Controlled hydrogel delivery of focal adhesion kinase inhibitor for decreased scar formation

Abstract

To provide topical biomaterial-based dressings that allow for targeted and controlled release of pharmacological inhibitors, and compositions of improving healing, particularly for amelioration of scarring.SOLUTION: A composition for promoting tissue healing comprises: a porous scaffold; and focal adhesion kinase (FAK) inhibitor disposed in pores of the scaffold, wherein the composition is configured to deliver a dose of FAK inhibitor effective to promote tissue healing at a controlled rate during a treatment time.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of U.S. Provisional Patent Application No. 62 / 672,513, filed May 16, 2018, and U.S. Provisional Patent Application No. 62 / 848,515, filed May 15, 2019, the entire contents of each of which are incorporated herein by reference.

[0002] (Incorporated by reference) The entire contents of all publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0003] (Statement regarding federally funded research) This invention was made with government support under Grant Nos. W81XWH-13-2-0052 and W81XWH-13-2-0054 awarded by the Department of Defense, Grant No. DE026914 awarded by the National Institutes of Health, and Grant No. WFUHS441011SR01 awarded by the Armed Forces Institute for Regenerative Medicine (AFIRM DoD). The United States Government has certain rights in the invention.

[0004] (Field) The present disclosure relates generally to treating wounds and promoting tissue healing of wounds. In particular, described herein are compositions comprising a porous scaffold and a focal adhesion kinase (FAK) inhibitor, as well as methods of making and using such compositions. [Background technology]

[0005] Cutaneous scarring represents a significant healthcare burden, with billions of dollars spent annually on wound and scar treatment. Unfortunately, current strategies to prevent and / or treat scarring have been largely unsuccessful due to a lack of a comprehensive understanding of the underlying pathophysiology. Recent reports from our laboratory have demonstrated the importance of mechanical stress in regulating biological processes related to hypertrophic scar (HTS) formation. In these studies, sustained mechanical loading applied to healing incisions led to a fibrotic response and HTS formation.

[0006] A key regulator of this process is focal adhesion kinase (FAK), a cytoplasmic tyrosine kinase that plays a central role in transmitting mechanical signals to initiate fibrotic responses. The integrin-FAK pathway also mediates tumor progression and metastasis in a variety of malignancies. Pharmacological inhibitors of FAK have emerged as anticancer chemotherapeutic agents in preclinical and clinical trials. Cancer and hypertrophic scars share similarities in that hyperproliferative fibroproliferative and extracellular matrix repair events underlie their pathogenesis.

[0007] In this context, FAK is also an intriguing and potentially promising target for anti-scarring therapy. Fibroblast-specific deletion of FAK in knockout mice showed a significant reduction in inflammation and fibrosis in an incision HTS model. However, keratinocyte-restricted FAK deficiency was associated with increased dermal protein degradation that may contribute to wound chronicity. In our previous study, inhibition of FAK with a small molecule inhibitor by local subcutaneous injection abolished the local inflammatory response and reduced scar development in an HTS mouse model.

[0008] Preclinical and clinical studies have shown that offloading mechanical strain using stress shielding devices in incisional and excisional wounds is effective in inhibiting scarring in large animals and humans. However, approaches that physically reduce mechanical strain are not applicable to large wounds resulting from extensive burns, traumatic blast injuries, or excisional skin surgery. Summary of the Invention [Problem to be solved by the invention]

[0009] Therefore, there is an urgent need for topical biomaterial-based dressings that allow for the targeted and controlled release of pharmacological inhibitors. Methods to improve healing, especially for scar repair, are of great interest, which is addressed by the present invention. [Means for solving the problem]

[0010] The present invention relates to the treatment of wounds and the promotion of tissue healing in wounds. In particular, described herein are compositions comprising a porous scaffold and a focal adhesion kinase (FAK) inhibitor, as well as methods of making and using such compositions.

[0011] One aspect of the present invention provides a composition for promoting tissue healing. In some embodiments, the composition may include a porous scaffold. In some embodiments, the composition may include a focal adhesion kinase (FAK) inhibitor disposed within the pores of the scaffold. In some embodiments, the composition is configured to deliver a dose of the FAK inhibitor effective to promote tissue healing at a controlled rate over a treatment period.

[0012] In some embodiments, the porous scaffold comprises a hydrogel film, hi some embodiments, the porous scaffold comprises a pullulan-collagen hydrogel.

[0013] In some embodiments, the FAK inhibitor comprises VS-6062 (PF-562271) or a benzenesulfonate salt of PF-562271. In some embodiments, the composition comprises about 1 μg / cm 2 ~about 500μg / cm 2 In some embodiments, the composition comprises about 40 μg / cm of a FAK inhibitor. 2 ~about 200μg / cm 2 Includes FAK inhibitors.

[0014] In some embodiments, the composition is configured for sustained release of the FAK inhibitor disposed within the pores of the scaffold, hi some embodiments, the composition is configured for sustained release of the FAK inhibitor disposed within the pores of the scaffold for a period of up to about 96 hours.

[0015] In some embodiments, the composition is prepared by molecular imprinting of the FAK inhibitor during formation of the porous scaffold. In some embodiments, the composition is prepared for rapid release of the FAK inhibitor. In some embodiments, the composition comprises a FAK inhibitor on the delivery surface of the composition. In some embodiments, the composition is further configured for rapid release of the FAK inhibitor from the delivery surface of the composition. In some embodiments, the composition is configured for rapid release of the FAK inhibitor from the delivery surface of the composition within about 24 hours.

[0016] In some embodiments, the composition is adapted to release substantially all of the FAK inhibitor during the treatment period, hi some embodiments, the composition is adapted to release from about 30% to about 75% of the FAK inhibitor during the treatment period.

[0017] In some embodiments, the composition comprises multiple layers, where one or more layers are configured for sustained release of the FAK inhibitor and one or more layers are configured for rapid release of the FAK inhibitor, hi some embodiments, the composition comprises layers configured for both sustained release of the FAK inhibitor and rapid release of the FAK inhibitor.

[0018] In some embodiments, the composition is configured to deliver a dose of the FAK inhibitor effective to reduce scarring in the tissue during the treatment period, hi some embodiments, the composition is configured to deliver a dose of the FAK inhibitor effective to promote hair growth in the tissue during the treatment period.

[0019] In some embodiments, the composition is configured as a wound dressing.

[0020] Yet another aspect of the invention provides a method of promoting tissue healing. In some embodiments, the method comprises placing a composition on a tissue surface comprising a porous scaffold and a focal adhesion kinase (FAK) inhibitor engineered for controlled release disposed within the pores of the scaffold. In some embodiments, the method comprises delivering a dose of the FAK inhibitor to the tissue surface effective to promote tissue healing at a controlled rate over a treatment period.

[0021] In some embodiments, the porous scaffold comprises a hydrogel film, hi some embodiments, the hydrogel comprises a pullulan-collagen hydrogel.

[0022] In some embodiments, the method further comprises reducing scarring in the tissue, hi some embodiments, the method further comprises promoting hair growth in the tissue.

[0023] In some embodiments, the composition is configured as a wound dressing.

[0024] In some embodiments, the FAK inhibitor is disposed on a delivery surface of the composition. In some embodiments, the controlled rate comprises a rapid release rate and a sustained release rate. In some embodiments, the delivering step of the method comprises delivering the dose of the FAK inhibitor to the tissue surface within about 24 hours after the placing step. In some embodiments, the dose of the FAK inhibitor is delivered to the tissue surface for a period of up to about 96 hours.

[0025] In some embodiments, the method further comprises removing the composition from the tissue surface. In some embodiments, the method further comprises repeating the placing and delivering steps at least three times. In some embodiments, the method further comprises repeating the placing and delivering steps at least ten times.

[0026] In some embodiments, the FAK inhibitor is VS-6062 (PF-562271) or the benzenesulfonate salt of PF-562271.

[0027] In some embodiments, the method comprises delivering from about 30% FAK inhibitor in the composition to about 75% FAK inhibitor in the composition to the tissue.

[0028] Yet another aspect of the invention provides a method of making a composition comprising a porous scaffold for treating a wound. In some embodiments, the method comprises forming a mixture comprising a focal adhesion kinase (FAK) inhibitor, pullulan, collagen, and a porogen. In some embodiments, the method comprises removing the porogen and / or dehydrating the mixture to produce a composition comprising a porous scaffold having a focal adhesion kinase (FAK) inhibitor tailored for controlled drug release disposed within the pores of the scaffold.

[0029] In some embodiments, the porous scaffold comprises a hydrogel, hi some embodiments, the porous scaffold comprises pullulan and collagen hydrogel.

[0030] In some embodiments, the method includes the step of depositing a FAK inhibitor on a surface of the composition.

[0031] In some embodiments, the method includes cross-linking pullulan, hi some embodiments, the method includes cross-linking pullulan with sodium trimetaphosphate (STMP) and / or sodium tripolyphosphate (STPP).

[0032] In some embodiments, the method includes the step of crystallizing the porogen prior to the removing step.

[0033] In some embodiments, the composition comprises a heterogeneous distribution of the FAK inhibitor.

[0034] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which: [Brief description of the drawings]

[0035] [Figure 1] 1A-1C show the construction and in vitro release profile of pullulan-collagen hydrogels releasing FAK inhibitor ("FAKI"). (FIG. 1A) Schematic of molecular imprinting of FAKI into pullulan-collagen hydrogel scaffold. (FIG. 1B) Digital photographs of dry pullulan-collagen hydrogel (top) and hydrogel swollen in PBS after water uptake (bottom). (FIG. 1C) In vitro release profile of FAKI from pullulan-collagen hydrogels containing 50 μg FAKI prepared by molecular imprinting (blue) and surface incorporation (red). N=3 experiments for each indicated time point. [Diagram 2] 2A-C show FAKI delivered via hydrogel accelerated wound healing in a splinted full-thickness excision wound model. (FIG. 2A) Digital photographs of splinted full-thickness excision wounds treated with or without FAKI hydrogel. (FIG. 2B) Quantification of wound closure days and time-dependent change in wound area after injury. (FIG. 2C) Groups: no scaffold or FAKI (control), pullulan-collagen hydrogel (hydrogel), and FAKI releasing pullulan-collagen hydrogel (H+FAKI). N=8 wounds per condition. Statistical differences exist at *p<0.05 versus control for each time point indicated. [Diagram 3]3A-H show reduction of profibrotic activity by FAKI treatment in a splinted full-thickness excision wound model. (FIG. 3A) Representative H&E and Masson's trichrome stained images of healed wounds. (FIG. 3B) Quantification of blue areas in Masson's trichrome stained images of healed wounds in control, hydrogel, and H+FAKI at 17 days post-injury. H&E: cytoplasm and extracellular matrix (red) and cell nuclei (black). Masson's trichrome stain: collagen (blue), cytoplasm and muscle (red) and cell nuclei (black). Scale bar=100 μm. (FIG. 3C) Immunofluorescence staining of α-SMA and (FIG. 3D) quantification of green areas in control, hydrogel, and H+FAKI at 17 days post-injury. α-SMA (green) and cell nuclei (blue). Scale bar=50 μm. (FIG. 3E) Representative Western blotting images and (FIG. 3F) quantification of FAK and p-FAK in wound lysates at 10 days post-injury. N=8 wounds per condition. (FIG. 3G) Immunofluorescence staining of p-Akt and (FIG. 3H) quantification of green areas in control, hydrogel, and H+FAKI at 17 days post-injury. p-Akt (green) and cell nuclei (blue). Scale bar=100 μm. For all analyses, statistical differences exist at *p<0.05 (control vs. H+FAKI), Δp<0.05 (hydrogel vs. H+FAKI), and #p<0.05 (control vs. hydrogel). [Figure 4] 4A-B show enhanced mechanical properties by FAKI treatment. Young's modulus (MPa) (FIG. 4A) and ultimate tensile stress (MPa) (FIG. 4B) in control, hydrogel, and H+FAKI groups at 3 weeks after injury. N=8 wounds per condition. For all analyses, statistical differences exist with *p<0.05 (control vs. H+FAKI) and #p<0.05 (control vs. hydrogel). [Figure 5-1] 5A-5B show improved wound healing and attenuated myofibroblast deposition by FAKI treatment in a contact burn wound model. (FIG. 5A) Digital photographs of full-thickness contact burn wounds treated with and without FAKI hydrogel. (FIG. 5B) Quantification of time-dependent changes in wound area after injury. [Figure 5-2] 5C-5D show improved wound healing and attenuated myofibroblast deposition with FAKI treatment in a contact burn wound model. (FIG. 5C) Immunofluorescent staining of α-SMA and (FIG. 5D) quantification of green areas in control, hydrogel, and H+FAKI at 24 days after debridement. N=7 wounds per condition. Scale bar=250 μm. For all analyses, statistical differences exist at *p<0.05 (control vs. H+FAKI) and Δp<0.05 (hydrogel vs. H+FAKI). [Figure 6] 6A-6B: Local subcutaneous injection of FAKI reduces scar formation in mouse incisions. (FIG. 6A) Masson's trichrome staining of dorsal incisions of mice treated with increasing concentrations of FAKI dissolved in 10% DMSO showed reduced scar lesions at 15 and 75 μM FAKI. N=5 wounds per condition. Scale bar=100 μm. No animals survived longer than 2 days with 150 μM FAKI injection. (FIG. 6B) Quantification of scar area (dashed lines) per condition. Statistical differences exist vs. control at *p<0.05. [Figure 7] 7A-7E show the setup and timeline used to analyze the effect of topical treatment of FAKI applied in a hydrogel device on deep skin wounds in a Red Duroc pig model, as described herein. FIG. 7A shows the area used for testing. FIG. 7B shows an example of a deep porcine wound with skin removal. FIG. 7C shows a cross-section of unwounded porcine skin. FIG. 7D shows a cross-section of wounded porcine skin with a wound depth of 0.06 inches (in). FIG. 7E shows the timeline from the day of injury (D0) to 180 days post-op (D180). [Figure 8]FIG. 8A-8C show that topical treatment of deep dermal wounds with FAKI hydrogel accelerates wound healing in Duroc pigs. N=8 wounds per condition. Initial wound size=25 cm2. FIG. 8A shows photos of wounds taken from post-operative day (POD) 2 through post-operative day 30 (POD30). After POD11, through POD21 and POD30, the wounds appear to be healing better. Wound area was measured over time. FIG. 8B shows results over time for wounds treated with FAKI hydrogel versus wounds treated with hydrogel or untreated. Wounds treated with FAKI in the hydrogel device show accelerated healing over POD21 and POD30 over wounds left untreated (control) or wounds treated with hydrogel alone. By POD 14, wounds treated with FAKI hydrogel were nearly completely closed, whereas untreated (control) wounds still had 20% open wounds, a statistically significant difference (indicated with an *). By POD 17, wounds treated with FAKI hydrogel were completely closed, whereas untreated (control) wounds still had approximately 10% open wounds, a statistically significant difference (indicated with an * in FIG. 8B). FIG. 8C shows that untreated wounds or wounds treated with hydrogel alone took 25 days to completely close, whereas wounds treated with FAKI in the hydrogel device closed by POD 14, a statistically significant result. [Figure 9]9A-9B show that topical treatment of deep dermal wounds in Duroc pigs with FAKI hydrogel significantly improves scar appearance. FIG. 9A shows photographs of wounds taken from post-op day (POD) 45 to post-op day 90. Wounds treated with FAKI in a hydrogel device (bottom row) show significantly improved appearance compared to wounds left untreated (top row) or treated with hydrogel alone (middle row) from POD 45 to POD 90. Furthermore, wounds treated with FAKI in a hydrogel device show significantly more hair within the wound area. FIG. 9B shows that wounds treated with topical application of FAKI in a hydrogel device have improved appearance at 90 days as assessed by blinded scar experts. Four blinded scar experts assessed scars using a visual analog scale (VAS). Untreated controls were scored 100, treatment with hydrogel alone was scored 95, and treatment with FAKI in the hydrogel device showed a significantly improved score of 60: treatment with FAKI in the hydrogel device had a visual analogue scale (VAS) and scores were assessed by four blinded scar experts (*p<0.001). [Figure 10]10A-10B show that topical treatment of deep dermal wounds in Duroc pigs with FAKI hydrogel improves scar appearance, and the improved scar appearance remains at 6 months. FIG. 10A shows photographs of wounds taken from post-op (POD) day 45 through post-op day 180 (POD180) (6 months). Wounds treated with FAKI in a hydrogel device show a significantly improved appearance compared to wounds left untreated or treated with hydrogel alone from POD 45 through POD 180. Additionally, wounds treated with FAKI in a hydrogel device show hair within the wound area. FIG. 10B shows that wounds treated with topical application of FAKI in a hydrogel device have an improved appearance when assessed by blinded scar experts. Four blinded scar experts assessed the scars using a visual analog scale (VAS). Untreated controls were scored 100, treatment with hydrogel alone was scored 95, and treatment with FAKI in a hydrogel device showed a significantly improved score of 60: treatment with FAKI hydrogel has ΔP<0.0001 (control vs. H+FAKI). [Figure 11] 11A-11C show that topical treatment of deep dermal wounds with FAKI hydrogel results in regeneration of hair follicles and skin appendages. FIG. 11A shows trichrome staining of a FAKI hydrogel-treated wound (bottom) compared to an untreated wound (top) and a wound treated with hydrogel alone (middle) at 90 days after surgery. Arrow 1 points to a hair follicle. Arrow 3 points to a hair follicle with surrounding glandular structures. Arrows 2 and 4-6 point to skin glands (sweat and sebaceous glands). FIG. 11B-11C show the results of counting the number of hair follicles (left graph) and sebaceous and sweat glands (right graph). Untreated wounds are on the left of each graph, wounds treated with hydrogel alone are in the center, and wounds treated with FAKI hydrogel are on the right. At 90 days post-surgery, wounds treated with FAKI hydrogel had significantly more hair follicles with an average of approximately 2.7 follicles, whereas wounds treated with hydrogel alone had an average of approximately 0.7 follicles and untreated wounds had approximately 0.6 follicles. [Figure 12] Figures 12A-C show that topical treatment of deep dermal wounds with FAKI hydrogel blocks myofibroblast recruitment and activation. Figure 12A shows staining of control and FAKI hydrogel treated wounds for alpha-smooth muscle actin. Figures 12B-C show results from blinded observers quantifying the amount of alpha-smooth muscle actin in randomly selected areas within the scar lesion on POD60 (Figure 12B) and POD90 (Figure 12C). Wounds treated with FAKI hydrogel showed significantly less alpha-smooth muscle actin compared to controls (*p<0.001). [Figure 13] 13A-13C show that FAKI hydrogel treatment results in better skin structure resembling normal unwounded skin. The left panel of FIG. 13A shows dermal collagen staining against a dark background. The right panel of FIG. 13A shows automated collagen fiber segmentation extracted by the CT-FIRE quantitative fiber analysis algorithm. Different colored fibers represent individual collagen fibers with different fiber metrics generated by computational extraction. FIG. 13B shows the length (pixels) of collagen fibrils as shown in FIG. 13A. The mean and median size of collagen fibers are significantly smaller in FAKI hydrogel compared to untreated controls. FIG. 13C shows the absolute number of individual collagen fibers of a particular type per image analyzed by the CT-FIRE algorithm. [Figure 14] 1 shows a device described herein for promoting tissue healing and reducing scarring, the device including a skin graft with a FAKI hydrogel scaffold. [Figure 15] Figures 15A-B show the safety of FAKI hydrogel delivered topically to the skin. Figure 15A shows negligible levels of systemic absorption following topical delivery of FAK hydrogel. Figure 15B shows that daily application of even high concentrations of FAKI solutions to unwounded skin did not result in skin irritation or sensitization. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0036] Compositions for promoting tissue healing, for example, for reversing scar formation at a wound site and forming new hair and skin appendages, as well as devices and methods using the compositions, are provided. The compositions include a scaffold, for example a porous scaffold, and an inhibitor of focal adhesion kinase (FAK) activity engineered for controlled drug release in an effective dose. The scaffold can be configured to deliver an effective dose of the FAK inhibitor to a delivery surface for promoting tissue healing at a controlled rate during the treatment time. In some examples, the composition is configured or contacted with the skin at the wound site for a period of time sufficient to promote tissue healing and / or reduce scarring, for example, compared to a wound not treated by the method of the present disclosure. Aspects of the present disclosure provide methods for treating injury, re-epithelializing wounds, preventing or reducing scarring during healing of skin wounds, forming new hair and skin appendages, in which an effective dose of a FAK inhibitor is contacted with the wound. In some embodiments, the subject is a human subject. In some embodiments, the injury is a cut, which may be an epidermal incision, a burn, a wound that may be open or closed, etc. Examples of open wounds include, but are not limited to, an incision, a laceration, an abrasion, a puncture wound, a penetration wound, a gunshot wound, and / or a stab wound. In some embodiments, the porous scaffold is a porous hydrogel, such as a pullulan-collagen hydrogel.

[0037] It is to be understood that this invention is not limited to the particular methodology, protocols, cell lines, animal species or genera, and reagents described, all of which may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is limited only by the appended claims.

[0038] definition Focal adhesion kinase (FAK), also known as cytoplasmic protein tyrosine kinase 2 (PTK2), is a cytosolic protein tyrosine kinase that is localized to focal adhesions that form between cells that attach to extracellular matrix components (see Andre et al. (1993) Biochem. Biophys. Res. Commun. 190:140-147). FAK is a highly conserved 125 kD non-receptor tyrosine kinase that is recruited as a participant in focal adhesion dynamics between cells and is involved in motility and cell survival. FAK is phosphorylated in response to integrin binding, growth factor stimulation, and the action of mitogenic neuropeptides. A 159 amino acid carboxy-terminal region, the focal adhesion targeting domain (FAT), has been shown to be responsible for targeting FAK to focal adhesions. Paxillin, a focal adhesion adaptor protein, binds to FAK through its carboxy-terminal domain that overlaps with the FAT domain. Between the amino and carboxy regions is the catalytic domain. Phosphorylation of the activation loop within this kinase domain is important for the kinase activity of FAK.

[0039] Exemplary inhibitors of FAK are shown below in Table 1 (Table 1A, Table 1B). An inhibitor of interest is PF-562271, which has a potent, ATP-competitive, selective and reversible IC of 1.5 nM. 50 Also of interest is PF-00562271, the benzenesulfonate salt of PF-562271.

[0040] Inhibitors of FAK Many FAK inhibitors are known and used in the art.

[0041] [Table 1A] [Table 1B]

[0042] Porous scaffold. A structure with pores or openings. The structure may comprise natural and / or synthetic polymers or copolymers. Examples of materials suitable for forming the porous scaffold include acrylic polymers, cellulose, chitosan, collagen, cellulose polymers, gelatin, gums, poly(lactic-co-glycolic acid) (PLGA), poly(L-lactide) / poly(ε-caprolactone), polyvinyl-based polymers, pullulan, and other polysaccharides. The pores may extend throughout the entire scaffold and extend from a first surface to a second surface of the scaffold, or at least the first surface may be closed. The pores may have small interconnected voids. The pores may be irregular or regular. Irregular pores may be irregular in terms of size (e.g., diameter) or length. Regular pores may be similar to each other in size, shape, or distribution. The pores, particularly the ordered pores, can be formed using a porogen or a mass of particles that are eluted after the scaffold has solidified. Examples of porogens that can be utilized include potassium chloride, sodium chloride, sucrose, glucose, lactose, sorbitol, xylitol, polyethylene glycol, polyvinylpyrrolidone, and polyvinyl alcohol. The pores, particularly the ordered pores, can be formed by laser drilling or 3D printing. The porosity can be optimized to retain and release the drug (e.g., FAK inhibitor) for a desired time or at a desired rate. The porosity can be optimized to retain and release the drug (e.g., FAK inhibitor) at a delivery surface on the porous scaffold. The porosity can be optimized to retain and release the drug (e.g., FAK inhibitor) from a delivery surface on the porous scaffold to a tissue surface. The porous scaffold is a hydrogel and can retain or be configured to retain a fluid such as water.

[0043] Hydrogels. Wound dressings are materials that are applied to wounds or incisions to cover and protect the wound. For example, carbohydrate-based hydrogels can be made using pullulan and collagen under conditions that provide cross-linking and pore formation. Collagen can be added to a mixture of pullulan, cross-linking agent, and pore-forming agent (porogen), where the collagen is provided at a concentration of at least about 1% and up to about 12.5% ​​by weight of pullulan. Collagen can be provided at a concentration of about 1%, about 2.5%, about 5%, about 7.5%, about 10%, usually about 2.5% to about 10%, and can be about 4% to about 6%, and the collagen is usually fibrous collagen, e.g., type I, II, III, etc. Crosslinkers of interest include sodium trimetaphosphate (STMP) or a combination of sodium trimetaphosphate and sodium tripolyphosphate (STMP / STPP), for example, in a weight / weight ratio of about 1:5, about 1:2, about 1:1, or about 1:2 with pullulan. Porogens of interest for in-gel crystallization include any suitable porogen, such as salts, for example, KCl, for example, in a weight / weight ratio of about 1:5, about 1:2, about 1:1, or about 1:2 with pullulan. For molecular imprinting, the FAK inhibitor can be added to the mixture at a concentration of about 100 μg / ml to about 100 mg / ml, and can be about 1 mg / ml to about 10 mg / ml. For surface incorporation, a solution of the FAK inhibitor in a suitable vehicle, for example, aqueous solution, ethanol, etc., can be applied to the surface after the gel is formed.

[0044] The composition can be injected and compressed to form a sheet, preferably having a thickness of at least about 1 mm and no more than about 5 mm, usually no more than about 3 mm, and can be about 1.75 mm to 2.5 mm, for example, about 2 mm thick.

[0045] Pores can be formed in the hydrogel by rapid drying due to phase inversion of the swollen hydrogel. Some embodiments therefore include a step of dehydrating the mixture or the swollen hydrogel. Dehydration results in localized supersaturation and crystallization of the porogen. Pullulan and collagen are forced to organize around the crystals in an interconnected network, resulting in the formation of a reticulated scaffold after dissolution of KCl. Some embodiments therefore include a step of removing the porogen. The addition of porogen enhances the viscoelasticity of the hydrogel. The improved scaffold porosity allows for greater fluid absorption, higher water-to-polymer ratios, and more efficient hydrogel behavior.

[0046] The films can be stored dry and are easily rehydrated in any suitable aqueous medium. The aqueous nature of the hydrogel matrix provides an ideal environment for cell growth and sustainability in wound healing.

[0047] The mechanical characteristics of the hydrogel include the average pore size and the porosity of the scaffold. In the case of pullulan-collagen hydrogels, both variables vary with the concentration of collagen present in the hydrogel. In the case of a hydrogel containing 5% collagen, the average pore size is usually in the range of about 25 μm to about 50 μm, about 30 μm to about 40 μm, and can be about 35 μm. In the case of a hydrogel containing 10% collagen, the average pore size is usually in the range of about 10 μm to about 25 μm, about 12 μm to about 18 μm, and can be about 15 μm. Those skilled in the art will easily determine suitable hydrogels with other collagen concentrations. The porosity of the scaffold is usually in the range of about 50% to about 85%, and can be in the range of about 70% to about 75%, and decreases as the concentration of collagen increases.

[0048] Pullulan. A polysaccharide polymer derived from the fungus Aureobasidium pullulans. Pullulan is a linear homopolysaccharide with α-(1-6) linked maltotriose units that exhibits water-holding capacity in the hydrogel state, making it an ideal therapeutic vehicle for both cells and biomolecules. In addition, pullulan contains multiple functional groups that allow cross-linking and delivery of genetic material and therapeutic cytokines. Furthermore, pullulan-based scaffolds have been shown to enhance the behavior of both endothelial and smooth muscle cells in vitro.

[0049] Collagen. As used herein, the term "collagen" refers to a composition in which at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95% or more of the protein present is collagen in triple helical structure. Collagen is found widely in vertebrate species and has been sequenced for many different species. Due to the high degree of sequence similarity between species, collagen from various species can be used for biomedical purposes, for example, between mammalian species. Typical commercially available animal sources include bovine Achilles tendon, calfskin, and bovine bone. In some embodiments, the collagen used in the preparation of the oriented thin film is type I, type II, or type III collagen, and is derived from any suitable source, typically a mammalian source, such as bovine, porcine, etc.

[0050] Collagen has a triple-stranded rope-like coiled structure. The major collagen in skin, tendons, and bone is collagen I, which contains two α-1 polypeptide chains and one α-2 chain. Cartilage collagen contains only one type of polypeptide chain, α-1. Fetal collagen also contains collagens with a distinctive structure. The genes for the interstitial collagens types I, II, and III show an unusual and distinctive structure with many relatively small exons (54 bp and 108 bp) in evolutionarily conserved positions along the length of the triple-helical gly-XY segment.

[0051] Collagen types include type I (COL1A1, COL1A2), type II (COL2A1), type III (COL3A1), type IV (COL4A1, COL4A2, COL4A3, COL4A4, COL4A5, COL4A6), type V (COL5A1, COL5A2, COL5A3), type VI (COL6A1, COL6A2, COL6A3), type VII (COL7A1), type VIII (COL8A1, COL8A2), type IX (COL9A1, COL9A2, COL9A3), type X (COL10A1), and type XI (COL11A1, COL11A2). ), XII (COL12A1), XIII (COL13A1), XIV (COL14A1), XV (COL15A1), XVI (COL16A1), XVII (COL17A1), XVIII (COL18A1), XIX (COL19A1), XX (COL20A1), XXI (COL21A1), XXII (COL22A1), XXIII (COL23A1), XXIV (COL24A1), XXV (COL25A1), XXVII (COL27A1), XXVIII (COL28A1). Those skilled in the art will appreciate that other collagens are similarly suitable for the methods of the present invention, including mammalian collagens, e.g., bovine, porcine, equine, etc. Additionally or alternatively, collagen can be synthetically produced.

[0052] Supports. A variety of solid supports or substrates can be used with the porous scaffold (hydrogel), including deformable supports. Deformable means that the shape of the support can be changed to some other shape. Examples of deformable solid supports include polyacrylamide, nylon, nitrocellulose, polypropylene, polyester films such as polyethylene terephthalate, PDMS (polydimethylsiloxane), etc., known in the art for making wound dressings.

[0053] Wound dressings. A wound dressing is a material that is applied to a wound or incision to cover and protect the wound. For example, a porous matrix such as a hydrogel to promote tissue healing (e.g., to reverse scar and / or keloid formation at the wound site) is one type of wound dressing. Generally, a porous matrix such as a hydrogel is configured to be removably secured to the skin or other tissue surface and may include one or more active agents (drugs). In other examples, the wound dressing or a portion of the wound dressing may be biodegradable. The hydrogel may have any suitable shape or size.

[0054] Additional criteria for a biologically active wound dressing may include one or more of rapid adhesion to the wound immediately after placement, adequate vapor transmission to limit evaporative fluid loss from the wound and avoid exudate collection between the wound and the dressing. A biologically active wound dressing may function as a skin substitute and may function as one or more of: a barrier to microorganisms, limiting the growth of microorganisms already present in the wound, being flexible, durable, and tear-resistant. Skin substitutes are generally tissue compatible, i.e., should not induce inflammation or a foreign body reaction in the wound that may lead to the formation of granulation tissue. An inner surface structure of the hydrogel film described herein may be provided to allow ingrowth of fibrovascular tissue. An outer surface structure may be provided to minimize fluid transmission and promote epithelialization.

[0055] In some instances, FAKI alone or a FAKI hydrogel (e.g., pullulan / collagen hydrogel) may be part of a graft. The term "graft" generally refers to a piece of skin that is removed from one part of the body and then moved (and attached) to a new site on the same (or different) body. FIG. 14 shows an example of a FAKI porous scaffold skin graft. The patient has a full thickness scalp burn. A piece of skin is taken from a harvest site (in this case the right thigh) and layered (in this case overlaid) with a porous scaffold containing FAKI. In this illustrative example, the graft also includes control areas that include areas that are not overlaid with a porous substrate containing FAKI and areas that are overlaid with a porous substrate that does not contain FAKI, although grafts for transplantation will more commonly be predominantly layered with a hydrogel containing FAKI along most or all sides.

[0056] The term "wound" generally refers to both open and closed wounds, as explained below. Wounds can be further classified as acute or chronic. Acute wounds are wounds that do not have an underlying healing defect and usually occur following surgery or trauma in healthy individuals, and heal rapidly and completely. In contrast, chronic wounds are wounds that have a loss of tissue integrity and result from prolonged or frequent injury or damage. As used herein, the term "skin wound" refers to a break in the skin.

[0057] The term "open wound" is usually classified according to the object that caused the wound. Open wounds include burns, incisions, lacerations, abrasions, puncture wounds, penetration wounds, gunshot wounds, etc. An incision or incision wound may be caused by a clean, sharp object such as a knife, razor, or glass shards. An incision involving only the epidermis may be classified as a cut. Lacerations are irregular wounds caused by blunt impact to soft tissue overlying hard tissue (e.g., laceration of the skin covering the skull) or laceration of the skin and other tissues (e.g., caused by childbirth). Lacerations may exhibit bridging because connective tissue or blood vessels cling to the underlying hard surface. Abrasions (abrasions) are superficial wounds in which the top layer of skin (epidermis) is scraped off, often caused by slipping and falling on a rough surface. Puncture wounds may be caused by an object that pierces the skin, such as a nail or needle. Penetration wounds may be caused by the penetration of an object, such as a knife, into the body. A gunshot wound may be caused by a bullet or similar projectile entering or passing through the body, so there may be two wounds, one at the entry site and one at the exit site, commonly known as a penetration.

[0058] The term "closed wound" refers to contusions, more commonly known as contusions, which are caused by blunt trauma that damages tissue under the skin; hematomas, also known as blood tumors, which are caused by blood collecting further under the skin due to damage to blood vessels; and crush injuries, which can be caused by great or large amounts of force applied over a prolonged period of time.

[0059] The term "scar" refers to an abnormal morphological structure resulting from a previous injury or wound (e.g., incision, excision, or trauma). Scars are composed of connective tissue, primarily collagen types 1 and 3 and a matrix of fibronectin. Scars may consist of abnormally organized collagen fibers (as seen in normal scars of the skin) or may be an abnormal accumulation of connective tissue (as seen in scars of the central nervous system or pathological scars of the skin). Types of scars include, but are not limited to, atrophic scars, hypertrophic scars, and keloid scars, as well as scar contractures. Atrophic scars are depressed flat as depressions or holes below the surrounding skin. Hypertrophic scars are elevated scars that remain within the boundaries of the original lesion and often contain excess collagen arranged in an abnormal pattern. Keloid scars are elevated scars that extend beyond the edges of the original wound and violate the surrounding normal skin in a site-specific manner and often contain collagen whorls arranged in an abnormal manner. A cicatricial contracture is a scar that crosses a joint or skin crease and is prone to shortening or contracture. It occurs when the scar is not fully mature, often tends to become hypertrophic, and typically results in disabling or functional disability.

[0060] A variety of conditions may cause scarring, including surgical wounds, burns, cuts, gunshot wounds, etc. Scars typically form as a result of facial plastic surgery, including, but not limited to, rhygienic surgery, blepharoplasty, rhinoplasty, otoplasty, genitoplasty, facelift, forehead lift, brow lift, facial scar revision, facial scar removal, laser surgery, skin resurfacing, wrinkle treatment, plasma skin resurfacing, facial fat grafting, skin tightening, tattoo removal, and hair replacement. Thus, the present disclosure is beneficial for patients undergoing facial plastic surgery, particularly for the treatment of scarring and bruising, by speeding wound healing and reducing scar formation. Scars also commonly form as a result of general plastic surgery procedures including, but not limited to, abdominoplasty, breast reduction, breast augmentation, body lift procedures, spider vein treatment, stretch mark treatment, liposuction, excess skin removal procedures, cellulite reduction treatments, body contouring, body resurfacing, and body implants.

[0061] "Administering" refers to the administration or application of a pharmaceutical treatment or formulation to a subject via a specific route, including, but not limited to, topical, intradermal injection, intravenous, systemic, subcutaneous, intramuscular, sublingual, and by inhalation or injection, irrigation or osmotic pump.

[0062] Compositions, Devices, and Methods Described herein is a composition for promoting reduced scarring of tissue during healing. The composition includes a porous scaffold and an effective dose of a focal adhesion kinase (FAK) inhibitor. The FAK inhibitor is fabricated for controlled drug release and disposed within the pores of the scaffold, and the scaffold is configured to deliver a dose of the FAK inhibitor to a delivery surface effective to promote tissue healing at a controlled rate during a treatment time. The porous scaffold may include a hydrogel film, such as a pullulan-hydrogel film. The composition may be configured to deliver a dose of the FAK inhibitor to a delivery surface effective to reduce scarring of tissue during a treatment time and / or promote hair growth in tissue during a treatment time. The composition may be configured as a wound dressing and may be flexible.

[0063] Provided herein is a method of promoting healing, e.g., repair, of scar and / or keloid formation, and / or of hair growth, comprising contacting a wound with an effective dose of a FAK inhibitor hydrogel as disclosed herein for a period of time sufficient to reduce scarring, form hair, or form skin appendages. Provided herein is a method of promoting tissue healing, comprising placing a composition comprising a porous scaffold and a focal adhesion kinase (FAK) inhibitor engineered for controlled drug release disposed within the pores of the scaffold, applying the composition to a tissue surface, and delivering a dose of a FAK inhibitor effective to promote tissue healing from the composition delivery surface to the tissue at a controlled rate during a treatment period. In some methods, the composition comprises a hydrogel, such as a pullulan-collagen hydrogel. In some methods, the dose of the FAK inhibitor is effective to reduce scarring of the tissue during the treatment period. In some methods, the FAK inhibitor is effective to reduce scarring of the tissue after the treatment period, e.g., by activation of healing pathways in the body. In some methods, the dose of FAK inhibitor is effective to promote hair growth in the tissue during (or after) the treatment time. For example, hair can continue to grow even after the porous scaffold is removed. In some methods, the composition is configured as a wound dressing. In some methods, the FAK inhibitor is disposed on a delivery surface. In some methods, the controlled rate includes a rapid release rate (for release to the tissue surface) and a sustained release rate (for release to the tissue surface). In some methods, the FAKI for rapid release is released to the tissue surface within about 12 hours, within about 24 hours, or between these times. In some methods, the FAKI for sustained release is released to the delivery surface for a period of time up to about 24 hours, 48 ​​hours, 72 hours, or 96 hours, or any length of time therebetween. In some methods, the FAKI for sustained release is released to the tissue surface for a period of time up to about 24 hours, 48 ​​hours, 72 hours, or 96 hours, or any length of time therebetween. Some methods include removing the composition from the tissue surface, for example, by lifting or pulling the composition from the tissue surface.Some methods include allowing the composition to biodegrade. Some methods include repeating the placing and delivering steps at least 2 times, at least 3 times, at least 10 times, at least 20 times, or at least 30 times (e.g., with new compositions). In some methods, the FAK inhibitor is VS-6062 (PF-562271) or a benzenesulfonate salt of PF-562271. In some methods, about 30% of the total FAKI present in the composition to about 75% of the total FAKI present in the composition is delivered.

[0064] The hydrogel can be maintained in contact with the wound (tissue surface) for a period of time, e.g., at least about 1 day, at least about 2 days, at least about 3 days, at least about 1 week, at least about 10 days, at least about 2 weeks, at least about 3 weeks, or more. For example, in some variations, it is desirable to apply the hydrogel to the wound site during an initial period, such as from about 1 day to about 3 days after injury, i.e., during the early part of the proliferation phase. The hydrogel can be applied to a fresh wound caused by a scar removal procedure. In some cases, the hydrogel is applied up to 7 days after injury, i.e., during the later part of the proliferation phase. For example, swelling and wound exudate may indicate that the hydrogel is applied more than 3 days after injury. In some applications, the first hydrogel can be applied within the initial period after injury, e.g., within the first 3 days, and then removed, after which a second hydrogel can be applied. The second hydrogel can be adapted to changes in the skin and tissue surrounding the wound that may occur after the initial period, such as a decrease in swelling and exudate.

[0065] Provided herein is a method of making a composition comprising a porous scaffold for treating wounds, comprising forming a mixture comprising a focal adhesion kinase (FAK) inhibitor, pullulan, collagen, and a porogen, and removing the porogen and / or dehydrating the mixture to produce a composition comprising a porous scaffold having a focal adhesion kinase (FAK) inhibitor configured for controlled drug release disposed within the pores of the porous scaffold. In some embodiments, the porous scaffold comprises a hydrogel, such as a pullulan and collagen hydrogel. Some methods include depositing a FAK inhibitor on a surface of the composition. Some such methods further include crosslinking the pullulan with sodium trimetaphosphate (STMP) and / or sodium tripolyphosphate (STPP) or another crosslinking agent.

[0066] Some methods include crystallizing the porogen prior to the removal step. For example, a salt porogen such as potassium or sodium, or another salt as described elsewhere herein, can be used. In some methods, the composition includes a non-uniform distribution of the FAK inhibitor. For example, the FAK inhibitor can be concentrated along the pores. The FAK inhibitor can also or instead be concentrated along a particular layer within a multi-layer structure or at an outer surface of the composition (e.g., an outer surface of a device).

[0067] The subject methods find use in any application in which treatment of a wound in a subject is desired. Generally, such subjects are "mammals" or "mammals," terms used broadly to describe organisms within the mammalian range, including carnivora (e.g., dogs and cats), rodents (e.g., mice, guinea pigs, and rats), and primates (e.g., humans, chimpanzees, and monkeys). In many embodiments, the subject is a human.

[0068] Thus, since the subject methods can be used to treat a wide range of open and closed skin wounds, the subject methods can be used to treat wounds caused from a variety of causes, for example, as a result of conditions such as disease states, physical trauma such as falls, scrapes, puncture wounds, gunshot wounds, surgical wounds, infections, wartime injuries such as bombs, bullets, shrapnel, etc. Similarly, the subject methods can treat wounds of various sizes. For example, the subject methods can be used on both deep tissue wounds, where certain wounds may have a depth that reaches the muscle, and shallow or superficial wounds. The wounds may be limited to the epidermis so as not to penetrate the dermis layer, or may be as deep as or deeper than the dermis, for example penetrating or penetrating the dermis, and may even penetrate or penetrating the subcutaneous tissue layer or deeper, for example penetrating or penetrating the muscle layer and below. For example, the subject methods may be used to debride wounds having depths ranging from about 0.005 mm to about 2.35 mm, eg, from about 0.007 mm to about 2.3 mm, eg, from about 0.01 mm to about 2 mm.

[0069] The wound dressing may include an effective dose of FAK inhibitor imprinted or surface applied to a pullulan / collagen hydrogel, which may be attached or adhered to a substrate, such as a breathable protective layer or other protective film. Alternatively, the dressing may be constructed separately from the protective dressing. The support is generally made of a flexible material capable of conforming to the movements of the human body, including, for example, various nonwoven fabrics, woven fabrics, spandex, flannel, or laminates of these materials with polyethylene films, polyethylene glycol terephthalate films, polyvinyl chloride films, ethylene-vinyl acetate copolymer films, polyurethane films, and the like. By "flexible" it is meant that the support can be substantially bent or folded without breaking, tearing, ripping, and the like. The support may be porous or nonporous, but is typically nonporous or impermeable to the hydrogel composition, the active agent, if used, and fluids, such as any fluids that may exude from the wound site.

[0070] The length and width dimensions of the support are typically substantially equal, including exactly equal, to the length and width dimensions of the associated patch composition. The support layer may typically have a thickness ranging from about 10 μm to about 1000 μm, although in certain embodiments, it may be less than about 10 μm and / or greater than 1000 μm.

[0071] In addition to the hydrogel and optional support layer, the dressing may also include a release film on the surface of the hydrogel composition layer opposite the backing that protects the patch layer from the environment. The release film may be of any suitable material, with typical release films including polyesters such as PET or PP.

[0072] The dressings may be of various shapes, with representative shapes including square, rectangular, oval, circular, triangular, etc. The size of the dressings may also vary, with many embodiments having a hydrogel size of about 1 cm. 2 Approximately 1000cm from the following 2 For example, in certain embodiments, the range is about 10 cm 2 ~about 300cm 2 For example, the range is about 20 cm. 2 ~about 200cm 2 For example, about 130 cm 2 ~Approx. 150cm 2 In certain embodiments, the surface area is sufficient to cover a substantial portion, or even the entire track, or even a substantial portion, or even the entire body of a subject. Thus, the surface area is in the range of about 1000 cm 2 ~about 5000cm 2 It should be noted that the above manufacturing protocols are merely representative. As noted above, any suitable protocol capable of manufacturing the subject hydrogel patch compositions may be used.

[0073] The devices and bandages described herein may have any suitable shape. For example, the device or bandage may be rectangular, square, circular, oval, toroidal, or a section or combination thereof. In many variations, the device is flexible and flat, allowing it to conformally rest against the skin. Of course, the devices and bandages may be any suitable size to address various wounds. In some variations, the devices and bandages may be cut from a roll or sheet of bandage immediately prior to use to ensure adequate coverage of the wound site. The devices and bandages may extend up to about 20 cm (about 8 inches) from the wound in some cases, and in other cases, the devices or bandages may extend about 2 cm, 4 cm, 6 cm, 8 cm, 10 cm, 12 cm, 14 cm, 16 cm, or 18 cm from the wound, where "about" modifies each distance. In yet other variations, the bandage may extend about 22 cm, about 24 cm, about 26 cm, or even more from the wound. In some variations, the device is made of a polymer, such as a shape memory polymer. Any suitable shape memory polymer can be used, such as a styrene-based, epoxy-based, or acrylate-based shape memory polymer.

[0074] Also described herein are kits for repairing scar and / or keloid formation and / or developing hair and skin appendages. In general, the kits include a unit dose of a hydrogel containing an effective amount of FAKI as described herein in a packaged combination. Multiple doses in the kit can be designed to be applied in parallel. For example, some kits can include one hydrogel to be applied during an initial period, such as the early part of the proliferative phase of wound healing up to 3 days after injury, and then removed, and a second hydrogel to be applied thereafter. Some kits can include at least one wound dressing, or at least one wound cleansing agent, or other components that are desired or suitable for wound healing applications. The kits can also include instructions for using the device and / or other components contained therein.

[0075] The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention, wherein such container or containers may bear a notice in the form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceutical or biological products, indicating approval by the agency of manufacture, use, or sale for human administration.

[0076] The following examples are set forth so as to provide one of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the following experiments are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.

[0077] All publications and patent applications cited in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0078] The invention has been described with respect to particular embodiments found or suggested by the inventors to comprise preferred modes for carrying out the invention. It will be appreciated by those skilled in the art, in light of this disclosure, that numerous modifications and variations can be made in the particular embodiments exemplified without departing from the intended scope of the invention.

[0079] experiment Controlled delivery of focal adhesion kinase inhibitors results in accelerated wound closure with reduced scar formation.

[0080] Scar formation after wound formation or trauma represents a significant medical burden that costs the economy billions of dollars annually. Activation of focal adhesion kinase (FAK) has been found to play a pivotal role in transducing mechanical signals to induce fibrotic responses and scar formation during wound repair. We previously showed that inhibition of FAK using local injection of a small molecule FAK inhibitor (FAKI) could attenuate scar development in a hypertrophic scar model. However, clinical translation of FAKI therapy has been difficult due to the lack of an effective drug delivery system for extensive burn, blast, and large excision injuries. To address this issue, we developed pullulan collagen-based hydrogels that deliver FAKI to excision and burn wounds in mice. Specifically, we developed two different drug-loaded hydrogels to rapidly or sustainedly release FAKI to treat burn and excision wounds, respectively. Controlled delivery of FAKI via pullulan collagen hydrogels accelerated wound healing and reduced collagen deposition and scar-forming myofibroblast activation in both wound healing models. Our study highlights a biomaterial-based drug delivery approach for wound and scar management that has important translational implications.

[0081] A pullulan-collagen based hydrogel system is used to deliver FAKI for wound healing. The efficacy of FAKI hydrogel therapy was evaluated in large and deep murine excision and burn wound models. Two different types of hydrogels were developed: i.) sustained release hydrogels, and ii.) fast release hydrogels. To develop sustained release hydrogels, molecular imprinting technology was used to incorporate FAKI into porous hydrogels, allowing for the slow and sustained release of FAKI into the wound environment over a period of days. These hydrogels were used for the controlled delivery of FAKI to splinted excision wounds in mice. However, burn wounds are associated with increased serous drainage, requiring more frequent dressing changes. To address this, we used a FAKI surface incorporation method that allows for the rapid release of FAKI within a few hours.

[0082] Herein, we report that FAKI significantly improved wound healing in murine wounds and attenuated fibrogenic activity after wound healing. Our study highlights biomaterial-based anti-scarring drug delivery approaches for wound and scar management.

[0083] In vitro drug release profile of FAKI from pullulan-collagen hydrogels. We previously reported the fabrication and characterization of soft, dermis-like pullulan-collagen hydrogel matrices capable of uptake of cells or small molecule drugs (Wong et al., 2011c and Figure 1A). The swelling and in vitro degradation behavior of the hydrogels was not altered by FAKI loading (swelling ratio 30.87 ± 0.84 without FAKI vs. 32.15 ± 0.56 with FAKI) (Figure 1B). Mass spectrometry confirmed that the structural integrity of FAKI was maintained after incorporation into the hydrogels.

[0084] Depending on the type of injury, two different drug release kinetics were employed to effectively deliver FAKI. Molecular imprinting technology resulted in sustained release kinetics that showed optimal local concentrations of FAKI during the first few days after wound formation (Figure 1C). Using a previously reported mouse HTS model (Aarabi et al., 2007; Wong et al., 2011a), this concentration of FAKI was effective in reducing scar formation when locally injected into the wound site daily for 10 consecutive days (Figure 6). The sustained release FAKI hydrogel was used in splinted excision wounds. As burn wounds produce a large amount of serous drainage after debridement that fouls the dressing, frequent changes of the hydrogel and bandages are necessary to prevent any infection, and a rapid release profile is desirable. To standardize the dose of FAKI applied to both wound types, a hydrogel scaffold prepared by a surface drug incorporation method that rapidly releases FAKI was employed.

[0085] Hydrogels fabricated by molecular imprinting released FAKI continuously over several days, with FAKI being almost completely released around day 4. The surface loading process was completed within 24 hours. Both types of hydrogels released approximately 40%-45% of the total amount initially loaded (Fig. 1C). In the case of the fast-releasing FAKI, the total amount of drug released within 24 hours did not exceed its skin toxicity level (Fig. 6). In the in vitro experiments, no degradation of the hydrogels could be visually observed. Because the hydrogels and dressings are frequently changed when used in vivo, our in vitro release studies up to the first 72 hours are biologically relevant.

[0086] FAKI delivered via hydrogel promotes excision wound healing. Splinted wounds in mice, which recapitulate the mechanism of wound healing via re-epithelialization as seen in humans (Wong et al., 2011b), were treated daily with sustained-release FAKI-loaded hydrogel until the wounds were completely closed. Beginning on day 0 (when the wound was created), the excision wounds were dosed with FAKI hydrogel, which continuously releases FAKI. The used hydrogel was removed and replaced with fresh hydrogel and adhesive bandage every other day. The excision wounds treated with FAKI healed significantly faster than untreated wounds, being completely closed on day 11 post-injury versus day 14 post-injury for the control wounds (P<0.05, Figure 2). Blank hydrogels also accelerated wound closure, suggesting that the scaffold alone may provide a beneficial wound healing environment for excision wounds, consistent with previous reports (Wong et al., 2011c; Wong et al., 2011d). Bulk degradation of the hydrogel, as assessed by visual inspection, did not occur within the time course tested, suggesting that no significant FAKI release accompanied scaffold degradation in vivo. All animals tolerated topical FAKI therapy, and all wounds showed no signs of infection.

[0087] FAKI delivered via hydrogels suppresses profibrotic activity that leads to scar formation in excision wounds. In the absence of a pro-scarring stimulus (e.g., mechanical strain), mouse skin typically does not produce an elevation in HTS, but profibrotic events following injury can be measured by the amount of collagen deposition and the presence of myofibroblasts after the wound has healed. At 17 days after injury, histological analysis of collagen staining demonstrated that collagen deposition was greatest in control wounds, with minimal amounts of collagen observed in FAKI-treated wounds. Protein expression of α-SMA, a marker of myofibroblast differentiation and fibrogenesis, was significantly decreased by FAKI treatment. These results were consistent with a decrease in phosphorylation levels of FAK (p-FAK) in wound tissue lysates collected at 10 days after injury (Figures 3E and 3F).

[0088] Interestingly, when FAKI dissolved in 10% DMSO was instilled daily at the wound site for 10 days, no p-FAK inhibition was detected, supporting the idea that the FAKI-imprinted hydrogel scaffold allows sustained and controlled delivery of FAKI to the local site for improved activity. Activation of Akt (p-Akt) and inhibition of FAKI-mediated Akt phosphorylation were also examined in tissues harvested on day 17. Akt activation was prominent in the epidermal layer during this repair phase, and locally delivered FAKI substantially inhibited Akt activation (Figures 3G and 3H).

[0089] The mechanical properties of healed excision wounds are improved by FAKI treatment. Skin scars formed after wound healing are often accompanied by altered structural integrity and weaker skin mechanical properties. We tested whether FAKI treatment could improve the mechanical profile of healed wounds. The Young's modulus of healed skin was significantly higher in wounds treated with hydrogel alone and FAKI hydrogel compared to controls (Figure 4), supporting the improved mechanical integrity of healed skin and therefore its enhanced ability to resist external mechanical stress. Young's modulus was highest with FAKI treatment. We also examined ultimate tensile strength (UTS), which measures the maximum stress that skin can withstand when stretched, and FAKI-treated skin showed the highest UTS. Taken together, FAKI is beneficial in improving the physical integrity of healed skin, possibly via regulating collagen deposition and extracellular matrix composition of the dermis during wound healing and repair processes.

[0090] FAKI delivered via hydrogel accelerates healing of contact burn wounds. The prolonged healing associated with severe deep skin burns may commonly lead to HTS formation. A murine full-thickness contact burn wound model was used to study the antifibrotic effects of FAKI and whether FAKI could accelerate healing of burn wounds. After debridement 72 hours after injury, burn wounds were treated with a hydrogel that rapidly releases FAKI. Unlike excision wounds, burn wounds showed increased drainage from the wound site and adjacent tissues until the wound re-epithelialized. As a result, more frequent dressing changes were required. The hydrogel and bandages were changed daily for the first 3 days after the first treatment, and every other day thereafter. Compared to hydrogel alone or control wounds, burn wounds treated with FAKI showed accelerated wound closure without complications (Figure 5). Similar to the observations in excision wounds, bulk degradation of the hydrogel did not occur. After complete closure, α-SMA expression in the wound lesion was measured, which was significantly reduced with FAKI treatment (Figure 5).

[0091] Local delivery of focal adhesion kinase inhibitors results in accelerated healing, reduced scarring, and regeneration of hair follicles and skin appendages.

[0092] Here we report that FAKI in pullulan-collagen devices significantly accelerated wound healing, reduced scar formation, and regenerated hair follicles and skin appendages in a large animal model known to be most similar to human skin. Red Duroc pigs can develop thick, permanent, human-like scars when deep skin wounds are formed. A sustained release FAKI hydrogel (shown in Figures 1A-1C and described elsewhere herein) was used on deep skin wounds in Red Duroc pigs. Figures 7A-7D show a setup to analyze the effect of topical treatment of FAKI applied in a hydrogel device on deep skin wounds in the Red Duroc pig model. Figure 7A shows an area along the back of the pig used for testing. Figure 7B shows the deep wound of the pig with the removal of the skin. Figure 7C shows a cross-section of intact pig skin. Figure 7D shows a cross-section of wounded pig skin with a wound depth of 0.06 inches (in). Figure 7E shows the timeline from the day of injury (D0) to postoperative day 180 (D180). FAKI in pullulan-collagen devices were placed on the wound and replaced every other day for the first 21 days. From day 22 to day 90, the FAKI pullulan-collagen devices were replaced twice weekly. From postoperative day 91 to postoperative day 180, the wound was allowed to heal without application of the pullulan-collagen device. Biopsy samples, photographs, and tissue collection were performed as indicated.

[0093] FAKI delivered via hydrogel significantly accelerates wound healing of deep dermal wounds. Figures 8A-8C show that topical treatment of deep dermal wounds in Duroc pigs with FAKI hydrogel accelerates wound healing. N = 8 wounds tested for each condition. Initial wound size = 25 cm 2FIG. 8A shows photographs of wounds taken from post-operative day (POD) 2 through post-operative day 30 (POD30) as described above in FIGS. 7A-7E. Wounds treated with FAKI hydrogel are visibly healed better on POD11, POD21, and POD30 compared to untreated wounds or wounds treated with hydrogel alone. In addition to a visibly improved appearance, wounds treated with FAKI hydrogel were significantly smaller. The remaining wound area of ​​wounds treated with FAKI delivered via hydrogel was compared to untreated control wounds and wounds treated with hydrogel alone. Wound size was measured and graphed as a % of original wound area, and the results are shown in FIG. 8B. Wounds treated with FAKI hydrogel were smaller compared to controls after POD 4, and by POD 14, nearly all of the FAKI hydrogel-treated wounds were closed, whereas approximately 20% of the untreated (control) wounds were not closed and remained open, a difference that was not statistically significant ( * ) By POD17, all of the FAKI hydrogel-treated wounds were closed, whereas approximately 10% of the untreated (control) wounds remained open, a statistically significant difference ( * ) Hydrogel alone accelerated wound healing, suggesting that the scaffold alone may provide a beneficial wound healing environment for excision wounds, as described above. Figure 8C shows that untreated wounds or wounds treated with hydrogel alone took 25 days to fully close, whereas wounds treated with FAKI in the hydrogel device closed by POD 14, a statistically significant result ( Δ ).

[0094] FAKI delivered via hydrogel significantly improves scar appearance. Figures 9A-9B and 10A-10B show that topical treatment of deep skin wounds with FAKI delivered via hydrogel significantly improves scar appearance at POD45, POD60, POD90 (3 months), and POD180 (6 months). Figure 9B shows results from visual analog scale (VAS) analysis of scars. Visual analog scale scores were assessed by four blinded scar experts for untreated controls, hydrogel treatment, and FAKI via hydrogel treatment. Scar formation in controls was arbitrarily set to 100, and results were normalized to controls. Wounds treated with FAKI via hydrogel showed significantly less scarring and better visual appearance, approximately 58 on a scale of 100, compared to untreated controls (Figure 9B, Δ P<0.001). Hydrogel treatment alone showed only slight improvement compared to untreated controls, a trend that persisted on POD180 (Fig. 10B, Δ P<0.001).

[0095] FAKI delivered via hydrogel leads to significant hair formation and regeneration of hair follicles and skin appendages. Surprisingly, deep skin wounds treated locally with FAKI delivered via hydrogel show significant hair growth not seen in control treatments or wounds treated with hydrogel alone, as shown in FIG. 10B. FIGS. 11A-11C show results from POD90. The image in FIG. 11A shows trichrome staining at POD90. Arrow 1 points to a hair follicle. Arrow 3 points to a hair follicle with surrounding glandular structures. Arrow 2 and arrows 4-6 point to skin glands (sweat and sebaceous glands). Wounds treated with FAKI delivered via hydrogel had significantly more hair follicles at POD90 (average of approximately 2.8 vs. 0.6 in controls, #P<0.01 (control vs. H+FAKI) and this difference remains on POD 180. Wounds treated with FAKI delivered via hydrogel had significantly more glands (sebaceous and sweat glands) on POD 90 (approximately 7 in hydrogel+FAKI treated wounds vs. 0.1 in controls, * P<0.001 (control vs. H+FAKI) and this difference remains at POD180 (approximate values ​​for control vs. X).

[0096] FAKI delivered via hydrogel suppresses scar-forming pro-fibrotic activity in excision wounds. Similar to what was shown previously for murine excision wounds, FIG. 12A shows that FAKI delivered via hydrogel significantly reduces the expression of alpha-smooth muscle activity (α-SMA) in wounds treated with FAKI delivered via hydrogel versus untreated controls at POD60 and POD90. FIG. 12B (POD60) shows that the level of α-SMA expression is significantly lower in treated wounds (about 2% of the area stained green (indicative of α-SMA) in treated samples versus about 28% in untreated controls; * p<0.001), and this trend remained at POD90 (Figure 12C). Alpha-smooth muscle activity (α-SMA) was quantified in randomly selected areas within the scar lesion by a blinded observer.

[0097] FAKI delivered via hydrogel results in dermal collagen structure resembling unwounded skin. Figures 13A-C show that wounds treated with FAKI delivered via hydrogel result in dermal collagen structure resembling unwounded skin. Figure 13A shows that tissue from pigs locally treated with FAKI delivered via hydrogel has shorter fibrils compared to either untreated control wounds or wounds treated with hydrogel (compare the third panel in Figure 13A with the top panel and the second panel). Rather, collagen fibrils from tissue from pigs locally treated with FAKI are much more similar to tissue from unwounded animals (bottom panel). Figure 13B shows the results of quantification of collagen fibril length from samples stained as shown in Figure 13A. Wounds locally treated with FAKI delivered via hydrogel showed significantly shorter fibrils compared to untreated control wounds (p<0.01). Figure 13C shows the number of collagen fibrils from panels stained as shown in Figure 13A. Wounds treated topically with FAKI delivered via hydrogel showed significantly more fibrils compared to untreated control wounds (p<0.05). The horizontal line in the middle of the bar graph represents the median and the X represents the mean. N=9 images for control, hydrogel, and hydrogel+FAKI. N=4 images for no wound.

[0098] FIG. 15A-B shows the safety of FAKI delivered locally via hydrogel. FIG. 15A-B shows the toxicology and pharmacokinetic studies of FAKI delivered locally via hydrogel. Systemic absorption of FAKI molecules was examined from serum at various time points after application, and the results are shown in FIG. 15A. Systemic absorption of locally delivered FAKI to 5%-7% of the total body surface was minimal (1 ng / ml=2 nM FAKI). FAKI hydrogel samples were applied daily to unwounded skin at high concentrations of FAKI (150 μM FAKI, 1.5 mM FAKI), and the results are shown in FIG. 15B. Daily application of FAKI at high concentrations did not result in skin irritation or sensitization.

[0099] Currently, clinically available therapies to inhibit abnormal healing and scarring associated with burn and traumatic wounds are weakly evidenced and minimally effective. The integrin / FAK mechanotransduction signaling pathway plays a central role in dermal scar formation after injury by mediating mechanical stress signals and promoting fibrotic processes through inflammatory mediators. Thus, inhibition of FAK signaling is a promising strategy for scar-free wound healing in large and deep dermal wounds. Newer generation small molecule inhibitors of FAK, including VS-6062 and the improved VS-6063 / defactinib, have proven effective in treating advanced solid tumors in clinical trials. Although VS-6062 showed favorable pharmacokinetics in these studies, it was discontinued as an anticancer agent due to potential drug-drug interactions when administered orally. When administered orally, major systemic toxicity with nausea / vomiting and gastrointestinal-related adverse effects was observed in these studies. This localized topical delivery of compounds avoids systemic absorption and subsequent toxicity and was used in our studies.

[0100] In a previous mouse study, intradermal injection of a small molecule FAK inhibitor (PF573228) suppressed inflammatory chemokine pathways and attenuated scarring in vivo in an incision HTS model. However, large areas of deep skin wounds are difficult to treat by dermal injections, and targeted local delivery of pharmacological agents is likely to be the preferred modality in the clinic. Biomaterial-based approaches for wound treatment have been extensively investigated in the past. Various chitosan or hyaluronic acid hydrogel composites have been tested on skin wounds and had beneficial effects on skin healing and regeneration.

[0101] Previously, our group reported that biodegradable pullulan-collagen skin hydrogel alone could enhance early wound healing in mouse wounds. Pullulan hydrogel is non-toxic, modifiable, and exhibits ideal water-retaining capacity. The porous matrix also possesses multiple functional groups that allow cross-linking, and may serve as a structured template for cell-based and / or biomolecule delivery for various skin healing disorders. This bioengineered matrix was used to deliver mesenchymal stem cells to high oxidative stress wounds to regenerate skin cell populations.

[0102] The findings of this study are the first to deliver an anti-scarring agent within a pullulan-collagen hydrogel for open wounds. The molecular imprinting technique to encapsulate FAKI combined with a proprietary hydrogel fabrication method allows for sustained release of FAKI from the hydrogel scaffold, precisely controlled for sustained release, rapid release, or a combination of the two. As shown herein, FAKI therapy significantly accelerated healing and reduced scar formation in full-thickness excision and burn wounds. The accelerated wound healing is likely due to the inhibitory effects of FAKI on early immune cell responses (e.g., proliferation and migration) and inflammation.

[0103] This study provides mechanistic insight into FAK-mediated mechanotransduction and antifibrotic therapeutics. It is demonstrated that FAKI inhibits AKT activation, suggesting that FAK may act through multiple downstream effector proteins to enhance fibrotic processes in healing skin (Figure 3). Pullulan-collagen hydrogel alone was shown to be beneficial in promoting healing, but had little or no effect on burn wounds.

[0104] We hypothesize that these differences in efficacy may be due to different physiological responses to different types of injury, as burn wounds are pathologically different from surgically excised wounds and often induce metabolic and inflammatory changes. Furthermore, the mechanical integrity of reconstructed skin after FAKI treatment is also improved. Direct investigation of mechanical integrity using tensile testing was used to calculate Young's modulus and ultimate tensile strength (Figure 4). FAKI-treated skin exhibited the highest Young's modulus as well as ultimate tensile strength, both of which are measures of the physical integrity of the healed skin. Thus, the ability of healed skin to withstand deformation and fracture in response to external forces or stresses is enhanced by FAKI treatment compared to hydrogel alone.

[0105] In conclusion, we show that FAKI therapy via pullulan-collagen hydrogel scaffolds is effective in reducing scar formation while promoting excision and burn wound healing in mouse models. Our biomaterial-based delivery of FAKI holds great promise as an effective therapeutic strategy for scar management in large, deep dermal wounds. The results of this study will enable translation of FAKI scar-reducing therapy in a highly translational preclinical large animal model for use in human clinical trials.

[0106] Materials and Methods Construction of biocompatible FAKI-releasing hydrogel scaffolds. FAKI, VS-6062 (formerly PF-562271), was supplied by Verastem, Inc. (Needham, MA). Porous pullulan-collagen hydrogels were fabricated as previously described (Wong et al., 2011c). FAKI-containing hydrogel constructs were prepared by either molecular imprinting (sustained release) or surface incorporation (fast release). Briefly, 1 g pullulan (Tokyo Chemical Industry Co., Ltd. (TCI), Tokyo, Japan) was mixed with 1 g trisodium trimetaphosphate (STMP) (Sigma-Aldrich, St. Louis, MO) and 1 g potassium chloride (Fisher Scientific, Hampton, NH). A collagen solution containing 50 mg of rat tail collagen type I and 5% (w / w) pullulan (Corning, Corning, NY) was added to the mixture. For molecular imprinting (FIG. 1A), 10 mg of VS-6062 (FAKI) in 1 ml of 5% dimethyl sulfoxide (DMSO) was added to the mixture. Sodium hydroxide (0.65 ml of a 1N solution) was then added, followed by deionized water to a total of 10 ml. The mixture was gently vortexed for 15 minutes for homogeneous distribution. Once the mixture had increased in viscosity due to cross-linking of pullulan, the mixture was poured onto a Teflon sheet and compressed to produce a 2 mm thick film. The film was dried and washed with water until the pH of the washing solution was 7.0. After this washing step, approximately 50% of the FAKI was present in the hydrogel. The swollen hydrogel was frozen at −80° C. and then lyophilized. For the surface incorporation method, blank hydrogels were first prepared, then 5 mg of FAKI dissolved in ethanol was evenly spread onto the dried hydrogels, followed by evaporation of the solvent.Finally, the hydrogel films were stored at 4°C in a moisture-free container until use.

[0107] Water uptake / swelling properties: Hydrogels constructed with and without FAKI were tested for swelling behavior in PBS. Freeze-dried hydrogels containing 5% collagen with or without FAKI were cut into circular patches using a 0.8 mm biopsy punch, weighed, and incubated in PBS at 25° C. Excess solution was gently removed and the swelling ratio was calculated using the following formula: Swelling ratio=(Wet weight of hydrogel-Dry weight of hydrogel) / Dry weight of hydrogel.

[0108] Extraction of FAKI from hydrogel: A weight of 2.5 mg of dried hydrogel was mixed with 0.5 ml of water and then homogenized using a Bullet Blender bead mill homogenizer (Next Advance, Averill Park, NY). The homogenate was diluted with 0.5 ml of methanol to extract FAKI, and then centrifuged at 5000 g for 5 min. A sample recovery of more than 90% was achieved, which was analyzed by HPLC and mass spectrometry (LC-MS) methods described below.

[0109] In vitro drug release profile for FAKI from pullulan-collagen hydrogels. To compare the two different template preparation methods, the in vitro release kinetics of FAKI was studied. A dry hydrogel weighing 2.5 mg containing 50 μg FAKI was placed in a 25000 MWCO dialysis membrane tube containing 1 ml PBS. After sealing both sides of the dialysis tube, it was submerged in 9 ml PBS in a vial. The vial was incubated at 37°C with gentle agitation. Small aliquots were periodically withdrawn to quantify and measure the completeness of FAKI released from the hydrogel.

[0110] LC-MS analysis: Experiments were performed on an AB SCIEX 4000 QTRAP® LC-MS / MS system. The HPLC equipment was a Shimadzu Prominence LC system equipped with a system controller CBM-20A, a binary LC-20AD pump, and a SIL-20AC autosampler. HPLC was performed using the following specifications: mobile phase: 90% methanol / 10% water / 0.1% formic acid, column: Dionex Acclaim 120 C8, 5 μm, 50×2.1 mm, flow rate: 0.3 ml / min, and injection volume: 10 μL. The HPLC was directly connected to an AB SCIEX 4000 QTRAP® triple quadrupole mass spectrometer with electrospray ionization. The mass spectrometer was operated in positive multiple reaction monitoring (MRM) mode, ion source: turbospray, resolution: Q1 unit, Q3 unit, mass MRM transition (m / z): 508.30>121.00.

[0111] Data Analysis: The structural integrity of FAKI was determined by molecular weight and HPLC retention time. Cumulative release from in vitro experiments performed in triplicate was expressed as mean ± SD.3.

[0112] Animals and splinted excision and contact burn wound models Animals (mice): All animal procedures were approved by the Stanford University Administrative Panel on Laboratory Animal Care and the U.S. Army Medical Research Command Animal Care and Use Review Office. Wild-type female C57BL / 6J mice aged 8-10 weeks (The Jackson Laboratory, Bar Harbor, ME) were used to create either splinted full-thickness excision wounds or third-degree burn wounds according to published methods (Wang et al., 2013; Wong et al., 2011b; Sultan et al., 2012).

[0113] Excision wound creation: For surgically created excision wounds, two full-thickness wounds (one on each side of the dorsal midline) were made using a 6 mm punch biopsy tool. A silicone ring surrounding the wound was sutured to the skin to prevent wound contraction (the primary means of wound healing in rodents) but allowed the wound to close by re-epithelialization (similar to human wound healing).

[0114] Burn wound creation: A contact burn wound was created on only the dorsal side with an aluminum rod heated to 100°C for 20 seconds in a hot water bath. The wound was immediately cooled with cold water for 30 seconds. To simulate the usual clinical setting of full-thickness human burns, necrotic tissue was excised along the wound edge to the level of the underlying subcutaneous fat 72 hours after the initial injury. In both models, wounded animals were randomly divided into three groups and received either a standard dressing (Tegaderm) or blank hydrogel or FAKI-loaded hydrogel. Dressings and hydrogels were changed every 2–3 days. Burn wounds were allowed to heal without splinting, and hydrogels and bandages were changed daily for the first 3 days and every other day thereafter. For some excision wounds, FAKI dissolved in 10% DMSO was instilled directly onto the wound site daily for 10 days, after which cell lysates were collected for Western blotting. All digital photographs were analyzed by two blinded investigators using the National Institutes of Health (NIH) ImageJ software.

[0115] Hypertrophic scar model and local subcutaneous injection of FAKI: Dorsal incisional wound formation and hypertrophic scar development in mice were performed as previously described (Aarabi et al., 2007; Wong et al., 2011a). Animals were randomly assigned to receive FAKI at concentrations of 15 μM, 75 μM, or 150 μM dissolved in 10% DMSO via daily local subcutaneous injection adjacent to the wound site. Animals receiving 150 μM FAKI did not survive longer than 2 days due to unidentified systemic complications. Specimens were harvested 14 days after injury for histology, and scar area was quantified (Figure 6).

[0116] Animals (mice): Red Duroc pigs were used to generate thick, permanent, human-like scars during deep dermal wounding according to published methods (Zhu KQ et al., Burns 2003 Nov; 29(7):649-664. Burns; Zhu KQ et al., Wound Repair Regen. 2007 Sep-Oct; 15 Suppl 1:32-39). Pig skin has been found to be most similar to human skin (e.g., similar epidermal thickness, similar dermal-epidermal thickness ratio, hair follicle and vascular pattern). Seven female Red Duroc pigs, aged 6-8 weeks and weighing 16-20 kg, were used. Multiple 5 x 5 cm, 0.07 inch excision wounds (Figure 7B and insets in Figure 7E) were created with a powered dermatome. Wounds were near full thickness in the center and deeper partial thickness toward the periphery. Wounds were randomly assigned to control (no treatment), hydrogel only (placebo), or hydrogel with FAKI therapeutic agent.

[0117] Histological and immunofluorescence staining. Specimens were taken 17 days after injury and 24 days after debridement for excision and burn wounds, respectively, and fixed in 4% paraformaldehyde, dehydrated, and embedded in paraffin. Hematoxylin & Eosin (H&E) staining and Masson's trichrome staining were performed according to the routine protocol. The percentage of blue area in the Masson's trichrome stained images was quantified using ImageJ. Immunofluorescence staining was performed using a primary antibody against mouse alpha smooth muscle actin (α-SMA). A secondary antibody conjugated to FITC was used, and the images were observed under a fluorescent microscope. The percentage of green area was quantified using ImageJ.

[0118] Western blotting. Western blotting was performed to study FAK expression and phosphorylation at tyrosine-397 in excision wound lysates. Proteins were collected and quantified using the BCA assay. Equal amounts of protein were loaded into each well of precast 4% gels and subjected to SDS-PAGE. After protein transfer, nitrocellulose membranes were incubated with primary antibodies against either FAK (Cell Signaling, Danvers, MA) or p-FAK (Abcam, Cambridge, MA), followed by incubation with HRP-conjugated secondary antibodies. Chemiluminescent detection of antibodies was performed according to the manufacturer's protocol. Band intensity was quantified using ImageJ.

[0119] CT-FIRE. Sections were stained for collagen and analyzed using CT-FIRE software. CT-FIRE software allows users to automatically extract collagen fibers from images and quantify the fibers using descriptive statistics including fiber angle, length, straightness, and width. The program reads image files supported by MATLAB® and extracts individual collagen fibers via a combinatorial method called CT-FIRE (called ctFIRE in the software implementation). The CT-FIRE approach has been described in Bredfeldt JS, Liu Y, Pehlke CA et al. (2014) "Computational segmentation of collagen fibers from second-harmonic generation images of breast cancer." J Biomed Opt 19:16007-16007, which incorporates the discrete curvelet transform for image denoising and enhancing fiber edge features (Candes E, Demanet L, Donoho D, Ying L. (2006) "Fast discrete curvelet transforms." Multiscale Modeling and Simulation. Simul 5:861-899) and the Fiber Extraction (FIRE) algorithm for the extraction of individual fibers. [3] It combines the advantages of.

[0120] Mechanical testing of skin. Three weeks after the creation of the splinted excision wound, wound tissue was collected and mounted on an MTS Bionix® tensile tester (MTS Systems, Eden Prairie, MN) for mechanical testing. The sample gauge distance was set to 10 mm, and the thickness and width of the samples were measured manually. The skin tissue was stretched at a constant speed of 100 m / s until fracture, after which the true stress vs. true strain curve was plotted. The ultimate tensile strength (UTS) was determined as the maximum stress achieved before fracture, and the strain corresponding to the UTS was defined as the critical strain. Young's modulus was calculated via linear least squares regression (R2≧0.99).

[0121] Data analysis. Results are reported as mean ± SD. Statistical differences were determined using one-way ANOVA with Tukey's post-hoc test for multiple comparisons. Differences were considered statistically significant at p < 0.05.

[0122] When a feature or element is referred to herein as being "on" another feature or element, the feature or element may be directly on the other feature or element, or there may be intervening features and / or elements present. In contrast, when a feature or element is referred to as being "directly on" another feature or element, there are no intervening features and / or elements present. Also, when a feature or element is referred to as being "connected," "attached," or "coupled" to another feature or element, the feature or element may be directly connected, attached, or coupled to the other feature or element, or there may be intervening features or elements present. In contrast, when a feature or element is referred to as being "directly connected," "directly attached," or "directly coupled" to another feature or element, there are no intervening features or elements present. Although described or illustrated with respect to one embodiment, the features and elements so described or illustrated may be applicable to other embodiments. It will also be understood by those skilled in the art that a reference to a structure or feature being located "adjacent" to another feature may have portions that overlap the adjacent feature or portions that underlie the adjacent feature.

[0123] The terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting of the invention. For example, as used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise. It will be further understood that as used herein, the terms "comprises" and / or "comprising" specify the presence of the specified features, steps, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups thereof. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items and may be abbreviated as " / ".

[0124] Spatially relative terms such as "under," "below," "lower," "over," "upper," and the like may be used herein for ease of description to describe the relationship of an element or feature shown in the figures to one or more other elements or features. It will be understood that the spatially relative terms are intended to encompass different orientations of the device in use or operation in addition to the orientation depicted in the figures. For example, if a device in the figures is inverted, an element described as "under" or "beneath" another element or feature would be oriented "over" the other element or feature. Thus, the exemplary term "under" can encompass both an orientation of over and under. The device may be otherwise oriented (rotated 90 degrees or in other orientations) and the spatially relative descriptors used herein may be interpreted accordingly. Similarly, terms such as "upwardly," "downwardly," "vertical," "horizontal," etc. are used herein for descriptive purposes only, unless specifically indicated otherwise.

[0125] Although various features / elements (including steps) may be described herein using the terms "first" and "second," these features / elements should not be limited by these terms unless the context dictates otherwise. These terms may be used to distinguish one feature / element from another. Thus, a first feature / element discussed below could be referred to as a second feature / element, and similarly, a second feature / element discussed below could be referred to as a first feature / element, without departing from the teachings of the present invention.

[0126] Throughout this specification and the claims that follow, unless the context otherwise requires, the word "comprise," as well as variants such as "comprises" and "comprising," mean that various components can be used together in methods and articles (e.g., apparatus and methods, including compositions and devices). For example, the term "comprising" will be understood to mean the inclusion of any specified element or step, but not the exclusion of any other element or step.

[0127] In general, any of the apparatus and methods described herein should be understood to be inclusive, while all or a subset of the components and / or steps may be exclusive and may be expressed as "consisting of" or "consisting essentially of" various components, steps, subcomponents, or substeps.

[0128] As used in this specification and claims, including those used in the examples, unless expressly indicated otherwise, all numbers may be read as if they are preceded by the word "about" or "approximately", even if not expressly shown. The phrase "about" or "approximately" may be used when describing a size and / or location to indicate that the described value and / or location is within a reasonable expected range of values ​​and / or locations. For example, a numerical value may have a value that is ±0.1% of the specified value (or range of values), ±1% of the specified value (or range of values), ±2% of the specified value (or range of values), ±5% of the specified value (or range of values), ±10% of the specified value (or range of values), etc. Any numerical value provided herein should also be understood to include values ​​about or approximately that value, unless the context indicates otherwise. For example, if the value "10" is disclosed, "about 10" is also disclosed. Any numerical range recited herein is intended to include all subranges contained therein. As would be well understood by one of ordinary skill in the art, when a value is disclosed, it is understood that "less than or equal to" that value, "more than or equal to" that value, and possible ranges between the values ​​are also disclosed. For example, when a value "X" is disclosed, "less than or equal to X" as well as "more than or equal to X" (e.g., when X is a numeric value) are also disclosed. It is also understood that throughout this application, data is presented in many different formats, and this data represents endpoints and starting points, as well as ranges for any combination of the data points. For example, when a specific data point "10" and a specific data point "15" are disclosed, it is understood that between 10 and 15, as well as greater than 10 and 15, greater than or equal to 10 and 15, less than 10 and 15, less than or equal to 10 and 15, and equal to 10 and 15 are considered to be disclosed. It is also understood that each unit between two specific units is also disclosed. For example, when 10 and 15 are disclosed, 11, 12, 13, and 14 are also disclosed.

[0129] Although various exemplary embodiments have been described above, any number of modifications may be made to the various embodiments without departing from the scope of the invention as set forth in the claims. For example, the order in which the various method steps described are performed may often be changed in alternative embodiments, and one or more method steps may be skipped entirely in other alternative embodiments. Optional features of the various device and system embodiments may be included in some embodiments and not included in others. Thus, the foregoing description has been provided primarily for illustrative purposes and should not be construed as limiting the scope of the invention, as set forth in the claims.

[0130] The examples and figures contained herein show, by way of illustration and not by way of limitation, specific embodiments in which the subject matter may be practiced. As noted above, other embodiments may be utilized and derived therefrom, and thus structural and logical substitutions and changes may be made without departing from the scope of the present disclosure. Such embodiments of the subject matter of the present invention may be referred to herein, individually or collectively, by the term "invention" merely for convenience, and without any intention of intentionally limiting the scope of the present application to any single invention or inventive concept, if in fact more than one is disclosed. Thus, while specific embodiments have been illustrated and described herein, any configuration calculated to achieve the same purpose may be substituted for the specific embodiments shown. This disclosure is intended to cover any and all adaptations or variations of the various embodiments. Combinations of the above embodiments, and other embodiments not specifically described herein, will be apparent to those of skill in the art upon reviewing the above description.

Claims

1. A composition for use in promoting tissue healing and forming new hair and skin appendages, comprising: a porous scaffold comprising a pullulan-collagen hydrogel; a focal adhesion kinase (FAK) inhibitor disposed in the pores of the scaffold; The composition is configured for sustained release of a FAK inhibitor disposed within the pores of the scaffold to deliver an effective dose of the FAK inhibitor to the tissue to promote tissue healing at a controlled rate over a treatment period. composition.

2. The composition for use according to claim 1, wherein the porous scaffold comprises a hydrogel film.

3. The composition for use according to claim 1, wherein the FAK inhibitor comprises VS-6062 (PF-562271) or a benzenesulfonate salt of PF-562271.

4. The composition for use according to claim 2 or 3, wherein the porous scaffold comprises a hydrogel film and the FAK inhibitor is present in the composition at a concentration of from about 1 μg / cm 2 to about 500 μg / cm 2 , or from about 40 μg / cm 2 to about 200 μg / cm 2 .

5. A composition for use as described in any one of claims 1 to 4, wherein the composition is configured to sustained release of the FAK inhibitor disposed in the pores of the scaffold for a period of up to about 96 hours.

6. A composition for use according to any one of claims 1 to 5, wherein the composition is prepared by molecular imprinting the FAK inhibitor during formation of the porous scaffold.

7. The composition for use according to any one of claims 1 to 6, wherein the composition is further manufactured to rapidly release the FAK inhibitor.

8. The composition for use described in claim 7, further comprising the FAK inhibitor on a delivery surface of the composition, the composition further configured for rapid release of the FAK inhibitor from the delivery surface of the composition.

9. The composition for use according to claim 8, wherein the composition is configured for rapid release of the FAK inhibitor from the delivery surface of the composition within about 24 hours.

10. A composition for use according to any one of claims 1 to 9, wherein the composition is adapted to release from about 30% to about 75% of the FAK inhibitor during the treatment time, or adapted to release substantially all of the FAK inhibitor during the treatment time.

11. The composition for use according to any one of claims 1 to 10, wherein the composition comprises multiple layers, one or more layers being tailored for sustained release of the FAK inhibitor and one or more layers being tailored for rapid release of the FAK inhibitor.

12. A composition for use according to any one of claims 1 to 10, wherein the composition comprises a layer configured for both sustained release of the FAK inhibitor and rapid release of the FAK inhibitor.

13. The composition for use according to any one of claims 1 to 12, wherein the composition is configured to deliver a dose of the FAK inhibitor effective to reduce scarring of the tissue during the treatment period.

14. A composition for use as described in any one of claims 1 to 13, wherein the composition is configured as a wound dressing.