monomer for binding water; a second monomer to impart mechanical properties to the hydrogel; a third monomer for attachment to a natural or synthetic peptide or protein (NSPP), and a fourth monomer to impart phase transition behavior; the first monomer is oligo(ethylene)glycol monomethyl ether methacrylate (OEGMA); the second monomer is hydroxyethyl methacrylate poly(lactic acid) (HEMA-PLA); the third monomer is N-acryloxysuccinimide (NAS), the fourth monomer is N-isopropylacrylamide (NIPAAm); the natural or synthetic peptide or protein (NSPP) is thymosin beta-4; A composition wherein the natural or synthetic peptide or protein (NSPP) is bound to a third monomer, thereby crosslinking the polymer and forming a hydrogel that contains water.

2. The polymer is a first monomer in an amount of 3 to 8 mol %, a second monomer in an amount of 5 to 9 mol %, a third monomer in an amount of at least 7 mol %, and 10. The composition of claim 1, further comprising a fourth monomer in an amount that comprises the remainder up to 100% of the composition of said polymer.

3. The polymer is oligo(ethylene)glycol monomethyl ether methacrylate (OEGMA) in an amount of 5 mol % as a first monomer; hydroxyethyl methacrylate poly(lactic acid) (HEMA-PLA) in an amount of 7 mol % as a second monomer; N-acryloxysuccinimide (NAS) as a third monomer in an amount greater than 7 mol %, and 3. The composition of claim 2, comprising N-isopropylacrylamide (NIPAAm) as a fourth monomer in an amount up to 81 mol %.

4. A method of making a hydrogel (excluding methods of surgery or treatment of humans) comprising mixing an aqueous solution of the composition of any one of claims 1 to 3 with an aqueous solution of the natural or synthetic peptide or protein (NSPP) as described above.

5. The method of claim 4 , wherein the hydrogel forms at body temperature.

6. Use of a composition according to any one of claims 1 to 3 in the manufacture of a hydrogel for the repair and / or reconstruction of soft body tissue; wound healing; temporary wrinkle reduction; temporary lifting of scar bases and promoting healing; supporting dermal connective tissue formation in scar tissue after surgical intervention and promoting healing; supporting dermal connective tissue formation in scar management after burns; supporting vascular ingrowth and promoting healing in acute skin defects accompanied by bleeding; filling surgically created skin cavities; supporting skin grafting procedures; filling prostheses; use as a filler without tissue inductive properties; supporting and repairing periodontal tissue after tooth extraction; or for temporarily lifting periodontal ligament tissue and / or supporting periodontal ligament tissue transplants (excluding methods of surgery or treatment of humans).

7. Use of a composition according to any one of claims 1 to 3 in the manufacture of a medicament for the repair and / or reconstruction of soft tissue.

8. 1. A kit for forming a hydrogel for soft tissue repair and / or reconstruction, comprising: polymers and natural or synthetic peptides or proteins (NSPPs), The polymer is a first monomer for binding water; a second monomer to impart mechanical properties to the hydrogel; a third monomer for attachment to a natural or synthetic peptide or protein (NSPP), and a fourth monomer to impart phase transition behavior; the first monomer is oligo(ethylene)glycol monomethyl ether methacrylate (OEGMA); the second monomer is hydroxyethyl methacrylate poly(lactic acid) (HEMA-PLA); the third monomer is N-acryloxysuccinimide (NAS), the fourth monomer is N-isopropylacrylamide (NIPAAm); The kit, wherein the natural or synthetic peptide or protein (NSPP) is thymosin beta-4.

9. 9. The kit of claim 8, further comprising water in a separate container.

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