Biocompatible materials

Abstract

The invention relates to the field of materials, in particular to a biocompatible material. The present invention provides compositions comprising a polymer and a natural or synthetic peptide or protein (NSPP). The composition forms a hydrogel with water. The composition can be used as a filler for cosmetic and therapeutic applications. Embodiments of the invention provide methods of using the compositions or hydrogels to treat certain conditions, as well as surgical kits for simultaneous or sequential administration of the components of the compositions to enable in situ formation of hydrogels.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is a divisional application with an application date of December 7, 2020, application number 202080006185.0 (PCT / AU2020 / 051332), and invention name “Biocompatible Materials”.

[0003] This application claims the agreed priority of Australian provisional patent applications 2019904817 (December 19, 2019) and 2020903462 (September 25, 2020), the disclosures of which are incorporated herein by reference in their respective entireties. Technical Field

[0004] The present invention relates to a biocompatible material which can be used for tissue regeneration and repair.

[0005] The present invention relates to a filler that supports the natural healing of damaged tissue without inducing any specific tissue formation. The purpose is to use the present invention to partially or completely fill or cover tissue cavities or defects to provide the required filling space with minimal foreign body reaction.

[0006] In one embodiment, the present invention relates to tissue conductive medical fillers. In one embodiment, the polymers of the present invention can be formulated into hydrogels. In another embodiment, the hydrogels are thermoresponsive. In another embodiment, the compositions disclosed herein have been developed for delivery in a flowable form that can be injected, cast or sprayed. In one embodiment, the compositions form a hydrogel after being applied to, on or near the body.

[0007] The present invention can be used in tissue engineering applications. This includes both cosmetic and therapeutic applications. The present invention can be used in dermatological applications as well as dental and orthopedic applications to treat chronic, acute or surgical defects. However, it should be understood that the present invention is not limited to these specific fields of use. Background Art

[0008] The following discussion of the prior art is provided to place the invention in the proper technical context and enable the advantages of the invention to be more fully understood. However, it should be understood that any discussion of the prior art throughout the specification should not be taken as an explicit or implicit admission that such prior art is well known in the art or forms part of the common general knowledge in the art, or that the prior art can be reasonably expected by a person skilled in the art to determine, understand and regard as relevant.

[0009] All patents and publications cited herein are incorporated by reference in their entirety.

[0010] WO 2013 / 091001 (PCT / AU2012 / 001566) relates to polymers, in particular polymers that can be used as hydrogels, and to the use of hydrogels for tissue repair or recovery. In particular, the polymers and hydrogels of WO 2013 / 091001 can be used for cartilage, in particular the repair or recovery of articular cartilage. The polymer comprises at least a monomer for binding water, a monomer for imparting mechanical properties and a monomer for binding to extracellular proteins. The hydrogel comprises a polymer comprising at least a monomer for binding water and a monomer for binding to extracellular proteins. The polymer is cross-linked to form a hydrogel by binding to the extracellular matrix protein.

[0011] WO 2017 / 035587 (PCT / AU2016 / 050817) discloses biocompatible materials that can be used for tissue regeneration and repair, wherein the bioactive polymer can be in the form of a hydrogel (e.g., a thermoresponsive hydrogel). The bioactive polymers of WO 2017 / 035587 and the resulting hydrogels can be used for the regeneration of bone tissue. Therefore, WO 2017 / 035587 discloses a method for treating bone defects in mammals, the method comprising applying a therapeutically effective amount of a hydrogel formed by a bioactive polymer to a mammal to treat the bone defect.

[0012] WO 2017 / 015703 (PCT / AU2016 / 050653) discloses a polymer comprising at least one antiseptic / analgesic / anti-inflammatory monomeric unit in combination with at least three other monomeric units, the three other monomeric units causing properties selected from the group consisting of: temperature activation, water solubility, mechanical strength, protein / polysaccharide bonding ability and combinations thereof. In particular, WO2017 / 015703 discloses a polymer wherein: the water-soluble monomeric unit is a hydrophilic ethylene glycol (OEGMA) moiety; the monomeric unit imparting mechanical strength is polylactide-co-2-hydroxy-ethyl methacrylate (PLA / HEMA); the protein-reactive monomeric unit is an N-acryloyloxysuccinimide (NAS) moiety; and the thermosetting monomeric unit is an N-isopropylacrylamide (NIPAAm) moiety. The antiseptic / analgesic / anti-inflammatory monomeric unit comprises a methacrylate derivative of salicylic acid (5-HMA or 4-HMA, or a combination thereof).

[0013] All three applications cited above are by the present applicant. It is against this background that the present invention was developed.

[0014] In one form, the present invention is embodied as a flowable filler, wherein the filler forms a viscous hydrogel when applied into the body (e.g., by injection) or onto the surface of the body (i.e., at 30-37° C.). The hydrogel is well tolerated in the body with minimal inflammatory response. The hydrogel is host tissue-conductive, but not inductive, as it exhibits regenerative properties only in the presence of active bleeding or other fluids containing regenerative biological components. The filler can be injected through a fine gauge needle (e.g., 21G). The hydrogel adheres to the injection site. The filler can be formulated in an aerosol for application by spraying. The hydrogel can be applied topically in a manner that produces a 3D structure layer by layer in the body or by a minimally invasive manner. The main innovative aspects of the filler of the present invention include minimal foreign body reaction, host tissue conductivity, mixing with blood, injectability, adhesion properties, layer-by-layer filling, and optimal degradation spectrum.

[0015] The present invention can be used in soft tissue applications, such as skin applications, and hard tissue applications, such as dentistry and orthopedics. For example, the present invention can be used in cosmetic applications, such as wrinkle reduction. The present invention can be used to promote scar healing. This includes scars from burns as well as postoperative scars. The present invention can also be used to manage chronic wounds, such as diabetic ulcers.

[0016] The present invention can also be used in dental applications. Tooth extraction is an inherently traumatic procedure that damages soft tissue, underlying bone and ultimately results in significant loss of jaw or alveolar bone. Clinically, loss of alveolar bone results in aesthetic and functional complications associated with future prosthetic replacement of the missing teeth. If the missing teeth are to be replaced with implant-supported restorations, complex bone grafting procedures are always required. In order to reduce or potentially eliminate complex bone grafting procedures, alveolar or ridge preservation techniques have been proposed.

[0017] Many techniques have been described in the literature, most of which involve placing a graft filling material within the tooth extraction socket immediately following tooth extraction. There are a number of commercially available products that can be used to fill extractions. Indeed, no currently available product is capable of restoring bone volume to pre-extraction levels or resulting in improved healing outcomes.

[0018] Clinical studies conducted on demineralized bone allografts, synthetic bioactive glass, and deproteinized bovine bone mineral (xenografts) have shown that even 6 to 9 months after administration, the graft particles are still surrounded by connective tissue or a woven bone environment. These findings suggest that healing is physically impeded by the graft particles. The resulting surgical site is an unpredictable surgical environment for implant placement, even after extended waiting times. Given the scope of the problems associated with bone loss following tooth extraction, there is a significant unmet need for scaffolds that improve bone healing outcomes in tooth extraction patients.

[0019] There is generally a need for compositions for tissue repair that are injectable at room temperature and form hydrogels at body temperature.

[0020] The composition of the present invention is intended to be used as a filler that supports the natural healing of damaged tissue without inducing the formation of any specific tissue. The purpose is to use the present invention to partially or completely fill tissue cavities, provide the required filling space and have minimal foreign body reaction. The present invention can adhere to the cavity and mix with blood to promote host soft tissue and hard tissue repair. In order to induce any specific tissue growth (e.g., soft or hard), the present invention can be used in combination with other active ingredients.

[0021] 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.

[0022] The purpose of the particularly preferred form of the present invention is to use PNPHO-co-TB4 of a specific concentration and a specific formulation as a filler material. The filler material does not have any tissue induction properties. The filler can be used in combination with other materials, for example, inactive to provide a 3D structure, or active to induce the formation of a specific tissue. The present invention can be injected, cast or sprayed. The concentration of the polymer and TB4 can be adjusted to form different forms of the present invention.

[0023] The present invention is intended for both soft and hard tissues. In terms of soft tissue, the present invention is added to bioactive ingredients (such as cells or fat grafts) for skin type applications; 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., non-active bone particles) or active compounds (e.g., growth factors) to promote the growth of both soft and hard tissues. The envisioned commercial form is an injectable or putty.

[0024] Although the present invention will be described below with reference to preferred embodiments thereof, those skilled in the art will appreciate that the spirit and scope of the present invention can be embodied in many other forms. Summary of the invention

[0025] In the present invention, the applicant optimized its proprietary smart polymer PNPHO to bond with thymosin β-4 to form a cell-friendly medical filler material.

[0026] 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 the polymer comprises:

[0027] a first monomer for binding water;

[0028] a second monomer for imparting mechanical properties to the hydrogel;

[0029] a third monomer for conjugation to a natural or synthetic peptide or protein (NSPP); and

[0030] a fourth monomer for imparting phase change behavior;

[0031] And wherein the natural or synthetic peptide or protein (NSPP) is thymosin beta-4 or a functional homologue thereof.

[0032] In one embodiment, the first monomer is selected from the group consisting of: polyethers, polyvinyl alcohol (PVA), poly(vinyl pyrrolidone) (PVP), poly(amino acids), and dextran.

[0033] In one embodiment, the polyether is selected from polyethylene glycol (PEG), oligo(ethylene glycol) (OEG) or macromers thereof, polyethylene oxide (PEO), polyethylene oxide-co-propylene oxide (PPO), copolymers of polyethylene oxide blocks or random copolymers thereof.

[0034] In one embodiment, the first monomer is oligo(ethylene glycol) monomethyl ether methacrylate (OEGMA).

[0035] In one embodiment, the second monomer is a methacrylate or a random copolymer comprising a methacrylate.

[0036] 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).

[0037] In one embodiment, the second monomer is hydroxyethyl methacrylate poly(lactic acid) (HEMA-PLA).

[0038] In one embodiment, the third monomer has an electrophilic functional group for binding to the NSPP.

[0039] In one embodiment, the third monomer is selected from the group consisting of: N-hydroxysulfosuccinimide (SNHS), N-hydroxyethoxylated succinimide (ENHS), and N-acryloyloxysuccinimide (NAS).

[0040] 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.

[0041] 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%.

[0042] In one embodiment, the polymer comprises the second monomer in an amount of about 5 to about 9 mol%.

[0043] In one embodiment, the polymer comprises the third monomer in an amount of at least about 7 mol%, eg, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 mol%.

[0044] 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 constituting the remainder of 100% of the polymer composition.

[0045] In one embodiment, the polymer comprises the 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%.

[0046] 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 wherein 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%.

[0047] 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%.

[0048] In one embodiment, the natural or synthetic peptide or protein (NSPP) is thymosin beta-4.

[0049] In one embodiment, the composition comprises substantially equimolar amounts of polymer and thymosin beta-4.

[0050] In one embodiment where the cavity is partially or completely filled, the concentration of the polymer is from about 100 mg / mL to about 300 mg / mL of the composition.

[0051] 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 the combination of the NSPP and a third monomer crosslinks the polymers to form a hydrogel comprising water therein.

[0052] According to a third aspect of the present invention, there is provided a method of preparing a hydrogel, the method comprising adding water to the composition of the first aspect of the present invention.

[0053] According to a fourth aspect of the present invention, there is provided a method for preparing a hydrogel, the method 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).

[0054] In one embodiment, the hydrogel is formed at body temperature. In one embodiment, the hydrogel is formed after the composition and the NSPP are administered to a mammal by injection or by aerosol administration.

[0055] According to a fifth aspect of the present invention, there is provided a composition according to the first aspect of the present invention for use in preparing a hydrogel for repair and / or restoration of both hard tissue and soft tissue. Hard tissue (also referred to as calcified tissue) is a mineralized tissue with a firm intercellular matrix; the hard tissue of humans is bone, enamel, dentin and cementum. Soft tissue includes tissues that connect, support or surround other structures and organs of the body and are not hard tissues (e.g., bones). Soft tissue includes tendons, ligaments, fascia, skin, fibrous tissue, fat and synovium (all connective tissues), as well as muscles, nerves and blood vessels (all not connective tissues).

[0056] According to a sixth aspect of the present invention, there is provided use of the composition according to the first aspect of the present invention in preparing a hydrogel for wound healing.

[0057] According to a seventh aspect of the present invention, there is provided use of the composition according to the first aspect of the present invention in the preparation of a hydrogel for temporary wrinkle reduction.

[0058] According to an eighth aspect of the present invention, there is provided use of the composition according to the first aspect of the present invention in preparing a hydrogel for temporarily elevating the scar base and promoting healing.

[0059] According to a ninth aspect of the present invention, there is provided use of the composition according to the first aspect of the present invention in preparing a hydrogel for supporting dermal connective tissue formation and promoting healing in scar tissue after surgical intervention.

[0060] According to a tenth aspect of the present invention, there is provided use of the composition according to the first aspect of the present invention in preparing a hydrogel for supporting the formation of dermal connective tissue in scar treatment after burns.

[0061] According to an eleventh aspect of the present invention, there is provided use of the composition according to the first aspect of the present invention in preparing a hydrogel for supporting blood vessel ingrowth and promoting healing in acute dermal defects accompanied by bleeding.

[0062] According to a twelfth aspect of the present invention, there is provided use of the composition according to the first aspect of the present invention in preparing a hydrogel for filling a dermal cavity created by surgery.

[0063] According to a thirteenth aspect of the present invention, there is provided use of the composition according to the first aspect of the present invention in preparing a hydrogel for supporting skin transplantation surgery.

[0064] According to a fourteenth aspect of the present invention, there is provided use of the composition according to the first aspect of the present invention in preparing a hydrogel for physical delivery of a bone graft substitute.

[0065] According to a fifteenth aspect of the present invention, there is provided use of the composition according to the first aspect of the present invention in preparing a hydrogel for filling a prosthesis.

[0066] According to a sixteenth aspect of the present invention, there is provided use of the composition according to the first aspect of the present invention in preparing a hydrogel for use as a filler without tissue inducing properties.

[0067] According to a seventeenth aspect of the present invention, there is provided use of the composition according to the first aspect of the present invention in preparing a hydrogel for supporting and repairing periodontal tissue after tooth extraction.

[0068] According to an eighteenth aspect of the present invention, there is provided use of the composition according to the first aspect of the present invention in preparing a hydrogel for temporarily elevating periodontal ligament tissue and / or supporting periodontal ligament tissue transplantation.

[0069] According to a nineteenth aspect of the present invention, there is provided use of the hydrogel according to the second aspect of the present invention in the manufacture of a medicament for tissue repair and / or restoration.

[0070] According to the twentieth aspect of the present invention, there is provided a method for tissue repair and / or restoration, the method comprising administering to a mammal a composition according to the first aspect of the present invention. In one embodiment, the administering step is performed by injection or by administering an aerosol, thereby forming a hydrogel at the body temperature of the mammal.

[0071] According to the twenty-first aspect of the present invention, there is provided a method of wound healing, the method comprising administering to a mammal a composition according to the first aspect of the present invention. In one embodiment, the administering step is performed by injection or by administering an aerosol, thereby forming a hydrogel at the body temperature of the mammal.

[0072] According to a twenty-second aspect of the present invention, there is provided a method of temporary wrinkle reduction, the method comprising administering to a mammal a composition according to the first aspect of the present invention. In one embodiment, the administering step is performed by injection or by administering an aerosol, thereby forming a hydrogel at the body temperature of the mammal.

[0073] According to the twenty-third aspect of the present invention, there is provided a method of temporarily elevating the base of a scar and promoting healing, the method comprising administering to a mammal a composition according to the first aspect of the present invention. In one embodiment, the administering step is performed by injection or by administering an aerosol, thereby forming a hydrogel at the body temperature of the mammal.

[0074] According to the twenty-fourth aspect of the present invention, there is provided a method for supporting dermal connective tissue formation and promoting healing in scar tissue after surgical intervention, the method comprising administering a composition according to the first aspect of the present invention to a mammal. In one embodiment, the administering step is performed by injection or by administering an aerosol, thereby forming a hydrogel at the body temperature of the mammal.

[0075] According to the twenty-fifth aspect of the present invention, there is provided a method for supporting dermal connective tissue formation in scar treatment after burns, the method comprising administering a composition according to the first aspect of the present invention to a mammal. In one embodiment, the administering step is performed by injection or by administering an aerosol, thereby forming a hydrogel at the body temperature of the mammal.

[0076] According to the twenty-sixth aspect of the present invention, there is provided a method for supporting vascular ingrowth and promoting healing in acute dermal defects with bleeding, the method comprising administering to a mammal a composition according to the first aspect of the present invention. In one embodiment, the administering step is performed by injection or by administering an aerosol, thereby forming a hydrogel at the body temperature of the mammal.

[0077] According to the twenty-seventh aspect of the present invention, there is provided a method for filling a surgically created dermal cavity, the method comprising administering to a mammal a composition according to the first aspect of the present invention. In one embodiment, the administering step is performed by injection or by administering an aerosol, thereby forming a hydrogel at the body temperature of the mammal.

[0078] According to the twenty-eighth aspect of the present invention, there is provided a method of supporting skin graft surgery, the method comprising administering to a mammal a composition according to the first aspect of the present invention. In one embodiment, the administering step is performed by injection or by administering an aerosol, thereby forming a hydrogel at the body temperature of the mammal.

[0079] According to the twenty-ninth aspect of the present invention, there is provided a method of physically delivering a bone graft substitute, the method comprising administering to a mammal a composition according to the first aspect of the present invention. In one embodiment, the administering step is performed by injection or by administering an aerosol, thereby forming a hydrogel at the body temperature of the mammal.

[0080] According to a thirtieth aspect of the present invention, there is provided a method of filling a prosthesis, the method 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 inducing properties. In one embodiment, the administering step is performed by injection or by administering an aerosol, thereby forming a hydrogel at the body temperature of the mammal.

[0081] According to the thirty-first aspect of the present invention, there is provided a method for supporting and repairing periodontal tissue after tooth extraction, the method comprising administering to a mammal a composition according to the first aspect of the present invention. In one embodiment, the administering step is performed by injection or by administering an aerosol, thereby forming a hydrogel at the body temperature of the mammal.

[0082] According to the thirty-second aspect of the present invention, there is provided a method for temporarily elevating periodontal ligament tissue and / or supporting a periodontal ligament tissue transplant, the method comprising administering to a mammal a composition according to the first aspect of the present invention. In one embodiment, the administering step is performed by injection or by administering an aerosol, thereby forming a hydrogel at the body temperature of the mammal.

[0083] According to the thirty-third aspect of the present invention, there is provided a method for tissue repair and / or restoration, the method comprising administering to a mammal a hydrogel according to the second aspect of the present invention. In one embodiment, the administering step is performed by injection or by administering an aerosol, thereby forming a hydrogel at the body temperature of the mammal.

[0084] According to a thirty-fourth aspect of the invention, there is provided a composition according to the first aspect of the invention for use in tissue repair and / or restoration. In one embodiment, the administering step is performed by injection or by aerosol administration, thereby forming a hydrogel at the body temperature of the mammal.

[0085] 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 performed by injection or by administering an aerosol, thereby forming a hydrogel at the body temperature of the mammal.

[0086] According to a thirty-sixth 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 performed by injection or by administering an aerosol, thereby forming a hydrogel at the body temperature of the mammal.

[0087] According to a thirty-seventh aspect of the present invention, there is provided a composition according to the first aspect of the present invention for use in temporarily elevating the base of a scar and promoting healing. In one embodiment, the administering step is performed by injection or by administering an aerosol, thereby forming a hydrogel at the body temperature of the mammal.

[0088] According to the 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 after surgical intervention. In one embodiment, the administering step is performed by injection or by administering an aerosol, thereby forming a hydrogel at the body temperature of a mammal.

[0089] According to the 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 scar treatment after burns. In one embodiment, the administering step is performed by injection or by administering an aerosol, thereby forming a hydrogel at the body temperature of a mammal.

[0090] According to the 40th 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 dermal defects with bleeding. In one embodiment, the step of applying is performed by injection or by applying an aerosol, thereby forming a hydrogel at the body temperature of a mammal.

[0091] According to a 41st 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 dermal cavity. In one embodiment, the administering step is performed by injection or by administering an aerosol, thereby forming a hydrogel at the body temperature of the mammal.

[0092] According to a 42nd 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 performed by injection or by administering an aerosol, thereby forming a hydrogel at the body temperature of the mammal.

[0093] 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 the physical delivery of a bone graft substitute. In one embodiment, the administering step is performed by injection or by administering an aerosol, thereby forming a hydrogel at the body temperature of the mammal.

[0094] According to a 44th 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 inducing properties. In one embodiment, the administering step is performed by injection or by administering an aerosol, thereby forming a hydrogel at the body temperature of a mammal.

[0095] According to a 45th 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 after tooth extraction. In one embodiment, the administering step is performed by injection or by administering an aerosol, thereby forming a hydrogel at the body temperature of a mammal.

[0096] According to the forty-sixth aspect of the present invention, there is provided a composition according to the first aspect of the present invention for use in temporarily elevating periodontal ligament tissue and / or supporting periodontal ligament tissue transplantation. In one embodiment, the administering step is performed by injection or by administering an aerosol, thereby forming a hydrogel at the body temperature of a mammal.

[0097] 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 restoration. In one embodiment, the administering step is performed by injection or by administering an aerosol, thereby forming the hydrogel at the body temperature of the mammal.

[0098] In all aspects and embodiments where the step of administering is performed by administering an aerosol, the aerosol can be administered to any opening of the body, such as, but not limited to, the nasal cavity, oral cavity, or an open wound.

[0099] According to the forty-eighth aspect of the present invention, there is provided a kit for forming a hydrogel, the kit 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 homologue thereof. In one embodiment, the kit further comprises water in a separate container.

[0100] According to the forty-ninth aspect of the present invention, a kit for forming a hydrogel is provided, comprising in respective containers: a natural or synthetic peptide or protein (NSPP); and a composition, wherein the composition 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 change behavior; wherein the natural or synthetic peptide or protein (NSPP) is thymosin B-4 or a functional homolog thereof; and wherein the NSPP is cross-linked with the second monomer so that a hydrogel can be formed when the composition comes into contact with water.

[0101] 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 such that the composition, NSPP, and water are mixed together upon dispensing.

[0102] In one embodiment, the NSPP and compositions are used as fillers to deliver bone graft substitutes in situ in patients in need of such treatment. The filler holds the BGS in place (binder) for at least 6 weeks (degradation) and provides a scaffold for cell ingrowth (osteoblasts).

[0103] In another embodiment, to form an aerosol for topical application, the concentration of the polymer in the composition is from about 5 mg / mL to about 70 mg / mL. For example, the polymer concentration in the composition is 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. Therefore, the polymer can be delivered via aerosol at very low concentrations and can be adhered to the surgical site and used accordingly.

[0104] Definitions and terminology

[0105] In describing and claiming the present invention, the following terms will be used in accordance with the definitions listed below. It should also be understood that the terms used herein are for the purpose of describing specific embodiments of the present invention only and are not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art to which the present invention belongs.

[0106] Unless the context clearly requires otherwise, throughout the specification and claims, the words "comprising," "including," and the like should be construed in an inclusive sense rather than an exclusive or exhaustive sense; that is, in the sense of "including but not limited to."

[0107] The terms "preferred" and "preferably" refer to embodiments of the present invention that may provide certain benefits in certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. In addition, the recitation of one or more preferred embodiments does not mean that other embodiments are not applicable, and is not intended to exclude other embodiments from the scope of the present invention.

[0108] As used herein, phrases defining limits of range or length, such as "1 to 5," refer to any integer from 1 to 5, i.e., 1, 2, 3, 4, and 5. In other words, any range defined by two expressly mentioned integers is intended to include and disclose any integers defining the limits as well as any integers included in the range.

[0109] Except in the operating examples or otherwise stated, all numerals expressing the amount of ingredients or reaction conditions used herein are understood to be modified by the term "about" in all cases. The examples are not intended to limit the scope of the invention. Hereinafter or unless otherwise stated, "%" means "% by weight", "ratio" means "weight ratio", and "part" means "part by weight".

[0110] 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. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0111] The term "room temperature" is intended to mean a temperature of about 20°C to about 25°C.

[0112] The term "animal" includes humans and non-humans, such as mammals, eg, horses, cows, pigs, sheep, cats, dogs, and the like.

[0113] As used herein, "implant" refers to an article or device that is placed completely or partially within the body of an animal, such as by a surgical procedure.

[0114] As used herein, the term "natural or synthetic peptide or protein" (or NSPP) refers to a protein or peptide that occurs naturally in the extracellular portion of animal tissues and that 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 similar functions to those naturally occurring proteins and peptides. For example, naturally occurring proteins and peptides are those that are typically found in the extracellular matrix (or ECM), which is a defining feature of animal connective tissue. Naturally occurring proteins that are typically found in the ECM include collagen, fibrin, fibronectin, and laminin (and their isoforms).

[0115] The NSPP used in the present invention is thymosin beta-4 or a functional homologue thereof.

[0116] The following abbreviations are used in this manual:

[0117] ECM Extracellular Matrix

[0118] EHNS N-Hydroxyethoxylated succinimide

[0119] HEMA Hydroxyethyl Methacrylate

[0120] LA lactic acid

[0121] NAS N-Acryloyloxysuccinimide

[0122] NIPAAm N-isopropylacrylamide

[0123] NSPP Natural or synthetic peptide or protein

[0124] OEG oligo(ethylene glycol)

[0125] OEGMA Oligo(ethylene glycol) monomethyl ether methacrylate

[0126] PBS Phosphate buffered saline

[0127] PEG Polyethylene glycol

[0128] PEO Polyethylene Oxide

[0129] PLA Poly(lactic acid)

[0130] PPO Polyethylene oxide-co-propylene oxide

[0131] PVA Polyvinyl Alcohol

[0132] PVP Poly(vinyl pyrrolidone)

[0133] PNPHO Poly(N-isopropylacrylamide-co-(N-acryloyloxysuccinimide)-co-(polylactide / 2-hydroxymethacrylate)-co-(oligo(ethylene glycol) / poly(NIPAAm-co-NAS-co-(HEMA-PLA)-co-OEGMA)

[0134] SNHS N-Hydroxysulfosuccinimide

[0135] TB4 Thymosin beta-4 / Thymosin beta-4

[0136] In an exemplary composition according to the invention, 140 mg / mL PNPHO (ratio 5:8(5):7:81) is used with 30 mg / mL thymosin beta-4; for the applicant's clinical trial purposes, this composition is labeled PNPHO-co-TB4 (or alternatively PNPHO-co-NSPP; or "TR001") and is referred to as such throughout the text of this specification. BRIEF DESCRIPTION OF THE DRAWINGS

[0137] Preferred embodiments of the present invention will now be described, by way of example only, with reference to the accompanying drawings, in which:

[0138] FIG. 1( a ) is a macroscopic image of a solution of the present invention (polymer and NSPP) that forms a hydrogel under simulated physiological conditions (PBS at 37° C.) and retains its structure after gelation.

[0139] Figure 1(b) is a macroscopic image of PNPHO-co-TB4 injection into a site with active bleeding, showing that hydrogel was immediately formed despite the presence of active bleeding at the defect site.

[0140] Figure 1(c) is a macroscopic image sequentially showing the PNPHO-co-TB4 solution and hydrogel formation in contact with the wound at body temperature.

[0141] Figure 1(d) is a macroscopic image of PNPHO-co-TB4 mixed with blood to form an adhesive hydrogel, which was used to fill a 3D area by layer-by-layer filling.

[0142] Figure 2(a) shows the synthetic preparation of PNPHO in DMF at 70 °C.

[0143] Figure 2(b) shows the PNPHO in CDCl3 1 H NMR spectrum; overlap of the approximately 2.9-3.0 ppm resonance of residual traces of DMF solvent with the NAS protons (e) resulted in an error in the original calculation based on the same data set (integral / area under each peak as a basis for the relative mol% of each monomer in the entire PNPHO polymer). Subsequent corrections confirmed the presence of the third monomer (NAS) in an amount greater than about 7 mol%.

[0144] Figure 3 Depicted are the individual solubilities of copolymers synthesized at different mole fractions of HEMA-PLA in aqueous solution at 4°C for lactic acid numbers of 3 (a) and 6 (b) (*, **, and *** indicate p<0.05, <0.01, and <0.001, respectively).

[0145] Figure 4 LCST measurements and comparison between 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 and shows the physical role of TB4 in accelerating the gelation kinetics.

[0146] Figure 5 Figure 3 shows the investigation of the scaffolding effect of PNPHO-co-TB4 on the integration of host tissues. Full-thickness dermal wounds were formed (a); PNPHO-co-TB4 and Integra were used for skin grafting (b); and the survival rate of grafts treated with Integra or PNPHO-co-TB4 at different time points (c).

[0147] Figure 6Evaluation of the inflammatory response to PNPHO-co-TB4 and direct comparison with Integra (dermal matrix gold standard) are shown; H&E staining of Integra-treated sites two weeks (a) and four weeks (b) after surgery; and H&E staining of PNPHO-co-TB4-treated sites two weeks (c) and four weeks (d) after transplantation. White arrows show PNPHO-co-TB4 structures, and black arrows show fibrous tissue formation around the implant.

[0148] Figure 7 The angiogenic response of PNPHO-co-TB4 and Integra treated sites in mice at different time points is shown. Blood vessel formation and ingrowth at the transplant site were determined by fluorescence radiant efficiency using an IVIS Lumina XR live imager. The angiogenic response was determined two (2) and four (4) weeks after transplant surgery using the AngioSense750 EX in vivo blood pool fluorescent imaging probe. This near-infrared fluorescent macromolecular probe persists in the vasculature and is able to image blood vessels and angiogenesis. At each time point after surgery, each mouse was injected with 2nmol AngioSense750EX in 100μL PBS. After 24 hours, the fluorescence radiant efficiency of each mouse was scanned (n=8). The radiant efficiency is used to indicate the density of new blood vessels in the wound area. The results showed that two weeks after surgery, the fluorescence radiant efficiency of the PNPHO-co-TB4 treated site was significantly higher than that of the Integra treated site (p<0.01). In contrast, angiogenesis signals were very low in both treatment groups at four (4) weeks post-surgery, indicating that vascularization was controlled and healing of the site was complete.

[0149] Figure 8 Shown is a histological evaluation of the PNPHO-co-TB4 treated site 2 weeks after skin graft surgery. White arrows show blood vessel ingrowth within the PNPHO-co-TB4 hydrogel structure and infiltration of fibroblasts into the injectable scaffold. Figure 8 (c) and Figure 8 The results in (d) show the formation of blood vessels within the PNPHO-co-TB4 structure. In addition, staining of skin biopsies showed obvious infiltration of host fibroblasts within the PNPHO-co-TB4 hydrogel structure; see Figure 8 (d).

[0150] Fig. 9Shown is Masson's trichrome staining of a skin graft site treated with PNPHO-co-TB4 4 weeks post-surgery. Black arrows indicate collagen fibers that were deposited from fibroblast ingrowth within the PNPHO-co-TB4 structure. To further determine fibroblast infiltration and skin extracellular matrix formation within the PNPHO-co-TB4 structure, the skin graft site was stained with Masson's trichrome 4 weeks post-transplantation. Fig. 9 The results in Figure 4 show collagen fiber formation within the PNPHO-co-TB4 construct 4 weeks after transplantation. This result confirms the infiltration of fibroblasts within the PNPHO-co-TB4 construct and its potential to integrate with host tissue and promote neodermal formation.

[0151] Fig.10 Masson's trichrome staining of a skin graft site treated with Integra 4 weeks after surgery is shown. Collagen fibril formation within PNPHO-co-TB4 constructs was significantly higher than that detected within Integra constructs. Results show that collagen formation was significantly reduced within Integra constructs compared to PNPHO-co-TB4.

[0152] Fig.11 Shown are the use of PNPHO-co-TB4 after tooth extraction, (a) extraction site with active bleeding, (b) injection of PNPHO-co-TB4 into the alveolar site via a 21G needle, (c) immediate gelation of PNPHO-co-TB4 at the site, and (d) a mixture of PNPHO-co-TB4 and blood at the site.

[0153] Fig.12 Depicted is the application of PNPHO-co-TB4 following tooth extraction in 10 patients. Clinical use of the device in the PET trial demonstrated that injection of PNPHO-co-TB4 into the alveolar site was successful in all ten patients, and the principal investigator did not report any device malfunctions.

[0154] Fig.13 Soft tissue regeneration and wound healing 7 days after surgery and PNPHO-co-TB4 treatment were shown. All ten patients treated with PNPHO-co-TB4 returned for the first follow-up one week after surgery. There were no reports of pain or discomfort in any patient. During the oral examination (one week after application), there were no signs of infection or inflammation at the site. In addition, the principal investigator examined wound closure and soft tissue formation. In all ten patients, wound closure was recorded and accelerated soft tissue formation was detected.

[0155] Fig.14H&E and Masson's trichrome staining of PNPHO-co-TB4 treated samples are shown.

[0156] Fig.15 Representative images of the plume patterns of formulations A) 17.5 mg / mL, B) 35 mg / mL, and C) 70 mg / mL are shown.

[0157] Fig.16 The maximum and minimum plume coverage diameters and derived ellipticity are depicted for the three formulations (mean ± SD, n = 3).

[0158] Fig.17 The deposition pattern of each formulation using the human nose model is shown.

[0159] Fig.18 The in vitro release of ciprofloxacin HCl from PNPHO and PNPHO-co-TB4 hydrogels at 37°C is shown, expressed as cumulative mass (results are indicative, n=1±SD). DETAILED DESCRIPTION

[0160] The present invention will now be described more fully with reference to the accompanying examples and drawings. However, it should be understood that the following description is illustrative only and should not be regarded in any way as limiting the generality of the present invention described above.

[0161] Reference will now be made in detail to certain embodiments of the present invention. Although the present invention will be described in conjunction with the embodiments, it will be understood that it is not intended to limit the present invention to those embodiments. On the contrary, the present invention is intended to cover substitutions, modifications and equivalents that may be included within the scope of the present invention as defined by the claims. Those skilled in the art will recognize that many methods and materials similar or equivalent to the methods and materials described herein may be used in the practice of the present invention. The present invention is by no means limited to the methods and materials described.

[0162] 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 evident from the text or drawings. All these different combinations constitute various alternative aspects of the invention.

[0163] Disclosed herein are uses of polymers that are tailored to tissue.The compositions of the present invention are preferably injectable.

[0164] 1. Polymer

[0165] As used herein, the term "polymer" refers to a macromolecule (polymer) composed of repeating structural units (monomers). These subunits are usually connected by covalent chemical bonds. The polymer can be a linear or branched polymer. Preferably, the polymer of the present invention is a copolymer comprising three or more different monomers.

[0166] 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 the NSPP.

[0167] As used herein, the term "monomer" refers to a structural unit that can be combined to form a polymer, but can itself be a polymer, or a derivative of a monomer or a polymer. The latter type of monomer is also referred to herein as a "macromer".

[0168] As used herein, a "macromer" is a polymer or oligomer whose molecules each have one end group that acts as a monomer molecule, so that each polymer or oligomer molecule contributes only a single monomer unit to the chain of the product polymer.

[0169] The polymer of the composition of the present invention 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.

[0170] 1.1. First monomer: water-binding monomer

[0171] As described above, the advantageous properties of the preferred hydrogels used herein can be attributed to the combination of the NSPPs with the specific components of the preferred polymers. One particularly advantageous property of these preferred polymers is their water binding ability. The presence of water in the hydrogel both provides an environment similar to the natural environment of the damaged tissue (which aids in tissue regeneration) and provides the hydrogel with the desired compressive resistance.

[0172] Therefore, the preferred polymers used herein should contain monomers or units capable of binding water to have the following ability: when the polymer is in contact with NSPP and water, it is capable of forming a hydrogel. In addition, the hydrogel formed thereby should have the desired compression resistance and resilience.

[0173] Those skilled in the art will appreciate that the water-binding monomers need to be present in the preferred polymers used in the present invention in a ratio sufficient to produce a polymer that meets these requirements. Typically, the ratio of water-binding 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 molar ratio of water-binding monomer to mechanical strength monomer. In fact, the water-binding monomers need not only render the polymer hydrophilic, but also render the polymer more water-binding.

[0174] Thus, preferred polymers for use in the present invention will have a water binding capacity of about 70% to about 500%, about 80% to about 400%, about 90% to 300%, or about 100% to 200%. For example, preferred polymers for use herein have a water binding capacity of 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 0%, 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%.

[0175] Suitable examples of water-binding monomers include those from which polymers such as polyethers (e.g., basic polyimides such as polyethylene glycol (PEG), oligo(ethylene glycol) (OEG), polyethylene oxide (PEO), polyethylene oxide-co-propylene oxide (PPO), co-polyethylene oxide blocks or random copolymers thereof, polyvinyl alcohol (PVA), poly(vinyl pyrrolidone) (PVP), poly(amino acids), and dextran. Polyethers, and more particularly oligo(oxyalkylenes) such as OEG are particularly preferred because they have the desired water-binding capacity, are easily synthesized and / or purchased, and are inert in the sense that they elicit minimal or no immune response in the tissue in which they are placed.

[0176] In addition, any of a variety of hydrophilic functional groups can be used to render the monomers (and thus the polymers formed from such monomers) water soluble. For example, water soluble functional groups such as phosphates, sulfates, quaternary amines, hydroxyls, amines, sulfonates, and carboxylates can be introduced into the monomers to render them water soluble.

[0177] Monomers may also react with other compounds to form "macromers." Thus, the first monomer may optionally be a macromer.

[0178] A preferred first monomer as the macromonomer is oligo(ethylene glycol) monomethyl ether methacrylate (OEGMA), which is a hydrophilic monomer composed of two hydrophilic monomers, ethylene glycol and methacrylate.

[0179] 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 %.

[0180] 1.2. Second monomer: monomer that imparts mechanical properties

[0181] As discussed above, the advantageous properties of preferred hydrogels used in the present invention can be attributed in part to the specific components that make up the polymer. In some embodiments, preferred polymers used in the present invention are capable of contributing additional mechanical properties to the hydrogel.

[0182] Those skilled in the art will appreciate that monomers capable of imparting mechanical properties to the hydrogel need to be present in the preferred polymer in a ratio sufficient to produce a hydrogel with the desired mechanical properties. Typically, the ratio 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 molar ratio of water-binding monomer to mechanical strength monomer. Suitable examples of monomers capable of imparting mechanical properties (e.g., compression resistance) to the hydrogel include methacrylates, such as hydroxyethyl methacrylate (HEMA), hydroxyethyl methacrylate poly(lactic acid) copolymers (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)).

[0183] The monomers may also react with other compounds to form a "macromer." A preferred second monomer as a macromer is hydroxyethyl methacrylate poly(lactic acid) (HEMA-PLA).

[0184] 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 %.

[0185] 1.3. The third monomer: NSPP binding monomer

[0186] As described above, the preferred hydrogel used in the present invention is formed by combining a polymer with a NSPP in the presence of water. In order to effectively combine the polymer with the NSPP, it is preferred that a monomer or unit having cross-linking ability is included in the polymer.

[0187] This cross-linking ability means that the polymer is able to bind to the NSPP (as discussed further below) and by doing so cross-link the NSPP to form a hydrogel comprising the NSPP. Alternatively, by a similar mechanism, the NSPP acts as a cross-linking agent, thereby cross-linking the polymer to form a hydrogel.

[0188] By utilizing polymer design wherein monomers having functional groups that bind to thymosin beta-4 are provided in the polymer, among other things, the inventors have recognized that the polymer does not need to be further crosslinked, such as by chemical or UV crosslinking, to form a hydrogel.

[0189] Additionally, by covalently binding the NSPP to the polymer, the NSPP is more effectively retained within the hydrogel network, meaning that once the hydrogel is applied to the repair site, the NSPP cannot easily migrate away from the site. This means that the structural integrity of the gel at the repair site is maintained (due to the mechanical properties of the NSPP as described above), and helps provide an environment at the repair site that closely mimics the natural environment of the tissue.

[0190] In order to produce a polymer capable of binding to a NSPP, one skilled in the art will appreciate that the monomer capable of binding to a NSPP needs to be present in the polymer of the invention in a ratio sufficient to crosslink with the NSPP so that a hydrogel can be formed in the presence of water. Typically, the ratio 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.

[0191] Monomers capable of binding to NSPPs typically have electrophilic or nucleophilic functional groups, such that, for example, nucleophilic functional groups on the NSPPs can react with electrophilic functional groups on the monomers to form covalent bonds. Preferably, the polymer comprises more than two NSPP binding monomers, such that the polymer combines with the NSPPs to form a cross-linked polymer product due to an electrophilic-nucleophilic reaction. Such a reaction is referred to as a "cross-linking reaction".

[0192] Thus, for example, if the NSPP has a nucleophilic functional group (e.g., an amine), the polymer may have an electrophilic functional group (e.g., 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-acryloyloxysuccinimide (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 thiol.

[0193] Preferably, the polymer comprises the 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%.

[0194] 1.4. The fourth monomer: phase change monomer

[0195] In another embodiment, the preferred polymer 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. In addition, these phase transition properties allow the preferred polymers used in the present invention to form hydrogels, various properties of which (e.g., viscosity) can be altered by changing factors such as pH and temperature.

[0196] Thermoresponsive injectable hydrogels are designed so that their lower critical solution temperature (LCST) is below body temperature. Therefore, gelation can be achieved simply by increasing the temperature of the hydrogel, for example by warming it to body temperature (which occurs when the hydrogel is administered into the body). A variety of thermoresponsive and 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 monomeric block or poly-NIPAAm) is particularly suitable because its LCST is 32°C, making it in gel form at body temperature.

[0197] In order to produce a thermoresponsive polymer, one skilled in the art will appreciate that the phase change monomers need to be present in the composition used in the present invention in a ratio sufficient to be able to change the viscosity of the hydrogel containing the polymer by exposing the hydrogel to different temperature and pH conditions. Typically, the ratio of "phase change" monomers in the polymer is at least about 9:1 molar ratio of phase change monomer: water binding monomer. This ratio can be increased to, for example, 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 molar ratio of phase change monomer: water binding monomer.

[0198] The viscosity of the preferred hydrogel used in the present invention at a relatively low temperature (e.g., 4° C.) makes the hydrogel injectable. Then, as the temperature rises, the hydrogel becomes more viscous, forming a gel with a desired viscosity at a temperature of about 37° C. This means that the preferred hydrogel used in the present invention can be easily applied to the repair site by, for example, injection or by aerosol application at a relatively low temperature. Then, by warming up to the body's natural temperature in vivo, the hydrogel is converted into a more viscous gel with desired strength and elasticity.

[0199] Preferably, the polymer comprises a fourth monomer in an amount constituting the remainder of 100% of the polymer composition. In one embodiment, the mol % of the fourth monomer may 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 %.

[0200] 1.5. Other polymer properties

[0201] Those skilled in the art will appreciate that by combining different types of monomers, polymers with a series of different properties can be produced. In addition, by introducing specific monomers or functional groups into pre-existing polymers, the performance of polymers can be changed. For example, the copolymerization of HEMA monomers and other monomers (such as methyl methacrylate) can be used to change performance such as swelling and mechanical properties. Monomers can also react with other compounds to form "macromolecular monomers" (as described above), which are then included in the preferred polymers used in the present invention. For example, HEMA can react with lactide to form HEMA-polylactic acid polymers (HEMA-PLA), which itself can be used as monomers in polymers of the present invention. In addition, monomers themselves can be a combination of monomer units, which are then introduced into polymers. An example of this type of monomer is oligomeric (ethylene glycol) monomethyl ether methyl methacrylate (OEGMA), which is a hydrophilic monomer consisting of two hydrophilic monomers ethylene glycol and methacrylate.

[0202] The preferred polymer used in the present invention can be further modified with one or more parts and / or functional groups. Any part or functional group can be used as required. In some embodiments, the polymer can be modified with polyethylene glycol (PEG), carbohydrate and / or the acyclic polyacetal derived from polysaccharide. In addition, as mentioned above, hydrophilic groups can be introduced into the monomer (and therefore polymer), to increase the water binding capacity of the polymer.

[0203] In terms of sequence, copolymers can be block copolymers, graft copolymers, random copolymers, blends, mixtures and / or adducts of any of the aforementioned and other polymers. Typically, the polymer used according to 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 biocompatible and biodegradable.

[0204] Preferred polymers for use in the present invention may also contain other monomers in their structure. For example, the monomer may be a polymer such as poly(vinyl alcohol) (PVA), polyesters, acrylic polymers and ionomers, or monomers of these.

[0205] If the desired polymer is biodegradable or absorbable, one or more monomers with biodegradable linkages can be used. Alternatively or additionally, monomers can be selected so that the reaction product therebetween produces a biodegradable linkage. For each method, monomers and / or linkages can be selected so that the resulting biodegradable polymer degrades or is absorbed in a desired time period, such as from about 6 hours to about 6 months. Preferably, monomers and / or linkages are selected so that the resulting product is nontoxic when the polymer degrades under physiological conditions.

[0206] Biodegradable links can be chemical or enzymatically hydrolyzable or absorbable. Illustrative, exemplary and non-restrictive chemically hydrolyzable biodegradable links include polymers, copolymers and oligomers of glycolide, lactide, caprolactone, dioxanone and trimethylene carbonate. Exemplary enzymatically hydrolyzable biodegradable links include peptide bonds that can be cut by metalloproteinases and collagenases. Other exemplary biodegradable links include polymers and copolymers of poly(hydroxy acids), poly(orthocarbonates), poly(anhydrides), poly(lactones), poly(amino acids), poly(carbonates) and poly(phosphonates).

[0207] The chemical hydrolysis of lactide in the present invention leads to an increase in the lower critical solution temperature (LCST) of the polymer (by reducing the overall hydrophobicity of the polymer), thereby improving its bioabsorption capacity.

[0208] 1.6. Preferred polymers

[0209] 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 %.

[0210] 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 %.

[0211] The polymer preferably comprises the 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 %.

[0212] The polymer preferably comprises a fourth monomer in an amount constituting the remainder of 100% of the polymer composition, e.g., about 60 to about 81 mol %, e.g., 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 %.

[0213] The percentages stated herein relate to the composition of the final polymer and not the amounts of feedstock used in forming the polymer.

[0214] In one embodiment, the polymer preferably comprises:

[0215] i. an amount of about 3 to about 8 mol % (eg, about 4 to about 6 mol %) of the first monomer;

[0216] ii. an amount of about 5 to about 9 mol % (eg, about 6 to about 8 mol %) of a second monomer;

[0217] iii. an amount of at least about 7 mol% of a third monomer; and

[0218] iv. a fourth monomer in an amount up to about 85 mol % (eg, up to about 81 mol %).

[0219] In another embodiment, the polymer preferably comprises:

[0220] i. an amount of about 5 mol% of the first monomer;

[0221] ii. an amount of about 7 mol% of a second monomer;

[0222] iii. a third monomer in an amount of about 7 mol%; and

[0223] iv. a fourth monomer in an amount of about 81 mol%.

[0224] In one embodiment, the preferred polymer used in the present invention is a polymer of formula (I):

[0225]

[0226] in

[0227] A is the first monomer (water-binding monomer);

[0228] B is the second monomer (a monomer capable of imparting mechanical properties to the hydrogel);

[0229] C is the third monomer (a monomer having a functional group that binds to the NSPP);

[0230] D is the fourth monomer (a monomer capable of imparting phase change properties to the hydrogel);

[0231] m is an integer from 1 to 20, for example, from 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;

[0232] n is an integer from 1 to 20, for example, from 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;

[0233] p is an integer from 1 to 20, such as from 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; and

[0234] q is an integer from 1 to 20, for example, from 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.

[0235] Preferably, the ratio of m:n:p:q is about 5:8(5):7:81. One skilled in the art will appreciate that monomers A, B, C and D may be present in the polymer in any order as long as the desired water binding, reinforcement and / or crosslinking capabilities are achieved.

[0236] 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.

[0237] An example of a polymer of formula (I) is a polymer of formula (Ia):

[0238]

[0239] wherein A is the water-binding monomer OEGMA, B is the reinforcing monomer HEMA-PLA, C is the cross-linking agent NAS, D is the phase-change monomer NIPAAm, and m, n, p, q, x and y are as defined above.

[0240] One skilled in the art will appreciate that monomers A, B, C and D may be present in the polymer in any order so long as the desired water binding, reinforcement and / or cross-linking capabilities are achieved.

[0241] It has also been found that some monomers (e.g., HEMA-PLA), polyesters (e.g., poly(lactic acid), poly(caprolactone), poly(glycolide) and their random copolymers (e.g., poly(glycolide-co-lactide) and poly(glycolide-co-caprolactone) and other biodegradable, biocompatible polymers can increase the LCST of the preferred polymers used in the present invention during the degradation of the biodegradable segment (e.g., PLA) in vivo, thereby leading to bioabsorption of the polymer. This provides another advantage that the polymers used in the present invention can be designed to be biodegradable in vivo.

[0242] The overall size of the preferred polymers used in the present invention may vary depending on factors such as the type of monomers introduced into the polymer, the type of NSPP sought to form the hydrogel, and the conditions under which the protein is coupled to the polymer. However, in general, the preferred polymers used in the present invention may be molecules of about 1 to about 100 kDa, about 5 to about 60 kDa, or about 30 kDa.

[0243] 1.7.PNPHO

[0244] Preferably the polymer is PHPHO.The polymer PNPHO preferably comprises OEGMA in an amount of about 3 to about 8 mol%, such as about 4 to about 7 mol%, or about 3, 4, 5, 6, 7 or 8 mol%.

[0245] The polymer preferably comprises HEMA-PLA in an amount of about 5 to about 9 mol%, such as about 6 to about 8 mol%, or about 3, 4, 5, 6, 7 or 8 mol%.

[0246] The polymer preferably comprises NAS 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%.

[0247] The polymer preferably comprises NIPAAm in an amount constituting the remainder of 100% of the polymer composition, e.g., about 64 to about 85 mol%, e.g., 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%.

[0248] The percentages stated herein relate to the composition of the final polymer and not the amounts of feedstock used in forming the polymer.

[0249] In one embodiment, preferably, the polymer comprises:

[0250] i. OEGMA in an amount of about 3 to about 8 mol % (e.g., about 4 to about 6 mol %);

[0251] ii. HEMA-PLA in an amount of about 5 to about 9 mol % (e.g., about 6 to about 8 mol %);

[0252] iii. NAS in an amount of at least about 7 mol%; and

[0253] iv. NIPAAm in an amount up to about 85 mol % (eg, up to about 81 mol %).

[0254] In another embodiment, the polymer comprises:

[0255] i. OEGMA in an amount of about 5 mol%;

[0256] ii. HEMA-PLA in an amount of about 7 mol%;

[0257] iii. NAS in an amount of about 7 mol%; and

[0258] iv. NIPAAm in an amount of about 81 mol%.

[0259] A preferred form of polymer PNPHO for use in the present application is the polymer of formula (Ia) as shown above.

[0260] Based on the previously defined formula I:

[0261] iA is oligo(ethylene glycol) monomethyl ether methacrylate OEGMA;

[0262] ii. B is hydroxyethyl methacrylate poly(lactic acid) (HEMA-PLA);

[0263] iii. C is N-acryloyloxysuccinimide (NAS); and

[0264] iv. D is N-isopropylacrylamide (NIPAAm).

[0265] In addition, 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.

[0266] One skilled in the art will appreciate that monomers A, B, C and D may be present in the polymer in any order so long as the desired water binding, reinforcement and / or cross-linking capabilities are achieved.

[0267] 1.8. Polymer synthesis

[0268] Those skilled in the art will be aware of suitable methods for synthesizing the preferred polymers used in the present invention. These include methods such as ring-opening polymerization, addition polymerization (including free radical polymerization) and condensation polymerization.

[0269] The formation of the preferred polymer PNPHO is described in the examples below.

[0270] 2. Composition for forming hydrogel

[0271] The present invention also relates to preferred compositions useful for forming the hydrogels used in the present invention.

[0272] The composition of the present invention comprises a polymer and a NSPP, wherein the polymer comprises:

[0273] i. a first water-binding monomer; and

[0274] ii. a second monomer that imparts mechanical properties;

[0275] iii. a third monomer as a NSPP binding monomer, comprising a functional group capable of binding to the NSPP;

[0276] iv. a fourth monomer capable of imparting phase change properties to the hydrogel;

[0277] wherein the natural or synthetic peptide or protein (NSPP) is thymosin beta-4 or a functional homologue thereof;

[0278] And wherein the combination of the NSPP and the second monomer crosslinks the polymer, thereby enabling the formation of a hydrogel when the composition is contacted with water.

[0279] As used herein, the term "composition" refers to a solid or liquid composition comprising the above-mentioned components. In some embodiments, other components, such as pharmaceutically acceptable excipients and bioactive agents (e.g., drugs, vitamins and minerals) may also be included in the preferred compositions used in the present invention to help repair and / or regenerate target bone tissue, and / or provide a method for achieving targeted delivery of bioactive compounds.

[0280] Generally, the amount of polymer in the compositions used in the present invention is an amount that allows the formation of a hydrogel.

[0281] In some embodiments, the amount of polymer in the composition is: about 1% w / w to about 90% w / w, about 2% w / w to about 80% w / w, about 4% w / w to about 70% w / w, about 5% w / w to about 60% w / w, about 5% w / w to about 50% w / w, about 6% w / w to about 40% w / w, about 7% w / w to about 30% w / w, or about 8% w / w to about 20% w / w.

[0282] 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 about 85% w / w.

[0283] In general, as the concentration of polymer in the composition increases, the firmness of the hydrogel increases.

[0284] Typically, the amount of NSPP in the compositions of the present invention is an amount that allows for the formation of a hydrogel.

[0285] In some embodiments, the amount of NSPP in the composition is about 0.01% w / w to about 60% w / w, about 1% w / w to about 50% w / w, about 1% w / w to about 40% w / w, about 5% w / w to about 30% w / w, about 5% w / w to about 20% w / w, or about 5% w / w to about 10% w / w.

[0286] 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.

[0287] The w / w % is based on the total weight of the composition before the composition is contacted with water.

[0288] In one embodiment, the composition comprises equimolar amounts of the polymer and thymosin beta-4 or a functional homologue thereof.

[0289] 2.1. Excipients and bioactive agents

[0290] 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 carriers, dispersion or suspension aids, granulating agents, surfactants, disintegrants, isotonic agents, thickeners or emulsifiers, preservatives, binders, lubricants, buffers, oils, etc. suitable for the desired particular dosage form. Remington (Gennaro, AR, Remington: The Science and Practice of Pharmacy, 21st edition (2006) Lippincott Williams & Wilkins) discloses a variety of excipients for formulating pharmaceutical compositions and known techniques for their preparation. Unless any conventional excipient is incompatible with a substance or its derivatives, for example by producing any undesirable biological effect or interacting in a deleterious manner with any other component of the pharmaceutical composition, its use is contemplated to be within the scope of the present invention.

[0291] Excipients such as coloring agents, coating agents, sweetening, flavoring and perfuming agents may be present in the composition, according to the judgment of the formulator.

[0292] Bioactive agents or pharmaceutical compounds that can be added to the 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, anti-tumor agents, local anesthetics, hormones, angiogenic agents, anti-angiogenic agents, growth factors (such as insulin-like growth factor-1 (IGF-1), basic fibroblast growth factor (bFGF) and transforming growth factor b (TGFb), antibodies, neurotransmitters, psychotropic drugs, anti-cancer drugs, chemotherapeutic drugs, drugs affecting reproductive organs, genes and oligonucleotides.

[0293] Compositions containing components such as excipients and / or bioactive agents can be prepared by combining the preferred polymers disclosed herein with the NSPP, drying the resulting composition, and then combining it with one or more other components. The resulting composition can be in a powder or other granulated form, to which water can be added to form a hydrogel according to the present invention. Hydrogels containing these components can therefore be prepared simply by adding the desired aqueous solvent to the composition.

[0294] The amounts of polymer, NSPP and bioactive agent present in the preferred compositions used in the present invention will necessarily depend on the particular drug and condition to be treated. One skilled in the art will know the appropriate agents and amounts for treating the condition.

[0295] Exemplary embodiments include fat grafts, demineralized bone matrix (DBM), autologous grafts (i.e., bioactive grafts). Such additives serve to impart tissue induction properties to the composite material. For example, low concentrations of growth factors are present in DBM / autologous grafts, etc.

[0296] Other exemplary embodiments include bone particles (from human or animal sources) and purely inert space fillers, such as glass beads. The role of such additives is to provide the composite with the desired 3D structure.

[0297] Those of ordinary skill in the art will appreciate that bone graft substitute refers to a broad range of particles including, but not limited to, synthetic calcium / phosphate particles, animal-derived bone particles, and unprocessed human bone.

[0298] 2.2. Natural or synthetic peptides or proteins (NSPP)

[0299] In the context of the present invention, NSPP is relevant because, as described above, it crosslinks the polymer, which enables the polymer to form a hydrogel. Preferred hydrogels 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 (e.g., strength and elasticity) to the hydrogel and provides an environment at the repair site that mimics the natural environment, thereby facilitating tissue repair and regeneration.

[0300] Importantly, the NSPP comprises side chains or other functional groups that are exposed to react with functional groups of the NSPP binding monomer, thereby allowing the NSPP to be bound to the polymer via the NSPP binding monomer. Examples of suitable side chains include glutamic acid or lysyl side chains.

[0301] 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 also includes apo and halo forms, post-translationally modified forms, and glycosylated or deglycosylated derivatives of NSPP. Such functionally active fragments and variants include, for example, those with conservative amino acid substitutions.

[0302] Preferably, the NSPP for use in the present invention will be obtained from a recombinant source, although it may also be extracted from a natural source or synthesized.

[0303] 2.3 Thymosin β-4

[0304] The preferred NSPP is thymosin beta-4. Thymosin beta-4 is a highly conserved, naturally occurring, water-soluble, regenerative peptide that is present in all tissues and all cell types except red blood cells. It is also present in blood and other body fluids, including tears, saliva, cerebrospinal fluid, and wound fluid.

[0305] Human thymosin beta-4 has the following sequence: SDKPDMAEIE KFDKSKLKKT ETQEKNPLPS KETIEQEKQAGES.

[0306] Thymosin beta-4 can alternatively be written as: 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.

[0307] For the purposes of the subsequent discussion, the terms thymosin beta-4 and TB4 are used synonymously; TB4 is the abbreviated form of thymosin beta-4.

[0308] According to an exemplary embodiment of the present invention, thymosin beta-4 is most preferably used with PHPHO in a molar ratio of about 1:1. However, this can be switched depending on the intended application. In an exemplary composition, 140 mg / mL PNPHO (ratio 5:8(5):7:81) is used with 30 mg / mL thymosin beta-4; for the applicant's clinical trial purposes, this composition is labeled "PNPHO-co-TB4".

[0309] 2.4 Functional homologues (isoforms) of thymosin β-4

[0310] Another preferred NSPP is a functional homolog of thymosin beta-4. Functional homologs of the above polypeptides are also suitable for use in the compositions and methods described herein. A functional homolog is a polypeptide that has sequence similarity to a reference polypeptide and performs one or more biochemical or physiological functions of the reference polypeptide.

[0311] Functional homologues and reference polypeptides can be naturally occurring polypeptides, and sequence similarities can be attributed to convergent or divergent evolutionary events. Therefore, functional homologues are sometimes referred to in the literature as homologues or orthologues or paralogues. Variants of naturally occurring functional homologues, such as polypeptides encoded by mutants of wild-type coding sequences, can themselves be functional homologues.

[0312] Functional homologues can also be produced by site-directed mutagenesis of the coding sequence of the polypeptide or by combining domains from the coding sequences of different naturally occurring polypeptides ("domain swapping"). Techniques for modifying genes encoding functional polypeptides described herein are known and include, among others, directed evolution techniques, site-directed mutagenesis techniques, and random mutagenesis techniques, and can be used to increase a specific activity of a polypeptide, alter substrate specificity, alter expression levels, alter subcellular localization, or modify polypeptide: polypeptide interactions in a desired manner. Such modified polypeptides are considered to be functional homologues. The term "functional homologue" is sometimes applied to nucleic acids encoding functional homologous polypeptides.

[0313] Functional homologues can be identified by analyzing nucleotide and polypeptide sequence comparisons. For example, queries can be made on databases of nucleotide or polypeptide sequences to identify homologues of polypeptides. Sequence analysis can involve BLAST, Reciprocal BLAST or PSI-BLAST analysis of non-redundant databases using amino acid sequences as reference sequences. In some cases, amino acid sequences are derived from nucleotide sequences. Those polypeptides with greater than 40% sequence identity in the database are candidates for further evaluation of suitability as polypeptides. Amino acid sequence similarity allows conservative amino acid substitutions, such as a hydrophobic residue is substituted with another hydrophobic residue, or a polar residue is substituted with another polar residue. If desired, these candidates can be manually checked to narrow the number of candidates to be further evaluated. Can be manually checked by selecting those candidates that appear to have domains (e.g., conservative functional domains) present in the polypeptide.

[0314] Conserved regions can be identified by locating regions in the primary amino acid sequence of a polypeptide that serve as repeats, form some secondary structure (e.g., helices and beta sheets), create positively or negatively charged domains, or represent protein motifs or domains; see, for example, the Pfam website at www.sanger.ac.uk / Software / Pfam / and pfam.janelia.org / , which describes consensus sequences for a variety of protein motifs and domains. The 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 identified by aligning sequences of the same or related polypeptides from closely related species. Closely related species are preferably from the same family. In some embodiments, alignment of sequences from two different species is sufficient.

[0315] Typically, polypeptides that exhibit at least about 40% amino acid sequence identity can be used to identify 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.

[0316] 3. Hydrogel

[0317] The present invention also relates to a hydrogel comprising a polymer according to the present invention, a NSPP and water, wherein the polymer comprises:

[0318] i. a first water-binding monomer;

[0319] ii. a second monomer that imparts mechanical properties;

[0320] iii. a third monomer as a NSPP binding monomer, comprising a functional group capable of binding to the NSPP;

[0321] iv. a fourth monomer capable of imparting phase change properties to the hydrogel;

[0322] wherein the natural or synthetic peptide or protein (NSPP) is thymosin beta-4 or a functional homologue thereof;

[0323] And wherein the combination of the NSPP and the third monomer cross-links the polymer to form a hydrogel containing water therein.

[0324] In one embodiment, the hydrogel comprises a polymer having the above-mentioned monomers so that the hydrogel can be phase-changed from a liquid state at a relatively low temperature to a gel state at body temperature. An example of a monomer for this purpose is NIPAAm. By using this monomer, thymosin β-4 can be converted from a liquid state to a gel state according to a temperature curve. Therefore, the advantage is that the preferred hydrogel used in the present invention can be easily applied by, for example, injection or aerosol at a relatively low temperature. After warming up to natural body temperature in vivo, the hydrogel is subsequently converted into a more viscous gel with desired strength and elasticity.

[0325] Having provided the above polymer compositions, a hydrogel can be formed by adding water to the composition in any manner known to those skilled in the art. Indeed, one advantage of the present invention is that the polymer does not need to be cross-linked in any manner prior to contact with the NSPP to form a hydrogel.

[0326] Cells

[0327] Preferred hydrogels for use in the present invention may also contain cells to assist in the repair and / or regeneration of target tissues.

[0328] Generally, the cells used according to the present invention are any type of cells. When encapsulated or fixed in the preferred hydrogel used in the present invention, the cells should be alive. The products of the present invention can effectively fix cells. For example, injecting / spraying a layer of hydrogel at the site, then adding the cells to the viscous hydrogel, and then covering with another layer of hydrogel, the desired result is achieved. In this way, encapsulation occurs in vivo rather than in vitro.

[0329] In some embodiments, cells that can be encapsulated in hydrogels 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 can be encapsulated in hydrogels include stem cells, totipotent cells, pluripotent cells, and / or embryonic stem cells.

[0330] In some embodiments, exemplary cells that can be encapsulated / fixed in hydrogels include, but are not limited to, primary cells and / or cell lines from any tissue. For example, 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, neurons, sperm cells, egg cells, hepatocytes, epithelial cells from the lungs, epithelial cells from the digestive tract, epithelial cells from the intestines, liver, epithelial cells from the skin, etc., and / or hybrids thereof can be encapsulated in preferred hydrogels used according to the present invention.

[0331] Exemplary mammalian cells that can be encapsulated in preferred hydrogels for use 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, HEK 293 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.

[0332] In some embodiments, it is desirable that the cells are evenly distributed throughout the hydrogel. Even distribution can help provide a more uniform tissue-like hydrogel, thereby providing a more uniform environment for encapsulated cells. In some embodiments, the cells are located on the surface of the hydrogel. In some embodiments, the cells are located inside the hydrogel. In some embodiments, the cells are layered within the hydrogel. In some embodiments, the hydrogel comprises different cell types.

[0333] In some embodiments, the conditions under which cells are encapsulated in a hydrogel are changed to maximize cell viability. In some embodiments, for example, cell viability increases with lower polymer concentrations. In some embodiments, cells at the edge of the hydrogel tend to have reduced viability relative to cells fully encapsulated in the hydrogel. In some embodiments, it may be necessary to adjust and / or change the conditions of the surrounding environment (e.g., pH, ionic strength, nutrient availability, temperature, oxygen availability, molar osmotic pressure concentration, etc.) to maximize cell viability.

[0334] In some embodiments, cell viability can be measured by monitoring one of many indicators of cell viability. In some embodiments, the indicator of cell viability includes but is not limited to: intracellular esterase activity, plasma membrane integrity, metabolic activity, gene expression and protein expression. As just one example, when cells are exposed to fluorescent esterase substrates (such as calcein AM), living cells emit green fluorescence due to the intracellular esterase activity of hydrolyzing the esterase substrate into a green fluorescent product. As another example, when cells are exposed to fluorescent nucleic acid stains (such as ethidium homodimer-1), dead cells emit red fluorescence due to their damaged plasma membrane and thus permeable high-affinity nucleic acid stains.

[0335] Typically, the number / amount of cells in the composition is an amount that allows the formation of a preferred hydrogel for use according to 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, from about 1.0% w / w to about 50% w / w, from about 1.0% w / w to about 40% w / w, from about 1.0% w / w to about 30% w / w, from about 1.0% w / w to about 20% w / w, from about 1.0% w / w to about 10% w / w, from about 5.0% w / w to about 20% w / w, or from about 5.0% w / w to about 10% w / w. In some embodiments, the number / amount of cells in the composition suitable for forming a hydrogel is about 5% w / w.

[0336] In some embodiments, the concentration of cells in the precursor solution suitable for forming a hydrogel ranges from about 10 to about 1 x 10 8 cells / mL, about 100 to about 1 x 10 7cells / mL, approximately 1 x 10 3 About 1 x 10 6 cells / mL, or approximately 1 x 10 4 About 1 x 10 5 In 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.

[0337] In some embodiments, a single hydrogel may contain 3, 4, 5, 10 or more types of cells. As just one example, 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.

[0338] 3.2. Culture medium

[0339] Any of the various cell culture media (including complex culture media and / or serum-free culture media) that can support the growth of one or more cell types or cell lines can be used to grow cells and / or maintain cells. Typically, cell culture media includes buffer, salt, energy source, amino acid (e.g., natural amino acid, non-natural amino acid, etc.), vitamins and / or trace elements. Cell culture media can optionally include a variety of other ingredients, including but not limited to carbon sources (e.g., natural sugars, non-natural sugars, etc.), cofactors, lipids, sugars, nucleosides, animal-derived components, hydrolyzates, hormones, growth factors, surfactants, indicators, minerals, activators of specific enzymes, activator inhibitors of specific enzymes, enzymes, organic matter and / or small molecule metabolites.

[0340] Cell culture media suitable for use according to 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 culture media: RPMI-1640 medium, Dulbecco's modified Eagle's medium, Minimum Essential Medium Eagle, F-12K medium, Iscove's modified Dulbecco's medium.

[0341] Those skilled in the art will recognize that the cells listed herein represent an exemplary, but not comprehensive, list of cells that may be encapsulated within a precursor solution (and thus ultimately within a hydrogel) according to the present invention.

[0342] 4. Application

[0343] The purpose of the present invention is to provide a filler that supports the natural healing of damaged tissue without inducing any specific tissue formation. The purpose is to use the present invention to fill tissue cavities or cover tissue defects to provide partial or complete required filling space and have minimal foreign body reaction.

[0344] Preferably, the composition of the present invention is administered to a subject (eg, a mammal) by injection or by spraying particles of the present invention via an aerosol or the like.

[0345] Uniquely, the compositions of the present invention can be used to partially or completely fill defects regardless of the shape and / or depth / size of the defect, and can be added in a layer-by-layer manner to build up the desired volume. The compositions of the present invention adhere to the site without the need for physical closure, mix with the blood in situ, and can be injected into deep tissues through a fine gauge needle. The interstitial space can be refilled with the compositions of the present invention at different time intervals as needed.

[0346] The present invention provides an injectable filler. After injection into the body, the composition forms a viscous hydrogel. Due to the inherent properties of the present invention, the composition of the present invention is well tolerated in vivo 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.

[0347] The composition of the present invention can be injected through a fine gauge needle. The composition of the present invention adheres to the injection site and can be injected in a minimally invasive manner in a manner that creates a 3D structure layer by layer in the body.

[0348] Tissue types to which the present application is applicable include skin tissue and periodontal tissue.

[0349] The uses of the composition of the present invention include: a) injectable dermal fillers to fill skin cavities to temporarily reduce skin wrinkles (cosmetic use); b) injectable dermal fillers to temporarily elevate scar bases and promote healing (therapeutic use); c) injectable dermal fillers to support dermal connective tissue formation and promote healing in scar tissue after surgical intervention (therapeutic use); d) injectable dermal fillers to support dermal connective tissue formation in scar treatment after burns (cosmetic / therapeutic use); e) ready-to-use dermal matrix to support vascular ingrowth and promote healing in acute dermal defects with bleeding (therapeutic use); f) ready-to-use dermal matrix to fill surgically created dermal cavities (therapeutic use); g) ready-to-use dermal matrix to support skin graft surgery (cosmetic and therapeutic use); h) carrier system to physically deliver bone graft substitutes (therapeutic use; see, for example, Expert Rev Med Devices., 2006 Jan; 3(1): 49-57); i) carrier systems for filling prostheses (e.g., cages) ex vivo and / or in vivo (therapeutic use); j) fillings without tissue induction properties; k) ready-to-use matrices to support and repair periodontal tissue after tooth extraction (therapeutic use); l) ready-to-use matrices to support periodontal ligament tissue transplants (therapeutic use); and m) injectable matrices for temporary elevation of periodontal ligament tissue (therapeutic use).

[0350] Advantages of preferred embodiments of the present invention include the following: a) cell-friendliness; b) injectability; c) host tissue adherence; d) lack of tissue-specific induction properties; and e) good tolerance in vivo (minimal immune response).

[0351] The hydrogels of the present invention preferably result in minimal foreign body reactions upon injection into animals.

[0352] The hydrogel of the present invention is preferably host tissue conductive. This means that it exhibits regenerative properties in the presence of active bleeding or other fluids containing regenerative biological components. The hydrogel of the present invention is preferably miscible with blood.

[0353] The composition of the present invention is preferably injectable. Preferably, it can be injected multiple times into the same site. The composition of the present invention preferably forms a hydrogel in situ after being administered to a mammal by injection.

[0354] The hydrogels of the present invention preferably exhibit good adhesion to host tissue. The adhesive properties allow for the gradual establishment of an underlying tissue bed to support healing. The adhesive properties also allow for the formation of 3D structures in a minimally invasive manner.

[0355] The hydrogel of the present invention can preferably be used for layer-by-layer filling, so that 3D cavities with different heights can be filled.

[0356] The hydrogels of the present invention preferably degrade in situ over a period of days, weeks or months to leave healthy tissue behind.

[0357] The present invention has been developed as a safe and easy to use biomaterial to act as an injectable scaffold. The present invention consists of a single homogeneous molecule composed of a synthetic smart polymer (PNPHO) conjugated to thymosin beta-4. The composition of the present invention is liquid at room temperature and can be injected directly into the desired clinical site. The composition of the present invention forms an elastic gel when exposed to body temperature, mixes with the blood and stabilizes the clot at the site. In addition, based on in vitro and in vivo studies, the absorption rate of the composition of the present invention is believed to be that the composition of the present invention is absorbed into the body in less than three months. Moreover, studies in a sheep osteotomy model and osteoblasts and pre-osteoblasts confirmed that the device supports vascular ingrowth and bone formation.

[0358] It is hypothesized that the composition of the present invention can be injected into the alveolar base after tooth extraction to mix with blood, stabilize clots and provide a uniform scaffold for vascular ingrowth and bone regeneration. The purpose is to enhance wound healing at the site and preserve alveolar bone after tooth extraction by applying the composition of the present invention directly into the extraction alveolar. To investigate this hypothesis, the applicant conducted a clinical trial involving ten participants with planned tooth extraction. In this clinical study, soft tissue closure (wound healing), the appearance of the extraction site, the health of the participants, and the quality of the underlying bone were studied.

[0359] Table 1 summarizes the applications of the compositions of the present invention and the existence of supporting data for these potential uses of the technology.

[0360] Table 1.

[0361] Possible applications of the compositions of the invention and current supportive preclinical results

[0362]

[0363]

[0364] 5. Kit

[0365] Various kits are disclosed herein that include one or more preferred hydrogels for use in the present invention. For example, the present invention provides a kit comprising a hydrogel for repairing or regenerating a bone defect and instructions. The kit may include a variety of different hydrogels.

[0366] The kit may optionally include polymers, cells, NSPPs, biologically active compounds, water, etc. The kit may include any of a variety of other components or reagents in any combination. All of the various combinations are not explicitly listed, but each combination is included within the scope of the present invention. Some exemplary kits provided according to the present invention are described in the following paragraphs.

[0367] According to certain embodiments of the present invention, the kit may include, for example:

[0368] i. a solution comprising a polymer, a solution comprising NSPP; and

[0369] ii. Instructions for forming a hydrogel from a solution.

[0370] According to another embodiment, the kit may comprise, for example:

[0371] i. a composition comprising a polymer and a NSPP; and

[0372] ii. Instructions for forming a hydrogel from the composition.

[0373] According to another embodiment, the kit may comprise, for example:

[0374] i. A composition comprising a polymer and a NSPP, one or both of which are in solid form; optionally, a solvent such as water or the like; and

[0375] ii. Instructions for forming a hydrogel from the composition.

[0376] The kit may further comprise other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, needles and syringes. An exemplary kit may be in the form of an aerosol, or have components that can be combined to form an aerosol or similar means for administering the compositions of the invention.

[0377] The kit generally includes instructions for use of the preferred hydrogels of the present invention. The instructions may, for example, include protocols for and / or describe conditions for: producing a hydrogel, administering the hydrogel to a subject in need thereof, producing a hydrogel assembly, etc. The kit generally includes one or more containers or vessels so that some or all of the various components and reagents can be separately contained. The kit may also include a device for closing the various containers in a relatively airtight manner for commercial sale, such as a plastic box, which may include instructions, packaging materials (e.g., polystyrene foam), etc.

[0378] 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 of a variety of materials, such as glass or plastic. The container holds a hydrogel or composition effective for treating a disease and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). The label or package insert indicates that the hydrogel or composition is used to treat a selected disease. 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.

[0379] Preferred embodiments of the present invention

[0380] 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 an aerosol form, the concentration of the composition is about 5 mg / mL to about 70 mg / mL.

[0381] The preferred NSPP for use in the present invention is thymosin beta-4. Alternatively, a functional homologue of thymosin beta-4 may be used.

[0382] Preferably, PNPHO is conjugated to thymosin beta-4 (or a functional homologue thereof), wherein both the protein / peptide and PNPHO have defined effects.

[0383] Thymosin beta-4 (or its functional homologue):

[0384] i. as a source of bioactive signaling for tissue regeneration; and

[0385] ii. Promote the formation of blood vessels in and around the hydrogel filler.

[0386] PNPHO polymers are chemically bonded to proteins / peptides to:

[0387] i. regulating the physicochemical properties of the biopolymer for tissue application;

[0388] ii. Rapidly imparting thermosetting properties to the hydrogel filler to confine it locally; and

[0389] iii. To impart bioabsorption properties to injectable hydrogels.

[0390] The combination of these two main segments leads to the formation of a new type of smart tissue filler with a series of properties that are beneficial to tissue regeneration and repair. The advantage of PNPHO polymer is that all its components are approved by the US FDA for biomedical applications.

[0391] The PNPHO polymer contains a thermoresponsive fraction (N-isopropylacrylamide) to induce hydrogel formation at body temperature, and together with lactide, ethylene glycol and N-acryloyloxysuccinimide segments, to give the product high mechanical strength, water solubility and amine reactivity, respectively. The molecular structure of the PNPHO polymer and the role of each segment are schematically shown in the schematic diagram drawn in the following examples.

[0392] Preferably, equimolar amounts of PNPHO and thymosin beta-4 are used, although one of ordinary skill in the art will appreciate that the molar ratios may vary according to each situation encountered in practice.

[0393] Example

[0394] Material

[0395] Chemicals were purchased from Sigma-Aldrich unless otherwise stated. Stannous 2-ethylhexanoate (Sn(Oct)2), N-isopropylacrylamide (NIPAAm), 2-hydroxyethyl methacrylate (HEMA), 4,4'-azobis(4-cyanovaleric acid) (ACVA), and N-acryloyloxysuccinimide (NAS) were used as received. Oligo(ethylene glycol) monomethyl ether methacrylate (OEGMA, M n =475) was purified by passing a solution of it in dichloromethane (1:1 by volume) through a neutral alumina column to remove inhibitors before use. D, L-lactide (LA) monomer was dried in vacuum at 40°C for 24 hours before use. Azobisisobutyronitrile (AIBN) was generously donated by the School of Chemistry in the University of Sydney, Australia.

[0396] Synthesis of HEMA-poly(lactide) (HEMA-PLA) macromonomer

[0397] HEMA-PLA macromonomer was synthesized by ring-opening polymerization of LA with the hydroxyl group of HEMA as initiator and Sn(Oct)2 as catalyst (see Scheme 1).

[0398]

[0399] Solution 1

[0400] Synthesis of HEMA-poly(lactide) (HEMA-PLA) macromonomer

[0401] LA and HEMA were mixed in a three-necked flask at 110° C. for 15 minutes in a nitrogen atmosphere. Subsequently, a mixture of 1 mol% Sn(Oct)2 (relative to the HEMA feed) in 1 ml of anhydrous toluene was added to the LA / HEMA solution. The resulting mixture was stirred at 300 rpm and 110° C. for 1 hour in a nitrogen atmosphere. After the reaction, the mixture was dissolved in tetrahydrofuran and precipitated in cold distilled water at 1° C. The precipitate formed was separated by centrifugation at 3000 rpm for 5 minutes.

[0402] Repeat the centrifugal cycle 3 times to remove all unreacted monomers and by-products (mainly salts). The precipitate is then dissolved in ethyl acetate. The suspended solid particles are removed from the solution by centrifugation at 6000 rpm for 5 minutes, and the supernatant is dried for 12 hours with MgSO4. The dried supernatant is filtered to remove the MgSO4 particles. The polymer solution is then dried under reduced pressure at 60°C, and the solvent residue is further removed at 40°C for 24 hours in a vacuum. The resulting viscous oil is stored in a refrigerator for further use.

[0403] The feed ratio of HEMA:LA was varied from 1:1.5 to 1:2.5 to obtain PLA / HEMA macromers with different lactic acid lengths. Two PLA / HEMA macromers with lactic acid lengths of 3 and 6 were synthesized using 1:1.5 and 1:2.5 molar ratios of HEMA and LA monomers, respectively.

[0404] Using the proton peak evidence from HEMA and LA 1 The synthesis of PLA / HEMA macromers was confirmed by H NMR spectroscopy. The total integration of the 5.2 ppm peak of the methine in lactic acid and the 5.7 ppm and 6.0 ppm peaks of HEMA was used to determine the total integration of the 5.2 ppm peak of the methine in lactic acid and the 5.7 ppm and 6.0 ppm peaks of HEMA. 1 The molar ratio of LA to HEMA in the PLA / HEMA macromer was calculated from the H NMR spectrum.

[0405] Synthesis of Poly(NIPAAm-co-NAS-co-(HEMA-PLA)-co-OEGMA)(PNPHO)

[0406] PNPHO was synthesized using method (1) or (2) as described below (see Scheme 2).

[0407]

[0408] Solution 2.

[0409] Synthesis of Poly(NIPAAm-co-NAS-co-(HEMA-PLA)-co-OEGMA)(PNPHO)

[0410] Method 1

[0411] PNPHO was synthesized by free radical polymerization using AIBN as initiator. NIPAAm (12mmol), NAS (1.0mmol), HEMA-PLA (0.57mmol), OEGMA (0.56mmol), AIBN (0.07mmol) and anhydrous N, N'-dimethylformamide (DMF) were loaded into a Schlenk flask equipped with a magnetic stirring bar and a rubber septum. The flask was deoxygenated by three freeze-pump-thaw cycles, then sealed, and then immersed in an oil bath preheated to 70°C to start polymerization. After 24 hours, the reaction mixture was cooled to room temperature, precipitated in ether, filtered, and then dried under vacuum. The polymer was purified twice by redissolving / reprecipitating with THF / ether, and finally dried under vacuum for 2 days.

[0412] Method 2

[0413] PNPHO was synthesized by free radical polymerization using ACVA as an initiator. The composition of the copolymers was varied by changing the length of lactic acid (3 and 6 in HEMA-PLA) and the molar ratio of HEMA-PLA (6, 8, and 11 mol%) and OEGMA (3, 5, and 8 mol%). Known amounts of NIPAAm, NAS, HEMA-PLA, OEGMA, ACVA (7.0 × 10 -5 mol) was dissolved in 13 ml of anhydrous N,N'-dimethylformamide in a round-bottomed one-necked flask. The system was then deoxygenated by purging with nitrogen for 15 minutes. The results also showed that it is feasible to deoxygenate the monomer solution by purging with nitrogen in the solution in a vacuum for 10 minutes. This technique provides a more efficient way to remove oxygen from large-scale solutions.

[0414] The reactor was then sealed and immersed in an oil bath at 70°C for 24 hours. The resulting polymer solution was then cooled at room temperature for 1 hour and precipitated in 250 mL of ether. The precipitate was then collected by filtering the suspension and dried in a vacuum for 6 hours. The dried powder was dissolved in tetrahydrofuran and precipitated in ether to further remove the residues of the macromonomer. The final powder was dried in a vacuum for at least 48 hours.

[0415] Table 2

[0416] PNPHO polymer (OEGMA:PLA / HEMA (LA length):NAS:NIPAAm format)

[0417] Monomer feed Final composition Gel time (min) 5：8(5)：3.5：83 4.8：7.9(5)：7：80 2.5±0.6 5：8(5)：7：80 4.6：7.8(5)：13.6：74 3.1±0.9 8：6(5)：3.5：82 8：5.4(5)：7：80 5.1±0.6 8：6(5)：7：80 8：5.1(5)：14：72 5.4±0.1 3：8(5)：3.5：72 4.8：7.9(5)：7：80 2.0±0.2 3：8(5)：7：80 4.6：7.8(5)：13.6：74 2.2±0.9

[0418] PNPHO Composition

[0419] PNPHO was synthesized according to the scheme shown in Figure 2(a). 1 The synthesis of the PNPHO copolymer was confirmed by H NMR spectroscopy, as shown in Figure 2(b). Characteristic proton peaks of NIPAAm (a and b), NAS (e), HEMA-PLA (f, h, k), and OEGMA (m and n) were detected. Regarding the NAS proton resonance (e), it was found that these overlapped with the resonances of about 2.9-3.0 ppm from residual traces of DMF solvent, resulting in errors in the original calculations based on the same data set (integral / area under each peak, as a basis for the relative mol % of each monomer in the entire PNPHO polymer). Subsequent corrections confirmed the presence of the third monomer (NAS) in an amount greater than about 7 mol %. Based on the NIPAAm (a), NAS (e / 2 - The final composition of the copolymer was calculated by integrating these peaks for each monomer in (f), HEMA-PLA (h) and OEGMA (n / 2). In this study, the copolymer is denoted as PNPHO and a subscript corresponding to the molar ratio of HEMA-PLA (lactic acid length) to OEGMA is added. For example, PNPHO 5:8(5):7:81 represents a copolymer synthesized with 8 mol% HEMA-PLA with a lactic acid length of 5 and 5 mol% OEGMA. A variety of copolymers were produced. These are shown in Table 2. Table 2 also provides data on their gel time and temperature.

[0420] Solubility of PNPHO in PBS

[0421] The monomer ratio of PNPHO is varied to obtain a series of compositions that are soluble in aqueous media (e.g., PBS) to develop injectable formulations. NIPAAm-based copolymers are soluble in aqueous solutions below their LCST due to the formation of hydrogen bonds between the polar groups of the copolymer and water molecules. In this study, the effects of lactic acid 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.

[0422] Figure 3 The results in show that increasing the lactic acid length in the range of 3 to 6 in the HEMA-PLA backbone has no significant effect on the solubility of PNPHO in PBS (p>0.05). Therefore, within the range studied, the hydrophobic nature of the side chains in the PNPHO backbone has little effect on the overall solubility of PNPHO in aqueous media. Therefore, by varying the lactic acid length, other properties of PNPHO, such as gelation behavior and mechanical properties, can be adjusted without affecting the solubility of PNPHO in aqueous media.

[0423] The solubility of PNPHO in PBS can be adjusted by varying both the hydrophobic and hydrophilic content. The PLA segment is the main hydrophobic backbone, while both NAS and HEMA monomers exhibit relatively limited hydrophilicity. Therefore, OEGMA was included in the synthesis of PNPHO to promote the hydrophilicity of the copolymer.

[0424] 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 solubility reduction was also due to a decrease in the concentration of the relatively hydrophilic segment NIPAAm in the copolymer (p < 0.05). Therefore, reducing the NIPAAm content in PNPHO significantly affects the hydration of the copolymer.

[0425] When more than 3 mol% (e.g. 1.5 mol%) of OEGMA is used as the hydrophilic segment, the solubility of PNPHO in water increases dramatically. The results show that copolymers with less than 3 mol% of OEGMA are insoluble in aqueous media. Figure 3 The results in show that the solubility of PNPHO copolymers with 6 mol% PLA-HEMA significantly increased by 2 and 3 times when the OEGMA concentration was increased from 3 mol% to 5 mol% and 8 mol%, respectively. However, in copolymers containing a higher molar ratio 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 attributed to the formation of copolymers with longer chains and higher Mw, which hinders the hydration and solubility of the copolymers in aqueous solution.

[0426] The effect of the concentration of water-soluble PNPHO copolymer on the injectability of its solution through an 18G needle was evaluated. A 150 mg / mL PNPHO solution in PBS was found to be injectable through an 18G needle, and this copolymer concentration 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.

[0427] Thymosin beta-4

[0428] Since the succinimide linker exhibits high reactivity and optimal accessibility to amino-containing compounds, it is reasonable to speculate that this polymer can be applied to TB4 with amino groups to prepare flowable hydrogels. To confirm this hypothesis, the feasibility of the reaction between the polymer and TB4 was investigated. A TB4 solution was formed in phosphate-buffered saline. 1000 μL of TB4 solution was thoroughly mixed with 140 mg of polymer at 4 °C for at least 24 h, and the mixture was then allowed to gel at 37 °C.

[0429] Gelation behavior (time and temperature)

[0430] The results in Table 2 show that the gelation time of the hydrogels prepared with the PNPHO compositions ranges from 2.5 to 5 minutes. It is important to note that the measurements were made in simulated physiological conditions (37°C incubator), and at this temperature, the gelation rate of the hydrogels in contact with body fluids and / or body surfaces is significantly accelerated. This gelation time is advantageous for clinical applications because it allows the product to be applied and at the same time prevents the applied product from being dislocated from the site. In addition, the gelation temperature of these PNPHO formulations is below 37°C, which allows clinical use. The high gelation temperature of the formulation is very convenient for clinicians to deliver the solution in vivo.

[0431] Conjugation efficiency of PNPHO

[0432] To test the conjugation efficiency of PNPHO, the TB4 synthetic peptide was used as a model system. The results showed that TB4 was effectively conjugated with different PNPHO preparations. The results show the efficient effect of PNPHO conjugated with synthetic or natural peptides or proteins. The two components (TB4 and PNPHO) interact at the molecular level through the succinimidyl ester group of PNPHO and the amino group of TB4. To evaluate this, the lower critical solution temperature (LCST) of PNPHO and PNPHO-co-TB4 was compared.

[0433] The LCST of the PNPHO polymer and the conjugated system (PNPHO-co-TB4) is driven by the chemical composition of PNPHO, TB4, and the ratio of hydrophilic to hydrophobic groups in the molecular structure. PNPHO and PNPHO-co-TB4 exhibit opposite solubility properties upon heating. This thermoresponsive behavior is due to the ability of the NIPAAm group and related side chains (e.g., NAS, PLA / HEMA, OEGMA, and TB4) to undergo a change from a dissolved curled-up sphere to a collapsed globule (i.e., from a hydrophilic to a hydrophobic state) when the temperature is raised above the LCST (Figure 1). Since the transition of the polymers was monitored during the temperature change of the NMR data acquisition, 1 H-NMR provides an accurate analysis of LCST. 1 H-NMR spectra were recorded in 1°C intervals over the temperature range of 10°C to 30°C. The NMR signals of the individual peaks decreased with increasing temperature and the transition of the conjugated system from coiled to spherical form (reverse solubility). The plot of the NMR peak area versus temperature determined the LCST.

[0434] Based on PNPHO polymer and PNPHO-co-TB4 1 H-NMR spectroscopy revealed that the LCST of the conjugated system was 2 to 4 °C lower than that of the PNPHO polymer ( Figure 4). To quantify the gelation temperature, DO / m (relative to the characteristic peak of OEGMA) was used. The change in the gelation temperature of the conjugated system compared to PNPHO confirms the presence of a chemical interaction between the two components. In addition, the reduction in gelation temperature increases the kinetics of gelation in vivo and, therefore, promotes adhesion of the product at the site of application.

[0435] Bioresorbable behavior of protein-PNPHO hydrogels

[0436] Clinical studies of PNPHO-co-TB4 have shown that the product is completely absorbed by the body within 3 months. Histological evaluation of the implant site showed no traces of the product three to four months after implantation.

[0437] Feasibility study

[0438] Table 3

[0439] Product configuration for feasibility study applications

[0440]

[0441] Samples from the batch were incubated on 90 mm tryptic soy agar plates at 37° C. The absence of bacterial growth was verified prior to batch release.

[0442] The sample sizes used in implementing this protocol are shown in Table 4 .

[0443] Table 4.

[0444] Sample size used in the test plan

[0445]

[0446]

[0447] The sample size selected for all tests to be performed was chosen with statistical validity in mind. A large number of animals / test articles and control groups were selected, for example, 6 replicates per group per time point, much higher than the 3 replicates typically used in preclinical proof-of-concept studies.

[0448] Table 5.

[0449] Test plan adopted

[0450]

[0451] The PNPHO-co-TB4 test articles are manufactured by the applicant according to the documented procedures and methods. All equipment, tools and materials used in the product manufacturing process have been approved by the applicant's QMS. The PNPHO-co-TB4 device is provided to the testing facility in a ready-to-use syringe. Each test article is for single use.

[0452] The following equipment / tools / materials are required to perform this testing protocol. Histochemical analysis, staining, and visualization were performed according to industry standards.

[0453] Table 6

[0454] Equipment / tools / materials required to carry out the test plan

[0455]

[0456]

[0457] result

[0458] The PNPHO-co-TB4 solution transforms from a liquid at room temperature (20 to 25°C) to a gel at body temperature (37°C) and maintains its structural stability after injection under simulated physiological conditions (Figure 1(a)). A live sheep osteotomy model was used to examine the injectability and adhesion of PNPHO-co-TB4 in the presence of active bleeding. The results in Figure 1(b) show that the product can be injected into the defect site and immediately forms a hydrogel that fills the cavity, mixes with the blood and stabilizes the clot. The formation of the hydrogel was achieved despite the presence of active bleeding at the site and subsequent dilution of the hydrogel by the blood. This is an important attribute of the hydrogel system of the present invention because it can be used for clinical applications of injectable systems and prevent device failure. Due to the presence of body fluids in in vivo injection, better heat transfer promotes the kinetics of hydrogel formation. For example, Figure 1(c) shows that the formation of hydrogel on the skin is slower. This characteristic behavior of the present invention can be used to form a 3D structure by applying the product layer by layer on an exposed wound (Figure 1(d)), which typically has a lower temperature than the core / deep tissue. The present invention adheres to and mixes with blood and host cells to support healing.

[0459] The ability of PNPHO-co-TB4 to support tissue regeneration is based on the physical scaffold formed after administration. It is suggested that the matrix supports cell infiltration and vascular ingrowth throughout the structure. To test this hypothesis, a mouse animal model was used to study the in vivo potential of PNPHO-co-TB4 to integrate with the host environment and support vascular ingrowth. In addition, PNPHO-co-TB4 was directly compared with the integrated regenerative dermal matrix (Integra) as a positive control.

[0460] Animal models and surgery

[0461] A full-thickness mouse animal model (n=20) was used; in each animal, two (2) full-thickness dermal defects were surgically created; see Figure 5(a). Wounds were treated with PNPHO-co-TB4 or Integra (positive control), and skin grafts were sutured in place, as shown in Figure 2. Figure 5 (b) As shown. Conventional dressings were then applied to the treated site, specifically Atrauman and IV3000. Angiogenesis and ingrowth at the transplant site were determined by fluorescence radiation efficiency using an IVIS Lumina XR in vivo imager. Defects were regularly examined for graft survival up to 8 weeks after treatment. In addition, animals were sacrificed at different time points; biopsies were collected to quantify host tissue integration, vascular ingrowth, and inflammatory responses.

[0462] Skin graft survival

[0463] The survival of the grafted skin was studied at various time points throughout the study. Successful skin grafts remain intact within the host dermis, whereas failed grafts wrinkle and slough off over time; see Figure 5 (c). The results of this study showed that within 1 week after surgery, 4 skin grafts treated with Integra failed, indicating a survival rate of 80%. This finding is consistent with previous studies that reported a survival rate of approximately 85% 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; the survival rate of skin grafts treated with PNPHO-co-TB4 was 100%. The efficacy of PNPHO-co-TB4 in supporting full-thickness skin grafts suggests that PNPHO-co-TB4 is well tolerated with minimal inflammatory response. Successful skin graft survival 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, animals were subjected to in vivo fluorescence imaging at different time points and histological assessment of the treated sites was performed.

[0464] Inflammatory response to the compositions of the invention

[0465] Skin biopsies were collected two (2) and four (4) weeks after transplantation to evaluate the biological behavior of PNPHO-co-TB4 by histochemistry. H&E staining of the Integra-treated sites showed mild inflammatory response to the Integra constructs at weeks 2 and 4; Figure 6 (a) and Figure 6(b) Multiple layers of fibrous tissue are visible around the Integra site, respectively. Previous studies have demonstrated similar inflammatory responses to constructs and the formation of fibrous tissue around Integra (Hori, Osada, Isago, & Sakurai, 2017; Y. Wang, et al., 2015).

[0466] on the contrary, Figure 6 (c) and Figure 6 H&E staining of skin biopsy from the PNPHO-co-TB4-treated site in (d) showed that a very narrow layer of fibrous tissue had formed around the PNPHO-co-TB4 two weeks after transplantation. Figure 6 The white arrows in (c) indicate the structure of the PNPHO-co-TB4 gel, and the black arrows indicate minimal fibrous tissue formation around the PNPHO-co-TB4 hydrogel. Recognizing that increased inflammation is associated with impaired wound remodeling, the reduced fibrous tissue formation around PNPHO-co-TB4 suggests that this material contributes to effective soft tissue repair.

[0467] Neovascularization

[0468] The IVIS Lumina XR in vivo imager was used to determine vascularization and ingrowth at the transplant site by fluorescence radiant efficiency. Two (2) and four (4) weeks after transplant surgery, 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 and is capable of imaging blood vessels and angiogenesis. At each time point after surgery, each mouse was injected with 2nmol AngioSense750EX in 100μL PBS. After 24 hours, the fluorescence radiant efficiency of each mouse was scanned (n=8).

[0469] The radiation efficiency is used to indicate the density of new blood vessels in the wound area. Figure 7 The results showed that the fluorescence radiation efficiency of the PNPHO-co-TB4 treated site was significantly higher than that of the Integra treated site at two weeks after surgery (p < 0.01). In contrast, the angiogenesis signal of both treatment groups was very low at four (4) weeks after surgery, indicating that the vascularization was controlled and the healing of the site was complete. Similar findings were reported in a previous study, indicating that the treated site showed no angiogenesis signal after four weeks (Yiwei Wang et al., 2015).

[0470] Vascular ingrowth and cellular infiltration in the compositions of the present invention

[0471] Histological evaluation of PNPHO-co-TB4 treated sites four (4) weeks after skin grafting confirmed the potential of PNPHO-co-TB4 to support vascular ingrowth and host cell infiltration. Figure 8 As previously reported, a thin layer of fibrous tissue was formed around the PNPHO-co-TB4 structure, indicating that the injectable hydrogel was well tolerated in vivo; Figure 8 (a) and Figure 8 (b). More importantly, this fibrous tissue lamina can be used as an indicator of the PNPHO-co-TB4 boundary in the dermis.

[0472] Figure 8 (c) and Figure 8 The results in (d) show the formation of blood vessels within the PNPHO-co-TB4 structure. In addition, staining of skin biopsies showed obvious infiltration of host fibroblasts within the PNPHO-co-TB4 hydrogel structure; Figure 8 (d) To further confirm the fibroblast infiltration and the formation of skin extracellular matrix within the structure of the composition of the present invention, the skin transplant site was stained with Masson's trichrome 4 weeks after transplantation. Fig. 9 The results in show collagen fiber formation in the composition of the present invention 4 weeks after the implantation surgery. The results confirm the fibroblast infiltration within the structure of the composition of the present invention and its potential to integrate with the host tissue and promote the formation of new dermis.

[0473] The formation of collagen fibers within the PNPHO-co-TB4 structure was significantly higher than that detected within the Integra structure. The results in the CCC showed that collagen formation was significantly reduced within the structure of Integra compared to the composition of the present invention. This result is also consistent with previous findings reported in the literature (Yiwei Wang et al., 2015).

[0474] The results of this study confirm the scaffolding role of PNPHO-co-TB4 in supporting host tissue integration, vascular ingrowth, cellular infiltration, and neodermis formation.

[0475] Technical Feasibility: Conclusion

[0476] PNPHO-co-TB4 has been successfully tested in an authoritative animal study. This authoritative animal study involved 40 full-thickness skin grafts and directly compared PNPHO-co-TB4 and the gold standard dermal template (IntegraDermal Matrix). During the study, all skin grafts treated with the composition of the present invention survived (100% survival rate), compared to an 82% success rate in the Integra group. In addition, the composition of the present invention accelerated the formation of a vascular network, minimized the inflammatory response, promoted skin cell infiltration within its structure, and formed new dermis and collagen fibers at the site. These results indicate that this technology has high potential in the treatment of post-burn scars.

[0477] Clinical Research - Research Device Description

[0478] The composition of the invention, PNPHO-co-TB4, is provided in a ready-to-use sterile syringe. The product is single-use and double-bagged. The composition of the invention is liquid at room temperature and can be injected directly into the desired clinical location. At body temperature, the composition of the invention forms a white elastic scaffold.

[0479] Device composition

[0480] The composition of the present invention is an injectable stent. The composition of the present invention has two main parts in its formulation: (1) a synthetic polymer (PNPHO) and (2) a synthetic non-human or animal-derived peptide, namely thymosin beta-4. The chemical bonding of these two parts results in the formation of a single uniform molecule (e.g., PNPHO-co-TB4).

[0481] The smart polymer is poly(N-isopropylacrylamide-co-(N-acryloxysuccinimide)-co-(polylactide / 2-hydroxymethacrylate)-co-(oligo(ethylene glycol), denoted as PNPHO. The specific formulation of PNPHO used in the composition of the present invention is PNPHO 5:8(5):7:81. Equimolar amounts of PNPHO and thymosin β-4 are used in the formulation of PNPHO-co-TB4.

[0482] Intended use of the device

[0483] PNPHO-co-TB4 is designed to promote bone regeneration. Specifically, PNPHO-co-TB4 is designed to reduce bone resorption after tooth extraction, thereby enhancing health outcomes for patients.

[0484] Clinical Research Plan-Research Purpose

[0485] The primary objective was to determine qualitative measures and analytical methods to further investigate the use of the composition of the invention. Specifically, the evaluation and characterization of the healing process and bone regeneration evaluated by histology and CT in the presence of the composition of the invention after tooth extraction compared to a historical (literature) control population. The secondary objective was to examine the in vivo properties of the composition of the invention in humans.

[0486] Clinical Study Design - Clinical Trial Endpoints

[0487] The clinical trial endpoints of this study are to determine qualitative measures and analytical methodology to further study the use of the composition of the present invention up to 3-4 months post-surgery. Specifically, the evaluation and identification of the healing process and bone regeneration evaluated by histology and CT in the presence of the composition of the present invention after tooth extraction compared to a historical (literature) control population. In addition, the clinical trial endpoints are designed to examine the in vivo characteristics of the composition of the present invention in humans 3-4 months post-surgery.

[0488] Data Quality Assurance

[0489] The clinical study was designed, constructed and monitored, and the data were generated, documented, recorded and reported in accordance with ISO 14155 according to SSR internal procedure requirements and under the applicant's ISO 13485 quality management system certification.

[0490] The management of the clinical study was outsourced to an independent CRO, Southern Start Research (SSR). All data points were 100% monitored by SSR throughout the trial. Monitoring visits for data quality assurance and quality control were performed at the intervals specified in Table 7.

[0491] Table 7.

[0492] Monitoring visit plan

[0493]

[0494] Participant Groups

[0495] Ten (10) patients participated in this study. A summary of patient demographics is shown in Table 8.

[0496] Table 8.

[0497] Overview of the demographics of participants in PET clinical studies

[0498] Number of participants N＝10 Number of male participants M＝10 Number of female participants F＝0 Number of participants with a history of smoking S＝2 Minimum age at surgery Min-age=28 Maximum age at surgery Max-age = 73 Median age of participants Med-age=51 Average age of participants Ave-age=53

[0499] Treatment plan

[0500] Table 9 summarizes the study procedures and plans for visits and follow-up. The protocol was executed as planned in the Clinical Investigation Plan (CIP). All participants followed the plan and were followed up as planned, except for participant #7. Due to personal circumstances, the participant changed his planned treatment, and therefore histology was not collected for this participant at visit 4.

[0501] Follow-up period

[0502] Study participants were followed for three (3) months following tooth extraction and treatment with the composition of the present invention.

[0503] Table 9.

[0504] Study Procedures and Data Collection Visits

[0505]

[0506] *Enrollment and Visit 1 (preoperative baseline) may occur during the same visit (after completion of the informed consent process).

[0507] #As part of the standard of care, histological examination of tissue removed from the implant site was performed in preparation for the dental implant procedure that coincided with Visit 4.

[0508] @A referral for an initial CT scan will be made at this visit and the procedure will be performed prior to Visit 1. The CT results will be documented on the CRF for Visit 1.

[0509] @@A referral for a second CT scan will be made at this visit and the procedure will be performed within two weeks prior to Visit 4. The CT results will be documented on the CRF at Visit 4.

[0510] The expected follow-up schedule and corresponding visit windows are summarized in Table 10. Time point zero (0) is the time of tooth extraction (and implantation of the combination of the invention for the treatment population).

[0511] Table 10.

[0512] Study Follow-up Window

[0513] Visit Window Start Target Window End Visits 1 and 2 - - - Visit 3 6 days 7 days 10 days Visit 4 2 months 3 months 4 months

[0514] Results—Disposition of Subjects and Study Devices

[0515] Ten patients were treated with the composition of the invention. Devices were implanted in all participants. No study devices were left in place after completion of the clinical study. All study devices were audited by the Southern Start Research (SSR) clinical research organization in accordance with the device accountability procedure.

[0516] All findings were assessed qualitatively; these included, but were not limited to, wound appearance at various time points after extraction, patient reports of pain and discomfort, radiographic appearance of the underlying bone with CT scans at three months postoperatively, visual appearance of the site during implant placement surgery, and histologic examination of the treated site at three to four months postoperatively.

[0517] Device availability

[0518] There were no reports of device malfunctions throughout the clinical study. The composition of the present invention is provided in a ready-to-use form; this eliminates the need for the clinician to perform pre-mixing and any other preparation steps prior to surgery. In particular, Fig.11 (b) shows that the composition of the present invention can be easily injected into the extraction site through a 21G needle. Subsequently, due to the hydrophilic nature of the product, the composition of the present invention mixes with the blood at the site and forms a hydrogel to stabilize the clot.

[0519] Clinical use of the device in a PET trial demonstrated that injection of the composition of the invention into the alveolar site was successful for all 10 patients, and no device failures were reported by the principal investigator. Fig.12 The results in show that for all 10 patients, the application and gelation success rate of the composition of the present invention was 100%.

[0520] Table 11.

[0521] Availability of the composition of the invention in 10 patients in a PET trial

[0522] patient# Injection / Administration Gelation Adhesion membrane Closed 001-001 success immediately success Not used Unclosed 001-002 success immediately success Not used Unclosed 001-003 success immediately success Not used Unclosed 001-004 success immediately success Not used Unclosed 001-005 success immediately success Not used Unclosed 001-006 success immediately success Not used Unclosed 001-007 success immediately success Not used Unclosed 001-008 success immediately success Not used Unclosed 001-009 success immediately success Not used Unclosed 001-010 success immediately success Not used Unclosed

[0523] A summary of the findings from the usability of the composition of the present invention is summarized in Table 11. According to the principal investigator, use of the composition of the present invention can save up to 45 minutes in the procedure. This can be achieved because use of the composition of the present invention after extraction eliminates the need for primary closure (e.g., use of a membrane and micro-suturing of the site).

[0524] Table 12.

[0525] Safety of the composition of the present invention after tooth extraction based on visual observation of the site and histochemical analysis

[0526]

[0527]

[0528] 1 Normal CT scan results indicate the absence of inflammation, infection, necrosis, hypertrophic bone growth, or hypertrophic fibrosis.

[0529] Device Safety and Wound Healing

[0530] All ten patients treated with the composition of the invention returned for the first follow-up one week after surgery. No pain or discomfort was reported by any patient. During the oral examination (one week after application), there were no signs of infection or inflammation at the site. In addition, the principal investigator examined wound closure and soft tissue formation. In all ten patients, wound closure was recorded and accelerated soft tissue formation was detected (results see Fig.12 ).

[0531] Three months after tooth extraction and treatment with the composition of the present invention, the patient underwent an implant placement procedure. At this time, a biopsy was collected from the injection site of the composition of the present invention. The samples were subjected to histochemical analysis by an independent laboratory (Sonic Clinical Trials Pty Ltd). The results of these histochemical analyses are summarized in Table 12. In all samples analyzed, there was no evidence of necrosis, foreign body type giant cells or foreign body reaction. These findings indicate that the composition of the present invention is well tolerated in vivo and has biocompatibility in vivo.

[0532] Indications for bone regeneration efficacy

[0533] Three months postoperatively, CT scan imaging of the site was used to study the healing progress and extension of bone resorption at the site. An independent CT scan report from Dr. Tom Huang of Envision Medical Imaging confirmed that bone resorption was minimized. In addition to CT scan imaging of the site, biopsies were collected from the injection site of the composition of the present invention during the implant placement procedure. The samples were fixed and sent for independent histochemical analysis. The pathological behavior and bone regeneration process of the site were analyzed using hematoxylin and eosin (H&E) and Masson's trichrome staining of the site. In all samples analyzed, histochemical analysis showed the formation of interconnected active trabeculae, a mixture of woven bone and lamellar bone as well as active osteoblasts and osteoclasts. Active periodontal bone remodeling was noted in all patients.

[0534] Table 13 lists all reports of H&E and Mason's trichrome stained histochemical specimens from independent laboratories.

[0535] Table 13.

[0536] Summary of findings from H&E and Mason's trichrome staining of sites tested with compounds of the invention

[0537]

[0538]

[0539] Aerosol-forming ability of PNPHO-co-TB4

[0540] Particle size analysis was performed for each formulation with different solid contents (PNPHO concentrations between 17.5 mg / mL and 140 mg / mL) as volume diameter distribution and derived parameters (d10, d50, d90 and %V>10). Aerosol droplets generated from all formulations exhibited a bimodal distribution regardless of the formulation concentration and test temperature. In general, the first peak (~50 μm) tended to increase with decreasing formulation concentration, while the second peak (~500-600 μm) increased with concentration. This indicates that most aerosol droplets are in the 10-100 μm range.

[0541] For d10, d50, and d90, the percent sizes for all diluted formulations were 21.4 ± 3.9 μm, 45.6 ± 9.4 μm, and 238.6 ± 181.5 μm (mean), respectively. The common median size for nasal delivery is between 30 and 120 μm (1), so all diluted formulations are within the specification for nasal delivery. Importantly, all formulations had less than 3% volume of droplets less than 10 um in diameter, indicating that they are suitable for nasal delivery and avoid lower airway deposition. The 90th percentile size showed the greatest variability, suggesting potential problems with coarse droplet emission, especially for more concentrated formulations.

[0542] Plume coverage

[0543] The FDA recommends characteristics of the spray pattern for nasal dosage forms. Fig.15 The plume patterns of the tested formulations (started at room temperature) are shown in . All formulations showed a broad coverage area, but the most obvious feature was the absence of deposition in the center of the plume as the formulation concentration increased.

[0544] Despite these differences, the D max and D min value( Fig.16 ) is constant, and the obtained ellipticity (~1) indicates the spray symmetry after aerosolization. The ellipticity of all formulations is within the FDA specification (1.00-1.30). In practice, the best choice for nasal delivery should be a trade-off between coverage area and improved residence time. If runoff occurs, a higher coverage area will not provide the best results.

[0545] Nasal deposition pattern

[0546] The deposition pattern of the formulation on the human nose model is as follows Fig.17As shown. Both tested formulations (17.5 mg / mL and 35 mg / mL) showed rapid adhesion, as the deposition pattern remained stable and relatively unchanged from start-up to up to 15 minutes. In addition, no dripping into the throat was observed. The adhesion of the formulations in a short period of time may indicate a higher residence time and bioavailability of the delivered cargo. Both formulations were also able to reach the olfactory region (upper part of the nasal region), which is important for delivery from the nose to the brain.

[0547] In vitro drug release from PNPHO-based hydrogels

[0548] Ciprofloxacin hydrochloride was used as a model drug to study 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 chain during the gelation process causes water to be expelled from the matrix. In order to evaluate the amount of ciprofloxacin hydrochloride that seeped out of PNPHO-co-TB4, the hydrogel was evaluated using a Snapwell device. Briefly, ciprofloxacin hydrochloride powder was dissolved (20 mg / mL) in a PNPHO-co-TB4 solution. Subsequently, 200 μL of the PNPHO-co-TB4 / ciprofloxacin hydrochloride formulation was placed in the top compartment of the snapwell and allowed to form a hydrogel while the snapwell was left standing on a flat surface at 37 ° C for 30 minutes to avoid polymer loss through the membrane. Next, the top liquid supernatant layer of the hydrogel was collected.

[0549] To further analyze the drug released from the gel over time, 2 mL of PBS was added to the basolateral compartment of each snapwell, and the plate was then incubated at 37°C under constant orbital shaking (60 rpm). At predetermined time points over 24 hours, samples (200 μL) were removed from the basolateral medium and replaced with an equal amount of fresh preheated PBS each time. Ciprofloxacin hydrochloride was quantified in all samples using a validated high performance liquid chromatography method (HPLC, Shimadzu, Sydney NSW, Australia).

[0550] Quantification of Ciprofloxacin Hydrochloride by HPLC

[0551] The quantification of ciprofloxacin hydrochloride was determined using an HPLC system consisting of an LC20AT pump, a SIL20AHT autosampler, and a 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 freshly prepared in needle washes with acetonitrile: water (50:50 v / v) every day, and linearity was confirmed within a concentration range of 0.05-100 μg / mL, with a regression value of >0.999.

[0552] The initial release of ciprofloxacin HCl from PNPHO hydrogels during the gelation process (30 min) under physiological conditions was evaluated. An average of 2.7 ± 1.4 μg and 28.9 ± 17.9 μg (n = 3) were found ( Fig.18 ; t = 0) of cyclohexanoic acid hydrochloride was released into the liquid top layer of PNPHO and PNPHO-co-TB4, respectively. It is important to note that the initial release of the tested drug from the hydrogels was negligible compared to the loading amount, i.e., 20 mg / mL, and 2.7 ± 1.4 μg and 28.9 ± 17.9 μg were released from PNPHO and PNPHO-co-TB4, respectively.

[0553] The relatively low percentage of ciprofloxacin released from the hydrogel after administration shows the high potential of the present invention in drug delivery applications. Fig.18 In the Figure 1, the release of ciprofloxacin hydrochloride from the hydrogel over time at 37°C for each formulation was expressed as cumulative mass. There was no significant difference between the amount of ciprofloxacin hydrochloride released from the two hydrogels, with 137.4±32.4μg (89.5±15.8%) and 133.7±42μg (65.3±10.5%) released from PNPHO and PNPHO-co-TB4, respectively, after 24 hours. These results confirm the sustained and controlled release characteristics of the drug from PNPHO-based hydrogels.

[0554] Overview of adverse events

[0555] 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; histology information was not collected for this participant at visit 4 because this participant elected to change his treatment plan, which did not include bone histology.

[0556] Conclusions on device availability

[0557] The composition of the present invention was administered to all ten patients without difficulty. No device (syringe) failures were reported in the study. No preparation and / or mixing of the device was required prior to use. The composition of the present invention formed a white hydrogel immediately after injection into the alveolar site. The product mixed with the blood and adhered to the extraction site. The product adhered to the site and no initial closure of the site was required to accommodate the device.

[0558] Device safety

[0559] No device-related adverse events or serious adverse events were reported in the study. All ten (10) patients returned for their first follow-up visit 7 days after tooth extraction, and nine (9) patients who underwent implant placement surgery reported no inflammation, infection, pain, or discomfort, during which native tissue was collected for histochemical analysis. H&E staining of the site indicated that the product was well tolerated in vivo and there were no signs of abnormal or foreign body-type giant cells at the site. No necrosis or necrotic tissue was found at the site. In addition, CT scans of the extraction site and the site treated with the composition of the present invention showed no bone abnormalities at the site.

[0560] Device efficacy

[0561] Seven days after tooth extraction and treatment with the composition of the invention, wound closure was found for all patients. Osteogenic and osteoclastic activity, active bone remodeling at the cellular level were noted. Active bone and bone remodeling were found at the treatment site, demonstrating the osteoconductive properties of the device. As hypothesized, the product did not induce bone formation and did not have any other phenotypic effects on the host tissue.

[0562] Evaluation of risks and benefits

[0563] The potential benefits of the devices and the identified residual risks have been evaluated on an individual or collective basis to determine whether they are acceptable when weighed against the potential benefits of the devices.

[0564] There is growing evidence that bone-anchored prostheses improve quality of life measures, including chewing, speech, and general health. However, economic barriers and access to oral surgeons are considered limitations to the widespread use of implant-fixed prostheses. From a clinical perspective, minimizing bone loss after tooth extraction is necessary to simplify and therefore improve access to bone-anchored prostheses.

[0565] Bone loss following tooth extraction is difficult for clinicians to address and places a financial burden on patients and health systems, especially in rural and socioeconomically disadvantaged areas. The fact that 1 in 2 patients requires a secondary implant procedure to increase bone volume for successful implant placement highlights the extent of this problem and challenge in the field of dentistry. The compositions of the present invention are easy-to-use materials that can maintain spinal bone volume after tooth extraction. Dentists appreciate the ease of use and predictability of the implant placement procedure. Patients will benefit because the use of the compositions of the present invention promotes healing of wounds following tooth extraction, accelerates bone healing, and can potentially prevent the need for secondary augmentation procedures.

[0566] The application of the composition of the present invention to a newly extracted tooth socket is simple and easy to use in clinical practice. Unlike all other bone substitutes, the composition of the present invention is delivered to the tooth socket as a liquid and forms an elastic matrix at the site. The main purpose of this pilot PET trial is to study the safety, usability and osteoconductivity of the composition of the present invention. Efficacy indicators including radiographic and histochemical analysis were collected. In summary, although the trial is not authoritative, PET confirmed the safety and usability of the device; no device failure was found, and no device-related adverse events were detected.

[0567] Taking into account the intended use, the intended users, the reasonably foreseeable misuse, the likely effects of use of the composition-device family of the invention, and taking into account the generally accepted state of the art, it can be concluded that the risk management process has determined that the potential device benefits outweigh the residual risks.

[0568] Clinical relevance of the composition of the invention

[0569] The composition of the present invention has been developed as a safe and easy to use biomaterial to fill tissue defects / cavities. The composition of the present invention is a single homogeneous molecule comprising a synthetic "smart" polymer (PNPHO) cross-linked with thymosin beta-4. The composition of the present invention is a liquid at room temperature and can be injected directly into the desired clinical location. The composition of the present invention forms an elastic gel when exposed to body temperature, mixes with blood and stabilizes the clot at the site. The composition of the present invention was administered to ten (10) patients. No device malfunction or device-related adverse events were reported or recorded while using the composition of the present invention. Wound healing was found one week after administration.

[0570] Three months post-surgery, no pathological abnormalities, such as inflammation, infection, or giant cells, were detected at the site of application. In addition, active bone remodeling and osteogenic and osteoclastic activity were detected at the site treated with the composition of the present invention, confirming the osteoconductive nature of the device. These findings suggest that the composition of the present invention has a high potential for wound healing in both soft and hard tissues, as the product does not induce any phenotypic effects, such as soft and hard tissue repair, at the site. However, it is believed that further health benefits can be achieved by improving the osteoinductive properties of the composition of the present invention.

[0571] Clinical Research Summary

[0572] This pilot trial involved the use of the PNPHO-co-TB4 scaffold of the invention for alveolar protection after tooth extraction in ten patients. In all patients, the applicant's device was successfully applied; no membrane or micro-suturing was required at the extraction site. This enabled the principal investigator to save time during the procedure. At follow-up, wound closure was found 7 days after extraction, and there were no signs of infection or inflammation in any of the patients. After three months of using the composition of the invention, tissue biopsies were collected from the site for histochemical analysis; the results showed that the product was completely absorbed and there were no signs of pathological abnormalities at the site. In addition, active bone remodeling was found at the site.

[0573] Although the invention has been described with reference to specific examples, it will be appreciated by those skilled in the art that the invention may be embodied in many other forms.

[0574] The applicants would like to thank Dr. Hui Ong and Dr. Dina Silva for their contributions in conducting the in vitro aerosol formation studies and benchtop drug release testing.

Claims

1. A composition 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 conjugation to a natural or synthetic peptide or protein (NSPP); and a fourth monomer for imparting phase change behavior; And wherein the natural or synthetic peptide or protein (NSPP) is thymosin beta-4 or a functional homologue thereof.

2. The composition of claim 1, wherein the first monomer is oligo(ethylene glycol) monomethyl ether methacrylate (OEGMA).

3. The composition of any one of the preceding claims, wherein the second monomer is hydroxyethyl methacrylate poly(lactic acid) (HEMA-PLA).

4. The composition of any one of the preceding claims, wherein the third monomer is N-acryloyloxysuccinimide (NAS).

5. The composition of any one of the preceding claims, wherein the fourth monomer is (N-isopropylacrylamide).

6. A composition according to any one of the preceding claims, wherein the polymer comprises: an amount of about 3 to about 8 mol % of the first monomer, an amount of about 5 to about 9 mol % of the second monomer, an amount of at least about 7 mol % of the third monomer, and The fourth monomer is present in an amount constituting the remainder of 100% of the polymer composition.

7. The composition of any of the preceding claims, wherein the polymer comprises the fourth monomer in an amount of about 60 to about 85 mol%.

8. The composition according to claim 1, in: The first monomer is OEGMA, The second monomer is HEMA-PLA, The third monomer is NAS, and The fourth monomer is NIPAAm, and wherein 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%, an amount greater than about 7 mol% NAS, and The amount is as high as about 85 mol % NIPAAm.

9. The composition of claim 8, wherein the polymer comprises: The amount is about 5 mol% of OEGMA, The amount of HEMA-PLA is about 7 mol%, an amount greater than about 7 mol% NAS, and The amount was as high as about 81 mol% NIPAAm.

10. The composition according to any one of the preceding claims, wherein the natural or synthetic peptide or protein (NSPP) is thymosin beta-4.

11. A composition according to any preceding claim comprising equimolar amounts of the polymer and thymosin beta-4.

12. The composition of any of the preceding claims, wherein the concentration of the polymer is from about 100 mg / mL to about 300 mg / mL of the composition.

13. A hydrogel comprising the composition of any one of claims 1 to 12 and water, wherein the combination of the NSPP and the third monomer crosslinks the polymers to form a hydrogel comprising water therein.

14. The hydrogel of claim 13, wherein the hydrogel is formed at body temperature after the composition and the NSPP are administered to a mammal by injection.

15. Use of the composition according to any one of claims 1 to 12 in the preparation of a hydrogel, wherein the hydrogel is used for: repair and / or restoration of hard or soft body tissues; wound healing; temporary wrinkle reduction; temporary elevation of scar base and promotion of healing; supporting dermal connective tissue formation and promoting healing in scar tissue after surgical intervention; supporting dermal connective tissue formation in scar treatment after burns; supporting blood vessel ingrowth and promoting healing in acute dermal defects with bleeding; filling surgically generated dermal cavities ； Support skin grafting procedures; Physical delivery of bone graft substitutes; Filling of prostheses; Use as a filling without tissue inducing properties; Support and repair of periodontal tissues after tooth extraction; or temporarily elevate the periodontal ligament tissue and / or support a periodontal ligament tissue graft.

16. A method for: repair and / or restoration of hard or soft body tissue; wound healing; temporary wrinkle reduction; temporary elevation of scar base and promotion of healing; supporting dermal connective tissue formation and promotion of healing in scar tissue following surgical intervention; supporting dermal connective tissue formation in scar management following burns; supporting vascular ingrowth and promotion of healing in acute dermal defects with hemorrhage; filling surgically created dermal cavities ； Support skin graft surgery; physically deliver bone graft substitutes; fill prostheses; For use as a filling without tissue inducing properties; to support and repair periodontal tissue after tooth extraction; or to temporarily elevate periodontal ligament tissue and / or support periodontal ligament tissue transplantation, the method comprising administering to a mammal a composition according to any one of claims 1 to 12.

17. The method of claim 16, wherein the administering step is performed by injection or by aerosol administration, thereby forming a hydrogel at the body temperature of the mammal.

18. A composition according to any one of claims 1 to 12 for use in: tissue repair and / or restoration; wound healing; temporary wrinkle reduction; temporary elevation of scar base and promotion of healing; supporting dermal connective tissue formation and promoting healing in scar tissue after surgical intervention; supporting dermal connective tissue formation in scar management after burns; supporting vascular ingrowth and promoting healing in acute dermal defects with bleeding; filling surgically created dermal cavities ； Support skin grafting procedures; Physical delivery of bone graft substitutes; Filling of prostheses; Use as a filling without tissue inducing properties; Support and repair of periodontal tissues after tooth extraction; or temporarily elevate the periodontal ligament tissue and / or support a periodontal ligament tissue graft.

19. A kit for forming a hydrogel, comprising in respective containers: Natural or synthetic peptides or proteins (NSPP); and Composition, Wherein the composition comprises: a first monomer for binding water; a second monomer for imparting mechanical properties to the hydrogel; a third monomer for conjugation to a natural or synthetic peptide or protein (NSPP); and a fourth monomer for imparting phase change behavior; wherein the natural or synthetic peptide or protein (NSPP) is thymosin beta-4 or a functional homologue thereof; And wherein the NSPP is cross-linked with the second monomer, thereby enabling the hydrogel to form when the composition is contacted with water.

20. The kit of claim 19, further comprising instructions for sequential or simultaneous administration of the components of the kit.

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