Therapeutic hydrogel compositions

Abstract

The present invention relates to hydrogel compositions that are useful in therapeutic applications. The invention further relates to methods for preparing these hydrogel compositions and to their use for therapeutic applications, in particular ocular and topical therapeutic applications.SOLUTION: Disclosed is a shear-thinning hydrogel composition comprising 0.1 to 5wt% (e.g., 0.1 to 2. 5wt%) of a microgel particle-forming polymer; and 0.5 to 100mM of monovalent and / or multivalent metal ion salts as crosslinkers. The microgel particle-forming polymer is dispersed in the aqueous vehicle and the hydrogel composition has a pH in the range of 3 to 8. The viscosity of the gel composition decreases when the gel is subjected to shear. Methods of making such compositions and medical uses of such compositions are also disclosed.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to hydrogel compositions that are useful for therapeutic applications. The present invention further relates to methods for preparing these hydrogel compositions and their use for therapeutic applications, particularly ophthalmic and topical therapeutic applications. [Background technology]

[0002] In 2018, the WHO reported that corneal opacity is the leading cause of blindness worldwide. Corneal infections caused by conditions such as bacterial keratitis result in the breakdown of collagen and extracellular matrix, forming scars. Treatment often requires the resolution of the infection using steroids and antibiotics. Unresolved corneal opacity can lead to the need for surgical corneal transplantation. Attempts have been made to manage corneal scarring through aggressive management of infection / inflammation, but the use of potent anti-scarring treatments has not been very successful. One limitation of current eye drop treatments is low viscosity or weak gelling materials, which do not significantly enhance drug retention time.

[0003] Corneal opacity is the leading cause of amblyopia worldwide, affecting an estimated 27.9 million people worldwide, either bilaterally or unilaterally. [1] Such opacities typically result from alterations of the complex, clear corneal tissue architecture, which is crucial for light refraction on the retina and subsequent neuro-visual processing. Corneal scarring commonly results from ocular infections from a variety of pathogens, including bacteria, parasites, fungi, viruses, and protozoa. In developed countries, devastating corneal infections are most commonly associated with prolonged contact lens wear and / or poor lens hygiene. [2~4]In corneal keratinitis, Pseudomonas aeruginosa is the predominant causative organism. In the case of gram-negative infections, such as Pseudomonas, the structural integrity of the cornea is compromised by multiple virulence factors, allowing the microorganism to invade the epithelial cells, resulting in the activation of numerous inflammatory pathways, followed by inflammation, angiogenesis, cellular changes, and degradative stromal processes. [5] leads to the breakdown of complex collagen fibrils [6] Continued inflammation leads to fibrosis and dysregulated remodeling of the interstitial tissue matrix with more widespread disorganized collagen fibrils and loss of optical transparency, resulting in impaired light refraction and visual loss.

[0004] Typically, transforming growth factor beta (TGFβ) is largely restricted to the epithelium in healthy corneas, whereas local trauma induces the production of cytokines, including TGFβ, in the epithelium and stroma. [7] In the injured cornea, when physiological recovery or the aid of exogenous drug treatments is insufficient to suppress the inflammatory response, disorganized fibril organization and dysregulated extracellular matrix (ECM) deposition lead to a fibrotic response and persistent corneal scarring accompanied by visual impairment. Mechanistically, TGFβ activates corneal fibroblasts (corneal stromal cells), resulting in their differentiation into myofibroblasts and promoting wound contraction via the excretion of ECM molecules, including collagen. [7] .

[0005] Currently, standard clinical treatment for patients with bacterial keratitis focuses initially on sterilizing the affected eye with intensive broad-spectrum antibiotic eye drops, followed by the addition of topical corticosteroids to reduce inflammation. [8~9] These range from intensive lubrication (to reduce the biomechanical trauma of the eyelid rubbing against the wound bed during blinking) to systemic medications (sub-antimicrobial doses of tetracycline for matrix metalloproteinase inhibition) in an attempt to promote tissue remodeling.

[10] ) or supplements (vitamin C used as an antioxidant and free radical scavenger

[11] Strategies to inhibit scar formation continue, ranging from the use of ophthalmic gels to the use of ophthalmic gels. Unfortunately, while effective in sterilizing the eye, patients are often left with a severe corneal haze that can cause vision loss if the haze obstructs the visual axis. Surgical procedures to treat non-responsive and large corneal defects include the application of amniotic membrane as a biologically active dressing that releases anti-inflammatory and anti-fibrotic factors to enhance re-epithelialization and wound healing during acute injury. [12~14] or, in cases where visually significant central corneal scarring is established, excision of the scar tissue and replacement with a donor cornea. The reproducibility and repeatability of clinical outcomes for amniotic membrane transplantation and corneal transplantation are associated with the risk of failure and rejection. [15~18] .

[0006] If the fibrotic response to injury and infection could be attenuated, optical clarity could be maximized, visual function preserved, and the need for surgery and transplantation could be eliminated. Such an innovation would have the potential to prevent permanent vision loss in millions of subjects. As discussed above, fibrosis is caused by elevated levels of TGFβ-1 activity, and therefore, it may be possible to prevent fibrosis using TGFβ antagonists. Decorin is a naturally occurring, leucine-rich, small, anti-fibrotic proteoglycan that is naturally found at high levels bound to collagen in the corneal stroma. 22 , when released, tightly regulate TGFβ activity by binding to growth factors and sequestering them in the ECM

[19] Decorin regulates numerous growth factors, including TGFβ {Zηανγ, 2007 #28}. [20~24] and directly inhibiting collagen fibrillogenesis. [25~28] Human recombinant (hr) decorin is now available in GMP form and has been shown to function in minimizing fibrosis in the brain and spinal cord. [29~31]To date, the effectiveness of soluble decorin applied to the ocular surface for in vivo treatment has not been reported. One possible reason for this is the relatively rapid clearance (in the range of minutes) from the corneal surface due to the relatively low viscosity of the eye drops. [32、33] ), which means that any effectiveness of decorin would be limited. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Oliva MS, Schottman T, Gulati M. Turning the tide of corneal blindness. Indian journal of ophthalmology.2012; 60(5): 423 pages [Non-patent document 2] Konda N, Motukupally SR, Garg P, Sharma S, All MH, Willcox MD, Microbial Analyses of Contact Lens-Associated Microbial Keratitis. Optometry and Vision Science. 2014; 91(1): 47-53 [Non-patent document 3] Stapleton F, Dart J, Seal D, Matheson M. Epidemiology of Pseudomonas aeruginosa keratitis in contact lens wearers. Epidemiology & Infection.1995;114(3):395-402 [Non-patent document 4] Wu YT-Y, Willcox M, Zhu H, Stapleton F. Contact lens hygiene compliance and lens case contamination: A review. Contact Lens and Anterior Eye. 2015; Volume 38 (Issue 5): Pages 307-16 [Non-Patent Document 5] O'brien T. Management of bacterial keratitis: beyond exorcism towards consideration of organism and host factors. Eye. 2003; vol. 17 (issue 8): p. 957 [Non-patent document 6] Willcox MD. Pseudomonas aeruginosa infection and inflammation during contact lens wear: a review. Optometry and Vision Science. 2007; 84(4): 273-278. [Non-Patent Document 7] Tandon A, Tovey JC, Sharma A, Gupta R, Mohan RR. Role of transforming growth factor Beta in corneal function, biology and Pathology. Current molecular medicine.2010; Vol. 10 (Issue 6): pp. 565-78 [Non-patent document 8] Allan BD, Dart JK. Strategies for the management of microbial keratitis. The British Journal of Ophthalmology. 1995;79(8):777-86 [Non-Patent Document 9] Gokhale NS. Medical management approach to infectious keratitis. Indian Journal of Ophthalmology. 2008; Volume 56 (No. 3): Pages 215~20

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[0008] In a first aspect of the present invention, (i) 0.1 to 5.0 wt % (e.g., 0.1 to 3.5 wt %, 0.1 to 2.5 wt %) of a microgel particle-forming polymer; and (ii) 0.5 to 100 mM of a monovalent and / or polyvalent metal ion salt as a cross-linking agent; 1. A shear thinning hydrogel composition comprising: Dispersed in an aqueous vehicle; Shear-thinning hydrogel compositions are provided that have a pH in the range of 3 to 8 and whose viscosity decreases when the hydrogel is exposed to shear.

[0009] In a further aspect of the present invention, the shear-thinning hydrogel composition is an ophthalmic hydrogel composition suitable for application to the eye. In a further aspect of the present invention, there is provided an ophthalmic hydrogel composition suitable for application to the eye, the ophthalmic hydrogel composition comprising, consisting essentially of, or consisting of a shear-thinning hydrogel composition as defined herein.

[0010] In another aspect, the shear-thinning hydrogel composition is a topical hydrogel composition suitable for application to a bodily surface. In a further aspect of the present invention, there is provided a topical hydrogel composition suitable for application to a bodily surface, the topical hydrogel composition comprising, consisting essentially of, or consisting of a shear-thinning hydrogel composition as defined herein.

[0011] In a further aspect, the present invention provides a method of making a shear-thinning hydrogel composition as defined herein, comprising the steps of: a) dissolving a microgel-forming polymer in an aqueous vehicle to form a polymer solution; b) mixing the microgel-forming polymer solution formed in step (a) with an aqueous solution of a monovalent or polyvalent metal ion salt at a temperature above the gelation temperature of the microgel particle-forming polymer; and c) cooling the mixture resulting from step b) to a temperature below the gelling temperature of the microgel particle-forming polymer. The present invention provides a method comprising:

[0012] A further aspect of the present invention is a method of making a shear-thinning hydrogel composition as defined herein, comprising the steps of: a) dissolving a microgel-forming polymer in an aqueous vehicle containing 0.5 to 100 mM of a monovalent and / or polyvalent metal ion salt as a crosslinker to form a polymer solution containing 0.1 to 5.0 wt % (e.g., 0.1 to 3.5 wt % or 0.1 to 2.5 wt %) of the microgel particle-forming polymer; b) mixing the microgel-forming polymer solution formed in step (a) at a temperature above the gelation temperature of the microgel particle-forming polymer; and c) cooling the mixture resulting from step b) under shear mixing to a temperature below the gelling temperature of the microgel particle-forming polymer. The present invention provides a method comprising:

[0013] A further aspect of the present invention provides a shear-thinning hydrogel composition obtainable by, obtained by or directly obtained by any of the preparative methods defined herein.

[0014] A further aspect of the present invention provides a shear-thinning hydrogel composition as defined herein for use in therapy.

[0015] A further aspect of the present invention provides a shear-thinning hydrogel composition as defined herein for ophthalmic or topical administration.

[0016] A further aspect of the present invention provides a shear-thinning hydrogel composition as defined herein for the inhibition of scarring.

[0017] A further aspect of the present invention provides a shear-thinning hydrogel composition as defined herein for use in the treatment of bacterial keratitis.

[0018] A further aspect of the present invention provides a shear-thinning hydrogel composition as defined herein for administration to a skin wound.

[0019] A further aspect of the present invention provides a shear-thinning hydrogel composition as defined herein for use in the treatment of glaucoma by administration to the eye.

[0020] In a further aspect, the present invention provides a composition according to the present invention for use as a medicament. Examples of suitable medical uses of the compositions of the present invention are further described below. Preferably, the compositions of the present invention can be used as a topical medicament.

[0021] In a further aspect, the present invention provides a composition according to the present invention for use in inhibiting scarring.

[0022] In one preferred embodiment of the invention, the composition according to the invention is for use in inhibiting scarring in the eye.

[0023] Embodiments of the present invention will be further described hereinafter with reference to the accompanying drawings. [Brief explanation of the drawings]

[0024] [Figure 1A]1A-1C are schematic diagrams of the processing and inherent material properties of gellan-based fluid hydrogel eye drops. (a) Schematic diagram of the production of a fluid gel, in which an initial sol is continuously processed under shear while being cooled to form a "ribbon-like" gelled entity, as shown using (i) transmission microscopy and (ii) scanning electron microscopy. [Figure 1B] 1A-1C are illustrations of the processing and intrinsic material properties of gellan-based fluid hydrogel eye drops.FIGS. 1A-1C are illustrations of the time-dependent viscosity profiles obtained for gellan eye drops, highlighting a degree of thixotropy.FIGS. 1B-1C are illustrations of the time-dependent viscosity profiles obtained for gellan eye drops, highlighting a degree of thixotropy.FIGS. [Figure 1C] Illustrated processing and intrinsic material properties of gellan-based fluid hydrogel eye drops. The fluid gel is being dispensed from the eye dropper packaging (the gel is dyed blue to be visible in the photograph). [Figure 1D]

[0023] Figure 1 illustrates the processing and intrinsic material properties of gellan-based fluid hydrogel eye drops. Small deformation rheological data obtained at a single frequency (1 Hz, 0.5% strain) as a function of time. The data show the development of an elastic network after shear, resulting in a transition from liquid to solid-like behavior. [Figure 1E]

[0023] Figure 1 illustrates the processing and inherent material properties of gellan-based fluid hydrogel eye drops. Figure 2 illustrates anterior segment OCT images showing the ocular surface before (top image) and after (bottom image) fluid gel application. The images show a uniform layer covering the entire ocular surface. [Figure 2]

[0023] Figures 1A and 1B are in vitro assays demonstrating the bioactivity of formulated eye drops. (a) Cumulative release curves for hr decorin-loaded eye drops over 4 hours (240 minutes). The fitted line follows the power function, y=0.7x0.7 (R2=0.99). (b) Collagen fibrillogenesis turbidity data for PBS control, collagen only, and collagen + hr decorin. (c) Collagen fibrillogenesis turbidity data using dose-response curves for collagen, collagen + hr decorin, collagen + fluid gel (FG) only, and collagen + hr decorin-loaded fluid gel (DecFG). [Figure 3]Measurement of corneal opacity area. (a) Representative photographs taken on days 2, 3, 9, 12, and 16 after Psaudomonas infection and treatment, and (b) graph showing the mean area of ​​opacity ± SEM (mm2) measured by two independent blinded ophthalmologists from photographs taken from each individual mouse per group (shown in panel a) (n=6; **p<0.01, ***p<0.001). [Figure 4A] (a) Representative images of DAPI+ cell nuclei (blue) in the cornea used to approximate the epithelium illustrating epithelial thickness and stratification (number of cell layers) in an untreated, intact eye showing normal, non-keratinized, stratified (approximately 5 layers) epithelium; an eye taken 2 days after infection with a thickened, edematous stroma with cellular infiltration; and an eye taken 16 days after treatment showing re-epithelialization with 2-3 layers of stratification with decreased stromal edema in group 1 (gentamicin and prednisolone), increased stratification in group 2 (GPFG), and fully mature epithelium in group 3 (GPDecFG) (Scale bar 100 μm). [Figure 4B] Corneal re-epithelialization. Quantification of corneal thickness ± SEM in the untreated intact group (n=6), eyes from each treatment group evaluated on days 2 and 16 (n=6 for each group). All quantifications were performed on blinded images blinded to the observer. [Figure 4C] Corneal re-epithelialization. Quantification of epithelial layer thickness ± SEM in the untreated intact group (n=6), eyes from each treatment group evaluated on days 2 and 16 (n=6 for each group). All quantifications were performed on blinded images blinded to the observer. [Figure 4D] Corneal re-epithelialization. Quantification of cellular epithelial stratification layers ± SEM in the untreated intact group (n=6), eyes from each treatment group evaluated on days 2 and 16 (n=6 for each group). All quantifications were performed on blinded images blinded to the observer. [Figure 5] Figure 1. Illustration of extracellular matrix levels in the cornea. Representative images of immunohistochemical staining using DAPI+ to stain cell nuclei (blue) in each case, with plots quantifying IR for (a) αSMA+ (green to stain myofibroblasts), (b) IR fibronectin+ (green to stain fibronectin in the ECM), and (c) laminin+ (red to stain laminin in the ECM). Analysis was attempted on intact eyes, eyes taken 2 days after infection, and eyes obtained 16 days later, using various eye drop treatments: i) gentamicin and prednisolone (GP), ii) gentamicin, prednisolone, and fluid gel (GPFG), and iii) gentamicin, prednisolone, and hrDecorin fluid gel (GPDecFG). All studies were performed using treatment groups of n = 6, and quantification was performed on blinded images (scale bar = 100 μm). [Figure 6] 1 is a diagram of the in vivo experimental design. FIG. 1 is a diagram of the experimental design for the in vivo Pseudomonas keratitis study comparing fluid gel eye drops with or without hr decorin with gentamicin and prednisolone eye drops alone. [Figure 7] 1A and 1B are graphs of the storage modulus (G'), representing the elastic structure in gellan microgel suspensions, as a function of initial gellan polymer concentration, determined using an amplitude sweep. (a) A strain sweep obtained at 1 Hz (20°C) for various polymer concentrations prepared at a processing speed of 500 rpm. (b) A strain sweep obtained at 1 Hz (20°C) for various polymer concentrations at a processing speed of 1000 rpm. [Figure 8] 8 is a comparison of storage modulus as a function of polymer concentration and processing rate. G′ obtained in the linear viscoelastic region (LVR) of the amplitude sweep shown in FIG. [Figure 9]FIG. 10 is a comparison of storage modulus for commercially available eye drops / ointments for the treatment of dry eye. Data obtained from amplitude sweeps performed using the same method as described for gellan suspensions. Again, values ​​were obtained in LVR. The dotted line represents G' for the optimized gellan formulation. [Figure 10] Figure 1 shows flow rate profiles illustrating the ease of application of gellan microgel suspensions as a function of initial gellan polymer concentration. (a) Viscosity sweeps obtained between 0.1 and 600 s at 20 °C for various polymer concentrations prepared at a processing speed of 500 rpm. (b) Viscosity sweeps obtained between 0.1 and 600 s at 20 °C for various polymer concentrations prepared at a processing speed of 1000 rpm. [Figure 11] 10 is a comparison of the viscosity of microgel suspensions at 1 s as a function of polymer concentration and processing speed. The instantaneous viscosity was obtained by measuring the value at 1 s using the sweep shown in FIG. [Figure 12]

[0023] Figure 1 shows a comparison of the viscosity at 1 s of commercially available eye drops / ointments for the treatment of dry eye. Data obtained from flow profiles attempted using the same method as described for gellan suspensions. The dotted line represents the viscosity of the optimized gellan formulation. [Figure 13] Figure 1 shows the storage modulus (G'), representing the elastic structure in gellan microgel suspensions, as a function of added cross-linker, determined using amplitude sweeps: (a) strain sweeps obtained at 1 Hz (20°C) for various cross-linker concentrations for a 0.9% (w / v) system, and (b) strain sweeps obtained at 1 Hz (20°C) for various cross-linker concentrations for a 1.8% (w / v) polymer concentration. [Figure 14] 8 is a comparison of storage modulus as a function of crosslinker and polymer concentration. G′ obtained in the linear viscoelastic region (LVR) of the amplitude sweep shown in FIG. [Figure 15]Flow rate profiles demonstrating the ease of application of gellan microgel suspensions as a function of crosslinker concentration. (Left) Viscosity sweeps obtained between 0.1 and 600 s at 20 °C for 0.9% (w / v) gellan systems prepared with various crosslinker concentrations. (Right) Viscosity sweeps obtained between 0.1 and 600 s at 20 °C for 1.8% (w / v) gellan systems prepared with various crosslinker concentrations. [Figure 16] Figure 3 shows a comparison of the viscosity of microgel suspensions at 1 s as a function of polymer and crosslinker concentration. The instantaneous viscosity was obtained by measuring the value at 1 s using the sweep shown in Figure 3. [Figure 17] Graph of the storage modulus (G'), representing the elastic structure in gellan microgel suspensions, as a function of the cooling rate applied during processing, determined using amplitude sweeps. (a) Graph of strain sweeps obtained at 1 Hz (20°C) for various cooling rates for a 0.9% (w / v) system prepared at a processing speed of 1000 rpm. (b) Graph of strain sweeps obtained at 1 Hz (20°C) for various cooling rates for a 1.8% (w / v) polymer concentration prepared at a processing speed of 1000 rpm. [Figure 18] 8 is a comparison of storage modulus as a function of cooling rate and polymer concentration. G′ obtained in the linear viscoelastic region (LVR) of the amplitude sweep shown in FIG. [Figure 19] Figure 1 shows flow rate profiles illustrating the ease of application of gellan microgel suspensions as a function of the cooling rate applied during processing. (a) Viscosity sweeps obtained at 20 °C between 0.1 and 600 s for a 0.9% (w / v) gellan system prepared at various cooling rates. (b) Viscosity sweeps obtained at 20 °C between 0.1 and 600 s for a 1.8% (w / v) gellan system prepared at various cooling rates. [Figure 20] 9 is a comparison of the viscosity of microgel suspensions at 1 s as a function of polymer concentration and cooling rate applied during processing. The instantaneous viscosity was obtained by measuring the value at 1 s using the sweep shown in FIG. [Figure 21] Graph of storage modulus (G'), representing the elastic structure in gellan microgel suspensions, as a function of applied mechanical shear during processing, determined using amplitude sweeps. (a) Strain sweeps obtained at 1 Hz (20°C) for various processing rates for a 0.9% (w / v) system; (b) Strain sweeps obtained at 1 Hz (20°C) for various processing rates for a 1.8% (w / v) polymer concentration. [Figure 22] 8 is a comparison of storage modulus as a function of processing speed and polymer concentration. G′ obtained in the linear viscoelastic region (LVR) of the amplitude sweep shown in FIG. [Figure 23] Figure 1 shows flow rate profiles illustrating the ease of application of gellan microgel suspensions as a function of the mechanical shear applied during processing. (a) Viscosity sweeps obtained between 0.1 and 600 s at 20 °C for 0.9% (w / v) gellan systems prepared at various processing rates. (b) Viscosity sweeps obtained between 0.1 and 600 s at 20 °C for 1.8% (w / v) gellan systems prepared at various processing rates. [Figure 24] 9 is a comparison of the viscosity of microgel suspensions at 1 s as a function of polymer concentration and processing speed during gelation. The instantaneous viscosity was obtained by measuring the value at 1 s using the sweep shown in FIG. [Figure 25]

[0023] Figure 1 shows that shear-thinning hydrogel compositions according to the present invention reduce the expression of scarring-associated markers in cultured fibroblasts. Administration of TGF-β to cultured human dermal fibroblasts increases the expression of α-smooth muscle actin, a marker of myofibroblasts associated with scarring. The graph shows the effect of treatment with experimental hydrogel compositions on this expression. Hydrogel compositions with or without the anti-fibrotic agent decorin were able to reduce the expression of α-smooth muscle actin, demonstrating their ability to inhibit scarring. [Figure 26] FIG. 1 shows amplitude sweep data obtained for agar, gellan, kappa-carrageenan and alginate. [Figure 27]FIG. 1 shows frequency sweep data obtained for agar, gellan, kappa-carrageenan and alginate. [Figure 28] FIG. 1 shows viscosity sweep data obtained for agar, gellan, kappa-carrageenan and alginate. [Figure 29] FIG. 1 illustrates standard curves obtained for shear-thinning hydrogel compositions according to the present invention incorporating the following active agents: penicillin-streptomycin; dexamethasone; proteinase K; ibuprofen; dextran; and blue dextran. [Figure 30] FIG. 1 illustrates curves obtained for shear-thinning hydrogel compositions according to the present invention incorporating the following active agents: penicillin-streptomycin; dexamethasone; proteinase K; ibuprofen; dextran; and blue dextran. [Figure 31] Figure 1 shows photographs illustrating the results of a zone of inhibition assay using shear-thinning hydrogel compositions according to the present invention comprising the polymers alginate or gellan in combination with an anti-infective (penicillin-streptomycin). These results demonstrate efficacy against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). A summary of the results is also provided in the accompanying table. The figure also includes a graph illustrating the results of a zone of inhibition assay using shear-thinning hydrogel compositions according to the present invention comprising alginate in combination with an alternative anti-infective (vancomycin). The antibacterial efficacy of vancomycin was tested against MRSA. [Figure 32] FIG. 1 shows photographs depicting the breakdown over time of an exemplary ECM molecule, fibrin (shown as a white gel in the photographs), under the action of the active agent, proteinase K, released from an alginate or gellan shear-thinning hydrogel composition according to the present invention. [Figure 33]1 illustrates the results of this study and is a graph comparing the absorbance at 405 nm (y-axis) for increasing concentrations of gellan fluid gel shear-thinning hydrogel compositions of the present invention incubated with collagen alone ("Collagen Only"), or decorin alone ("hrDecolin"), or with ("DecFG") or without ("FG") human recombinant decorin (Galacorin™). [Figure 34] FIG. 1 depicts a mouse model used to test the effects of compositions of the present invention on experimental bacterial keratitis. [Figure 35] Graphs showing the area of ​​opacity associated with different treatments at different time points, the percentage of α-smooth muscle actin pixels above the threshold for the various control and treatment groups investigated, the percentage of fibronectin pixels above the threshold for the various control and treatment groups investigated, and the percentage of laminin pixels above the threshold for the various control and treatment groups investigated. [Figure 36] 1 shows the results of a study of intraocular pressure in hypertensive rats, with treated rats shown as dashed lines and untreated controls shown as solid black lines. The results were analyzed using a two-way analysis of variance with Sidak's multiple comparison test, and showed that the composition of the present invention (p<0.05) significantly reduced intraocular pressure in intraocular hypertensive rats by D28 compared to controls. DETAILED DESCRIPTION OF THE INVENTION

[0025] definition The term "hydrogel" is used herein to refer to a gel formed from hydrophilic polymers dispersed in an aqueous vehicle.

[0026] The term "aqueous vehicle" is used herein to refer to water or a water-based fluid (eg, a buffer solution such as phosphate buffered saline, or a physiological fluid such as serum).

[0027] The term "microgel" is used herein to refer to microscopic particles of gel formed from a network of microscopic filaments of polymer.

[0028] The term "shear-thinning" is used herein to define the hydrogel compositions of the present invention. This terminology is well understood in the art and refers to a hydrogel composition that has a viscosity that decreases when shear force is applied to the hydrogel. Shear-thinning hydrogel compositions of the present invention have a "resting" viscosity (when no shear force is applied) and a lower viscosity when shear force is applied. This property of the hydrogel composition allows the hydrogel to flow and be administered to the body when shear force is applied (e.g., by applying force to a tube or dispenser containing the hydrogel composition of the present invention). When applied under shear, the viscosity of the hydrogel composition increases when the applied shear force is removed. Typically, the hydrogel compositions of the present invention will have a viscosity below 1 Pa.s when subjected to shear force for administration of the hydrogel composition. A viscosity below 1 Pa.s will allow the hydrogel composition to flow. As a result, the static viscosity will typically be greater than 1 Pa.s, such as greater than 2 Pa.s, greater than 3 Pa.s, or greater than 4 Pa.s.

[0029] References to "treating" or "treatment" should be understood to include prevention as well as alleviation of established symptoms of a condition. "Treating" or "treatment" of a condition, disorder, or condition therefore includes: (1) preventing or delaying the onset of clinical symptoms of the condition, disorder, or condition that develops in a human suffering from or susceptible to the condition, disorder, or condition but who is not experiencing or exhibiting clinical or subclinical symptoms of the condition, disorder, or condition; (2) arresting the condition, disorder, or condition, i.e., arresting, reducing, or delaying the onset of the disease or its recurrence (in the case of maintenance treatment) or at least one clinical or subclinical symptom thereof; or (3) palliating or attenuating the disease, i.e., causing amelioration of the condition, disorder, or condition or at least one clinical or subclinical symptom thereof.

[0030] A "therapeutically effective amount" means the amount of a compound that, when administered to a mammal for treating a disease, is sufficient for such treatment to affect the disease. The "therapeutically effective amount" will vary depending on the compound, the disease and its severity, and the age, weight, etc., of the mammal being treated.

[0031] Throughout the description and claims of this specification, the words "comprises" and "containing" and variations thereof mean "including but not limited to" and are not intended to (and do not) exclude other additions, components, numbers, or steps. Throughout the description and claims of this specification, the singular includes the plural unless the context requires otherwise. In particular, where the indefinite article is used, the specification should be understood as contemplating both the plural and the singular unless the context requires otherwise.

[0032] The reader's attention is directed to all papers and documents filed contemporaneously with or prior to this application in connection with this application and publicly accessible herewith, the contents of all such papers and documents being incorporated herein by reference.

[0033] Hydrogel Compositions of the Present Invention In a further aspect of the present invention, (i) 0.1 to 5.0 wt % (e.g., 0.1 to 3.5 wt % or 0.1 to 2.5 wt %) of a microgel particle-forming polymer; and (ii) 0.5 to 100 mM of a monovalent and / or polyvalent metal ion salt as a cross-linking agent; 1. A shear thinning hydrogel composition comprising: Dispersed in an aqueous vehicle; Shear-thinning hydrogel compositions are provided that have a pH in the range of 3 to 8 and whose viscosity decreases when the hydrogel is exposed to shear.

[0034] The hydrogel compositions of the present invention are shear thinning, meaning that the viscosity of the composition decreases when the hydrogel is exposed to shear. This property allows the hydrogel to decrease in viscosity and flow when shear force is applied, thereby allowing the hydrogel to be dispensed and administered, for example, from an eye dropper to a tube, by applying shear force (e.g., by squeezing the end of the eye dropper or tube). Once administered, as the shear force applied to the hydrogel subsides, the viscosity of the hydrogel increases, forming a thicker gel that can remain at the administration site for an extended period of time.

[0035] Typically, the hydrogel compositions of the present invention will have a viscosity of less than 1 Pa s when subjected to shear forces for administration of the hydrogel composition. At a viscosity of less than 1 Pa s, the hydrogel composition will be able to flow. As a result, the resting viscosity will typically be greater than 1 Pa s, e.g., greater than 2 Pa s, greater than 3 Pa s, or greater than 4 Pa ​​s.

[0036] In one embodiment, the shear-thinning hydrogel composition of the present invention does not include collagen and / or fibrin.

[0037] The microgel particle-forming polymer can be any polymer capable of forming microgel particles in an aqueous vehicle. The microgel particles formed by the microgel particle-forming polymer can have any suitable morphology (e.g., they can be linear filaments or regularly or irregularly shaped particles) and / or particle size. The formation of microgel particles, as opposed to a macrogel structure, promotes the desired shear-thinning characteristic. Without wishing to be bound by any particular theory, it is hypothesized that in the absence of shear or at low levels of shear, the microgel particles bond together, substantially preventing bulk flow of the hydrogel. However, upon application of shear force, the interactions between adjacent microgel particles weaken, reducing viscosity and allowing the hydrogel composition to flow. When the applied shear force is removed, the interactions between adjacent microgel particles then reform, such that viscosity again increases and the ability to flow easily is hindered.

[0038] Preferably, the hydrogel composition comprises 0.5 to 5.0 wt% of the microgel particle-forming polymer. In one embodiment, the hydrogel composition comprises 0.5 to 3.5 wt% of the microgel particle-forming polymer. In one embodiment, the hydrogel composition comprises 0.5 to 2.5 wt% of the microgel particle-forming polymer. In one embodiment, the hydrogel composition comprises 0.8 to 1.8 wt% of the microgel particle-forming polymer. In a further embodiment, the hydrogel composition comprises 0.8 to 1.0 wt% (e.g., 0.9 wt%) of the microgel particle-forming polymer.

[0039] Preferably, the microgel particle-forming polymer is a microgel particle-forming polymer of one or more polysaccharides. In one embodiment, the microgel particle-forming polymer is selected from one or more of the following group: gellan, alginate, carrageenan (e.g., iota-carrageenan, kappa-carrageenan), agar, agarose, or chitosan. In a particular embodiment, the microgel particle-forming polymer is selected from one or more of the following group: agar, gellan, alginate, or carrageenan. In a particular embodiment, the microgel particle-forming polymer is selected from one or more of the following group: gellan, alginate, or carrageenan. In a more particular embodiment, the microgel particle-forming polymer is selected from gellan or alginate. In yet another embodiment, the microgel particle-forming polymer is gellan. In yet another embodiment, the microgel particle-forming polymer is alginate.

[0040] In an alternative embodiment, the microgel particle-forming polymer is gelatin.

[0041] Preferably, the hydrogel composition is transparent or translucent, hi one particular embodiment, the hydrogel composition is transparent.

[0042] In one embodiment, the hydrogel composition is transparent or translucent, and the microgel particle-forming polymer is selected from gellan, alginate, and / or carrageenan. In a further embodiment, the hydrogel composition is transparent, and the microgel particle-forming polymer is selected from gellan, alginate, and / or carrageenan. In a particular embodiment, the hydrogel composition is transparent, and the microgel particle-forming polymer is gellan or alginate. In a further embodiment, the hydrogel composition is transparent, and the microgel particle-forming polymer is gellan.

[0043] Gellan (also called gellan gum) is a water-soluble, anionic polysaccharide produced by the bacterium Sphingomonas elodea. It is commercially available in a low-acyl form under the trade name Kelco gel CG LA (Azelis, UK).

[0044] The hydrogel composition contains 5 to 100 mM of a monovalent and / or polyvalent metal ion salt as a crosslinker. The metal ion salt can be added as a component to the composition, but can also be present in other components of the composition, such as a buffer (e.g., phosphate-buffered saline) or any physiological fluid present in the composition, such as serum.

[0045] Preferably, the hydrogel composition comprises 5-40 mM of a monovalent and / or polyvalent metal ion salt as a crosslinker. In one embodiment, the hydrogel composition comprises 5-30 mM of a monovalent and / or polyvalent metal ion salt as a crosslinker. In another embodiment, the hydrogel composition comprises 5-20 mM of a monovalent and / or polyvalent metal ion salt as a crosslinker. In yet another embodiment, the hydrogel composition comprises 5-15 mM of a monovalent and / or polyvalent metal ion salt as a crosslinker. In yet another embodiment, the hydrogel composition comprises 8-12 mM (e.g., 10 mM) of a monovalent and / or polyvalent metal ion salt as a crosslinker.

[0046] In one particular embodiment of the invention, the microgel particle-forming polymer is gellan and the composition comprises 0.5-40 mM, 5-15 mM, 8-12 mM or 10 mM of a monovalent metal ion salt (e.g., NaCl) as a cross-linking agent.

[0047] In a further embodiment of the invention, the microgel particle-forming polymer is alginate and the composition comprises 0.5 to 40 mM, 5 to 15 mM, 8 to 12 mM or 10 mM of a polyvalent metal ion salt (e.g., Ca 2+ salt) as a cross-linking agent.

[0048] Preferably, the hydrogel composition has a pH in the range of 6 to 8. In one embodiment, the hydrogel composition has a pH in the range of 6.5 to 8. In a further embodiment, the hydrogel composition has a pH in the range of 7 to 7.5 (e.g., pH 7.4).

[0049] Preferably, the hydrogel compositions of the present invention have a rest viscosity (i.e., viscosity at zero shear) of 1 Pa.s or greater (e.g., 1 Pa.s to 200 Pa.s or 1 Pa.s to 100 Pa.s). More preferably, the rest viscosity will be 2 Pa.s or greater (e.g., 2 Pa.s to 200 Pa.s or 2 Pa.s to 100 Pa.s), 3 Pa.s or greater (e.g., 3 Pa.s to 200 Pa.s or 3 Pa.s to 100 Pa.s), 4 Pa.s or greater (e.g., 4 Pa.s to 200 Pa.s or 4 Pa.s to 100 Pa.s), or 5 Pa.s or greater (e.g., 5 Pa.s to 200 Pa.s or 5 Pa.s to 100 Pa.s).

[0050] The viscosity decreases when the hydrogel composition is subjected to shear force. Preferably, the viscosity decreases to a value below the resting viscosity at which the gel flows and can be administered. Typically, the viscosity will decrease to a value less than 1 Pa.s when shear force is applied.

[0051] In one embodiment, the hydrogel composition has a resting viscosity of 1 Pa.s or greater (eg, 1 Pa.s to 200 Pa.s or 1 Pa.s to 100 Pa.s) and when subjected to shear forces, the viscosity decreases to less than 1 Pa.s.

[0052] In another embodiment, the hydrogel composition has a resting viscosity of 2 Pa.s or greater (e.g., 2 Pa.s to 200 Pa.s or 2 Pa.s to 100 Pa.s), and when subjected to shear forces, the viscosity decreases to less than 2 Pa.s (e.g., less than 1 Pa.s).

[0053] In another embodiment, the hydrogel composition has a resting viscosity of 3 Pa.s or greater (e.g., 3 Pa.s to 200 Pa.s or 3 Pa.s to 100 Pa.s), and when subjected to shear forces, the viscosity decreases to less than 3 Pa.s (e.g., less than 1 Pa.s).

[0054] In another embodiment, the hydrogel composition has a resting viscosity of 4 Pa.s or greater (e.g., 4 Pa.s to 200 Pa.s or 4 Pa.s to 100 Pa.s), and when subjected to shear forces, the viscosity decreases to less than 4 Pa.s (e.g., less than 1 Pa.s).

[0055] In another embodiment, the hydrogel composition has a resting viscosity of 5 Pa.s or greater (e.g., 5 Pa.s to 200 Pa.s or 5 Pa.s to 100 Pa.s), and when subjected to shear forces, the viscosity decreases to less than 5 Pa.s (e.g., less than 1 Pa.s).

[0056] For the avoidance of doubt, all viscosity values ​​given herein are given at normal ambient temperature of 20°C. The viscosity of the hydrogel compositions of the present invention can be determined using standard techniques known in the art. For example, viscosity profiles can be obtained at 20°C using an AR-G2 (TA Instruments, UK) rheometer equipped with sandblasted parallel plates (40 mm, 1 mm gap height).

[0057] Preferably, the hydrogel has a modulus of elasticity of 5 Pa to 40 Pa at zero shear.

[0058] The elastic modulus of the hydrogels of the present invention can be determined by techniques known in the art.

[0059] Specific Embodiments In one particular embodiment of the present invention, the shear-thinning hydrogel composition comprises: (I) 0.1–5.0 wt % of a microgel particle-forming polymer (e.g., gellan); 0.5 to 40 mM of a monovalent metal ion salt (e.g., NaCl) or a polyvalent metal ion salt (e.g., Ca) as a crosslinker 2+ ) and having a pH of 3.5 to 8; (II) 0.1–5.0 wt % of a microgel particle-forming polymer (e.g., gellan); 0.5 to 40 mM of a monovalent metal ion salt (e.g., NaCl) or a polyvalent metal ion salt (e.g., Ca) as a crosslinker 2+ ) and having a pH of 6 to 8; (III) 0.1 to 5.0 wt % of a microgel particle-forming polymer (e.g., gellan); 0.5 to 40 mM of a monovalent metal ion salt (e.g., NaCl) or a polyvalent metal ion salt (e.g., Ca) as a crosslinker 2+ ) and having a pH of 6.5 to 7.5; (IV) 0.1–3.5 wt % of a microgel particle-forming polymer (e.g., gellan); 0.5 to 40 mM of a monovalent metal ion salt (e.g., NaCl) or a polyvalent metal ion salt (e.g., Ca) as a crosslinker 2+ ) and having a pH of 3.5 to 8; (V) 0.1–3.5 wt % of a microgel particle-forming polymer (e.g., gellan); 0.5 to 40 mM of a monovalent metal ion salt (e.g., NaCl) or a polyvalent metal ion salt (e.g., Ca) as a crosslinker 2+ ) and having a pH of 6 to 8; (VI) 0.1–3.5 wt % of a microgel particle-forming polymer (e.g., gellan); 0.5 to 40 mM of a monovalent metal ion salt (e.g., NaCl) or a polyvalent metal ion salt (e.g., Ca) as a crosslinker 2+ ) and having a pH of 6.5 to 7.5; (1) 0.1–2.5 wt % of a microgel particle-forming polymer (e.g., gellan); 0.5 to 40 mM of a monovalent metal ion salt (e.g., NaCl) or a polyvalent metal ion salt (e.g., Ca) as a crosslinker 2+ ) and having a pH of 3.5 to 8; (2) 0.1–2.5 wt % of a microgel particle-forming polymer (e.g., gellan); 0.5 to 40 mM of a monovalent metal ion salt (e.g., NaCl) or a polyvalent metal ion salt (e.g., Ca) as a crosslinker2+ ) and having a pH of 6 to 8; (3) 0.1–2.5 wt % microgel particle-forming polymer (e.g., gellan); 0.5 to 40 mM of a monovalent metal ion salt (e.g., NaCl) or a polyvalent metal ion salt (e.g., Ca) as a crosslinker 2+ ) and having a pH of 6.5 to 7.5; (4) 0.5–2.0 wt % microgel particle-forming polymer (e.g., gellan); 0.5 to 40 mM of a monovalent metal ion salt (e.g., NaCl) or a polyvalent metal ion salt (e.g., Ca) as a crosslinker 2+ ) and having a pH of 3.5 to 8; (5) 0.8–1.8 wt % microgel particle-forming polymer (e.g., gellan); 0.5 to 40 mM of a monovalent metal ion salt (e.g., NaCl) or a polyvalent metal ion salt (e.g., Ca) as a crosslinker 2+ ) and having a pH of 6 to 8; (6) 0.8–1.0 wt % microgel particle-forming polymer (e.g., gellan); 0.5 to 40 mM of a monovalent metal ion salt (e.g., NaCl) or a polyvalent metal ion salt (e.g., Ca) as a crosslinker 2+ ) and having a pH of 6.5 to 7.5; (7) 0.5–2.5 wt % microgel particle-forming polymer (e.g., gellan); 0.5 to 40 mM of a monovalent metal ion salt (e.g., NaCl) or a polyvalent metal ion salt (e.g., Ca) as a crosslinker 2+ ) and having a pH of 3.5 to 8; (8) 0.5–2.5 wt % microgel particle-forming polymer (e.g., gellan); 5 to 20 mM monovalent metal ion salt (e.g., NaCl) or polyvalent metal ion salt (e.g., Ca) as a crosslinker 2+ ) and having a pH of 6 to 8; (9) 0.5–2.5 wt % microgel particle-forming polymer (e.g., gellan); 5 to 15 mM monovalent metal ion salt (e.g., NaCl) or polyvalent metal ion salt (e.g., Ca) as a crosslinker 2+ ) and having a pH of 6 to 8; (10) 0.5–2.5 wt % microgel particle-forming polymer (e.g., gellan); 8 to 12 mM (e.g., 10 mM) of a monovalent metal ion salt (e.g., NaCl) or a polyvalent metal ion salt (e.g., Ca) as a crosslinker 2+ ) and having a pH of 6 to 8; (11) 0.5–2.5 wt % microgel particle-forming polymer (e.g., gellan); 0.5 to 40 mM of a monovalent metal ion salt (e.g., NaCl) or a polyvalent metal ion salt (e.g., Ca) as a crosslinker 2+ ) and having a pH of 6 to 8; (12) 0.8–1.8 wt % microgel particle-forming polymer (e.g., gellan); 5 to 20 mM monovalent metal ion salt (e.g., NaCl) or polyvalent metal ion salt (e.g., Ca) as a crosslinker 2+ ) and having a pH of 6 to 8; (13) 0.8–1.0 wt % microgel particle-forming polymer (e.g., gellan); 5 to 15 mM monovalent metal ion salt (e.g., NaCl) or polyvalent metal ion salt (e.g., Ca) as a crosslinker 2+ ) and having a pH of 6 to 8; (14) 0.8–1.0 wt % microgel particle-forming polymer (e.g., gellan); 8 to 12 mM (e.g., 10 mM) of a monovalent metal ion salt (e.g., NaCl) or a polyvalent metal ion salt (e.g., Ca) as a crosslinker 2+ ) and has a pH of 6 to 8 Examples include:

[0060] therapeutic agent In certain embodiments of the present invention, the hydrogel composition may further comprise one or more pharmacologically active agents. Any suitable pharmacologically active agent may be present. For example, the hydrogel composition may comprise one or more pharmacologically active agents selected from the group consisting of antifibrotic agents; anti-infective agents; pain relief agents; anti-inflammatory agents; antiproliferative agents; keratolytic agents; extracellular matrix modifying agents; cell-cell junction modifying agents; basement membrane modifying agents; and pigmentation modifying agents. The antifibrotic agent may be decorin. In the context of the present invention, it should be understood that when decorin is incorporated into the hydrogel composition of the present invention, the decorin may be present as an active agent incorporated into the hydrogel, rather than as a component of the hydrogel itself.

[0061] The hydrogel composition can include any suitable amount of pharmacologically active agent, for example, the hydrogel composition can include 0.01 to 50 wt% of the pharmacologically active agent.

[0062] In one embodiment, the hydrogel composition comprises decorin, optionally in an amount of 0.1-1.0 mg / ml; 0.1-0.5 mg / ml; 0.1-0.4 mg / ml; or 0.2-0.3 mg / ml.

[0063] In a further embodiment, the hydrogel composition comprises any one of the hydrogel compositions defined in paragraphs (1) to (14) above, optionally with decorin in an amount of 0.1 to 1.0 mg / ml; 0.1 to 0.5 mg / ml; 0.1 to 0.4 mg / ml; or 0.2 to 0.3 mg / ml.

[0064] In one embodiment of a composition of the invention that includes an anti-infective, such as the antibiotic gentamicin, the anti-infective may be present in an amount of 1-5 mg / ml. For example, the anti-infective, such as gentamicin, may be present in an amount of 1-4 mg / ml, 1-3 mg / ml, or 1-2 mg / ml. The anti-infective, such as gentamicin, may be present in an amount of 2-4 mg / ml, or 2.5-3.5 mg / ml.

[0065] In one embodiment of a composition of the invention comprising an anti-inflammatory agent, such as the steroid prednisolone, the anti-inflammatory agent may be present in an amount of 0.5 to 250 mg / ml. Suitably, the anti-inflammatory agent, such as prednisolone, may be present in an amount of 1.25 to 170 mg / ml, for example, 1.25 to 50 mg / ml, or 1.25 to 10 mg / ml.

[0066] Ophthalmic composition In a further aspect, the present invention provides an ophthalmic hydrogel composition suitable for administration to the eye, wherein the ophthalmic hydrogel composition is a shear-thinning hydrogel composition as previously defined herein.

[0067] In a further aspect of the present invention, there is provided an ophthalmic hydrogel composition suitable for application to the eye, the ophthalmic hydrogel composition comprising, consisting essentially of, or consisting of a shear-thinning hydrogel composition as previously defined herein.

[0068] The ophthalmic hydrogel compositions of the present invention are compatible with ocular application.

[0069] Topical Compositions In a further aspect, the present invention provides a hydrogel composition suitable for topical administration, wherein the ophthalmic hydrogel composition is a shear-thinning hydrogel composition as previously defined herein.

[0070] In a further aspect of the present invention, there is provided a topical hydrogel composition suitable for topical application to the body, the topical hydrogel composition comprising, consisting essentially of, or consisting of a shear-thinning hydrogel composition as previously defined herein.

[0071] Methods for preparing the hydrogel compositions of the present invention The present invention provides a method of making a shear-thinning hydrogel composition as defined herein, comprising: a) dissolving a microgel particle-forming polymer in an aqueous vehicle to form a polymer solution; b) mixing the microgel particle-forming polymer solution formed in step (a) with an aqueous solution of a monovalent or polyvalent metal ion salt at a temperature above the gelation temperature of the microgel particle-forming polymer; and c) cooling the mixture resulting from step b) under shear mixing to a temperature below the gelling temperature of the microgel particle-forming polymer. The present invention provides a method comprising:

[0072] Preferably, step (a) is carried out by heating the microgel particle-forming polymer and aqueous vehicle to a temperature above the gelation temperature of the microgel particle-forming polymer. For example, in embodiments where the microgel particle-forming polymer is gellan, the gellan / aqueous vehicle mixture can be heated to 60-90°C (e.g., 70°C) to dissolve the gellan polymer.

[0073] It is understood that the amount of polymer that dissolves will depend on the amount of polymer required in the hydrogel composition (ie, it will be within the limits previously defined for the hydrogel composition).

[0074] In step b), the solution formed in step a) is preferably maintained at a temperature above the gelation temperature for the microgel particle-forming polymer and mixed with an aqueous solution of a monovalent or polyvalent metal ion salt. Preferably, in step b), the solution from step a) is continuously stirred before, during, and / or after the addition of the monovalent or polyvalent metal ion salt solution. For example, the mixture is mixed at a speed of 50 to 2000 revolutions per minute (rpm) to ensure sufficient mixing. In one embodiment, a mixing speed of 300 to 900 rpm or 500 to 800 rpm can be used. Those skilled in the art will appreciate that mixing speeds and mixing equipment can be varied to provide the desired level of shear / agitation.

[0075] In one embodiment in which the microgel particle-forming polymer is gellan, the gellan / aqueous vehicle solution from step a) can be cooled, for example, to a temperature of 35-50°C (e.g., 40°C) prior to mixing with the monovalent cation solution.

[0076] It should be understood that the amount of monovalent or polyvalent metal ion salt solution added will depend on the amount of metal ion salt required in the final hydrogel composition (i.e., it will be within the limits previously defined for the hydrogel composition).

[0077] In step c), the mixture from step b) is cooled to a temperature below the gelling temperature for the microgel particle-forming polymer so that microgel particles form in the hydrogel composition. Preferably, the mixture from step b) is cooled gradually with constant mixing. In one embodiment, the mixture from step b) is cooled at a constant cooling rate with continuous stirring / shear. Cooling under stirring / shear can be continued until the mixture reaches ambient temperature (e.g., 20°C), at which point the final hydrogel composition can be collected and stored, for example, under refrigerated conditions.

[0078] The cooling rate used in step c) and the amount of shear / agitation applied can be varied. For example, cooling rates of 0.2-4°C / min, 0.5-3°C / min, 0.5-2°C / min, 0.5-1.5°C / min, or 1°C / min can be used. The amount of shear applied can be, for example, 50-2000 rpm, 300-900 rpm, or 400-500 (e.g., 450) rpm. Any suitable device can be used to provide the required agitation / shear. In the accompanying examples, a rotational rheometer (AR-G2, TA Instruments, UK) equipped with a cup and blade geometry (cup: 35 mm diameter, blade: 28 mm diameter) is used to provide the required shear.

[0079] The pharmacologically active agents are: i) During step (a) ii) during step b); or iii) during step c), the time when the mixture from step b) is at a temperature above the gelation temperature of the microgel particle-forming polymer; And can be added.

[0080] Preferably, the pharmacologically active agent is added to the mixture in step b) or step c) of the method. Preferably, the pharmacologically active agent is added during step c) when the mixture is at a temperature above the gelation temperature for the microgel particle-forming polymer. Most preferably, the mixture from step b) is cooled to a temperature above the gelation temperature for the microgel particle-forming polymer, the pharmacologically active agent is added and thoroughly mixed to form a mixture, which is then further cooled to a temperature below the gelation temperature for the microgel particle-forming polymer.

[0081] Preferably, the pharmacologically active agent is added to the mixture in either step b) or step c) in the form of a solution.

[0082] In one embodiment, the pharmacologically active agent is decorin.

[0083] A further aspect of the present invention is a method of making a shear-thinning hydrogel composition as defined herein, comprising the steps of: a) dissolving a microgel particle-forming polymer in an aqueous vehicle containing 0.5 to 100 mM of a monovalent and / or polyvalent metal ion salt as a crosslinker; b) mixing the microgel-forming polymer solution formed in step (a) at a temperature above the gelation temperature of the microgel particle-forming polymer; and c) cooling the mixture resulting from step b) to a temperature below the gelling temperature of the microgel particle-forming polymer. The present invention provides a method comprising:

[0084] In this aspect of the invention, the method is the same as the previous method defined above, except that the microgel particle-forming polymer is dissolved directly in an aqueous vehicle containing 0.5 to 100 mM of a monovalent and / or polyvalent metal ion salt as a crosslinker. The conditions and variables for steps a), b), and c) above apply equally to this variation of the method.

[0085] A further aspect of the present invention provides a shear thinning gel composition obtainable by, obtained by or directly obtained by any of the preparative methods defined herein.

[0086] Medical uses of the compositions of the present invention and methods of treatment using the compositions of the present invention One aspect of the present invention provides a composition of the present invention for use as a medicine. The composition of the present invention is suitable for medical use in inhibiting scarring (as provided in a further aspect of the present invention), and in preventing and / or treating infection, pain, inflammation, and proliferative disorders. Compositions utilized for such medical uses may optionally contain an active agent selected from the group consisting of antifibrotic agents, anti-infective agents, analgesics, anti-inflammatory agents, antiproliferative agents, keratolytic agents, extracellular matrix modifying agents, cell-cell junction modifying agents, basement membrane modifying agents, biological lubricants, and pigmentation modifying agents.

[0087] Without deviating from the above, it has been found that compositions of the invention that do not contain a pharmacologically active agent can also be used successfully to inhibit scarring, and such use is illustrated in the data presented herein.

[0088] It should be understood that the composition of the present invention is also suitable for use in medical treatment methods.For example, the composition of the present invention can be used in a method selected from the group consisting of: a method for inhibiting scarring; a method for preventing and / or treating infection; a method for preventing and / or treating pain; a method for preventing and / or treating inflammation; a method for preventing and / or treating proliferation disorders; a method for preventing and / or treating hyperpigmentation; a method for preventing and / or treating hypopigmentation; a method for inducing corneal dissolution; a method requiring modification of extracellular matrix; a method requiring modification of intercellular junction; and a method requiring modification of basement membrane.

[0089] In practicing such methods, the compositions of the present invention may be administered, as needed, to a subject in need of inhibition of scarring; a subject in need of prevention and / or treatment of infection; a subject in need of prevention and / or treatment of pain; a subject in need of prevention and / or treatment of inflammation; a subject in need of prevention and / or treatment of a proliferative disorder; a subject in need of prevention and / or treatment of hyperpigmentation; a subject in need of prevention and / or treatment of hypopigmentation; a subject in need of corneal dissolution; a subject in need of modification of the extracellular matrix; a subject in need of modification of intercellular junctions; and a subject in need of modification of the basement membrane.

[0090] As noted above, compositions utilized in such methods of treatment may optionally include an active agent selected from the group consisting of anti-fibrotic agents; anti-infective agents; analgesics; anti-inflammatory agents; anti-proliferative agents; keratolytic agents; extracellular matrix modifying agents; intercellular junction modifying agents; basement membrane modifying agents; biological lubricants, and pigmentation modifying agents.

[0091] Methods for inhibiting scarring may involve the administration of compositions of the invention that do not contain pharmacologically active agents.

[0092] Unless the context requires otherwise, discussions presented in this disclosure regarding medical uses of the compositions of the invention should also be considered applicable to methods of treatment utilizing the compositions of the invention. Similarly, discussions presented in this disclosure regarding methods of treatment utilizing the compositions of the invention should also be considered applicable to medical uses of the compositions of the invention.

[0093] Inhibition of scarring In many clinical contexts, scarring is recognized to have deleterious effects: for example, scarring of the eye can be associated with vision loss and risk of blindness, while scarring in the skin can be associated with reduced mobility, discomfort, and disfigurement, which can cause psychological difficulties.

[0094] Scarring can also lead to complications and therefore reduced effectiveness of surgical procedures. By way of example only, scarring following surgical insertion of a stent (e.g., for the treatment of glaucoma) can completely or partially block the passageway in the stent, thus rendering the surgery ineffective.

[0095] It will be understood that "inhibition of scarring" encompasses both partial inhibition of scarring and complete inhibition of scarring. Preferred values ​​relating to the extent to which scarring may be inhibited by the present invention are further described below.

[0096] The compositions of the present invention may be useful in inhibiting scarring or fibrosis in a number of body sites. By way of example only, the compositions of the present invention may be used to inhibit scarring in the eye, scarring in the skin, scarring in muscles or tendons, scarring in nerves, fibrosis in internal organs such as the liver or lungs, or the formation of adhesions, such as surgical or omental adhesions.

[0097] The scarring in the eye that can be inhibited by the medical use of the composition of the present invention can include scarring of the cornea, scarring of the retina, scarring of the ocular surface, and scarring in or around the optic nerve.While the composition of the present invention is suitable for topical use, it should be understood that topically administered drugs can have an effect on internal structures.Therefore, the composition administered to the surface of the eye can be effective in inhibiting scarring within the eye.

[0098] Ocular scarring that can be inhibited by the medical use of the compositions of the present invention can also include scarring associated with infection, such as keratitis. Such keratitis can result from bacterial, viral, parasitic, or fungal infections. The compositions and methods of the present invention have shown particular utility in inhibiting scarring associated with bacterial keratitis.

[0099] Keratitis can also occur as a result of injury or disorders including autoimmune diseases such as rheumatoid arthritis or Sjogren's syndrome, and the compositions and methods of the present invention can also be used to inhibit scarring associated with keratitis resulting from these causes.

[0100] Scarring in the eye that may be inhibited by medical use of the compositions of the invention may also include scarring associated with surgery, such as surgery for the treatment of glaucoma (e.g., by insertion of a stent), and surgical procedures such as LASIK or LASEK surgery, as well as scarring associated with accidental injury.

[0101] Preferably, compositions of the present invention for use in inhibiting scarring may comprise gellan. Surprisingly, compositions of the present invention comprising gellan are able to effectively inhibit scarring even in the absence of pharmacologically active agents, such as active antifibrotic agents. Nevertheless, the incorporation of antifibrotic agents into compositions of the present invention exhibits beneficial properties in inhibiting scarring. By way of example only, decorin represents one example of such an antifibrotic agent suitable for incorporation into compositions of the present invention for use in inhibiting scarring.

[0102] Those skilled in the art will be aware of a number of suitable methodologies that allow for the identification and quantification of scarring. These methodologies can also be used to identify inhibition of scarring. Thus, they can be used to explain effective medical uses of the compositions of the invention, to identify therapeutically effective doses of antifibrotic agents, and to identify and / or select antifibrotic agents to be incorporated into the compositions of the invention.

[0103] Those skilled in the art will be aware that there are numerous parameters by which inhibition of scarring in the eye can be assessed. Examples of these are further discussed in the Examples. Some of these, such as induction of myofibroblasts or ECM components, are general to body sites outside the eye, while others are specific to the eye.

[0104] For example, scarring in the eye can be indicated by an increase in corneal opacity. Such an increase in corneal opacity can be represented by an increase in the area of ​​the opaque cornea. Thus, inhibition of scarring can be indicated by a decrease in corneal opacity compared to a suitable control. Such a reduction in corneal opacity can be represented by a decrease in the area of ​​the opaque cornea.

[0105] The ability of compositions of the present invention, including the anti-fibrotic agent decorin, to reduce corneal haze and maintain such reduction over time is demonstrated in the data presented in the Examples.

[0106] The composition of the present invention can be used to inhibit scarring associated with skin wounds.Suitable skin wounds are selected from the group consisting of burns; incisions; excisions; abrasions; chronic wounds; and wounds resulting from physical reactions to irritants.Examples of this latter category include systemic chemical and / or allergic reactions that cause severe skin blisters and peeling, and genetic disorders that result in impaired skin structure and homeostasis.These reactions or disorders can lead to dramatically increased risk and severity of skin blistering, peeling, and wounds (even from relatively minor contact).Examples of such disorders include epidermolysis bullosa (e.g., epidermolysis bullosa simplex, junctional epidermolysis bullosa, or dystrophic epidermolysis bullosa) and Kindler's syndrome.The composition or method of the present invention is suitable for use in inhibiting scarring in subjects with such disorders.

[0107] Other parameters indicative of scarring may be common to many different tissues. For example, scarring in many body sites can be indicated by an increased presence of myofibroblasts. Such an increase can be indicated by an increase in α-smooth muscle actin expression. Thus, inhibition of scarring can be indicated by a decrease in the number of myofibroblasts compared to a suitable control. This decrease in the number of myofibroblasts can be indicated by a decrease in α-smooth muscle actin expression.

[0108] Myofibroblasts arise at the site of injury and are involved in the progression of the scarring response. They can be characterized by the expression of alpha-smooth muscle actin (α-sma). Myofibroblasts can have a number of adverse effects on scar formation, including causing contraction in the healed area. The compositions of the present invention are capable of inhibiting α-sma expression when evaluated in vitro and in vivo.

[0109] As further discussed in the Examples, compositions of the present invention (with or without the anti-fibrotic agent decorin) are capable of inhibiting myofibroblast differentiation in vivo in experimental models of bacterial keratitis. Compositions, particularly those incorporating decorin, are also able to maintain this reduced differentiation over time.

[0110] Myofibroblast differentiation can increase in response to the action of TGF-β1, a fibrotic growth factor that induces α-sma expression. The Examples provide details of in vitro studies (in human dermal fibroblasts) that demonstrate the ability of compositions of the present invention to block this increase in α-sma expression. This demonstrates that the beneficial inhibition of scarring achieved by compositions of the present invention is not limited to the eye. Furthermore, the inhibition of scarring is observed even in the absence of an active antifibrotic agent, and therefore appears to be an antifibrotic effect of the gel composition itself.

[0111] Fibrosis is also associated with the expression and deposition of ECM components. The amount of deposited ECM can increase in scarring, and the structure of the ECM can differ from that observed in undamaged control tissue. The data presented in the examples demonstrate that treatment with the compositions of the present invention results in tissue whose ECM component structure more closely resembles that of undamaged tissue, thereby demonstrating the usefulness of these compositions in inhibiting scarring.

[0112] The compositions of the present invention are suitable for use at the site of a surgical incision to inhibit scarring that may otherwise accompany the healing of such surgical wounds.

[0113] Suitable antifibrotic agents for incorporation into the compositions of the present invention may be capable of achieving at least 5% inhibition of fibrosis compared to a suitable control agent. For example, suitable antifibrotic agents may be capable of achieving at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% inhibition compared to a suitable control agent. Suitable antifibrotic agents for incorporation into the compositions of the present invention may be capable of achieving substantially complete inhibition of scarring compared to a suitable control agent.

[0114] Similarly, medical uses of compositions of the invention, or methods of treatment using such compositions to inhibit scarring, may achieve at least 5% inhibition compared to a suitable control. For example, such medical uses or methods of treatment may be able to achieve at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% inhibition compared to a suitable control. Medical uses or methods of treatment of the invention may achieve substantially complete inhibition of scarring compared to a suitable control.

[0115] The selection of a suitable control may be readily determined by one of skill in the art. By way of example only, a suitable control for evaluation of the ability of a composition of the invention to inhibit scarring in the eye may be provided by a recognized standard of care or an experimental surrogate thereof.

[0116] Active Agents Suitable for Incorporation in Compositions of the Invention Compositions of the present invention intended for medical use or use in methods of treatment may contain additional active agents. Suitable active agents can be selected with respect to the intended medical use. However, by way of example, suitable active agents can be selected from the group consisting of antifibrotic agents; anti-infective agents; analgesics; anti-inflammatory agents; antiproliferative agents; keratolytic agents; extracellular matrix modifying agents; intercellular junction modifying agents; basement membrane modifying agents; biological lubricants, and pigmentation modifying agents. For the avoidance of doubt, compositions of the present invention may suitably contain more than one active agent. When a composition contains more than one active agent, the active agents may be more than one active agent within a particular class of active agent (e.g., two or more antifibrotic agents), or a combination of agents selected from two or more different classes (e.g., an antifibrotic agent and an anti-infective agent, or an antifibrotic agent and an analgesic agent).

[0117] Examples of anti-fibrotic agents that can be incorporated into the compositions of the present invention are discussed in more detail below.

[0118] By way of example only, anti-infective agents suitable for incorporation as active agents into the compositions of the present invention may be antibacterial, antiviral, antifungal, or anti-helminth agents. In the case of antibacterial agents, suitable anti-infective agents may be antibiotics, such as gentamicin, penicillin, streptomycin (optionally in combination as penicillin-streptomycin), or vancomycin. Many other suitable examples of antibacterial agents that can be incorporated into the compositions of the present invention include additional antibiotics and will be known to those skilled in the art.

[0119] The composition of the present invention comprising an anti-infective agent can be used in a method for preventing and / or treating infection. Therefore, it should be understood that such a composition can be administered to a subject in need of prevention and / or treatment of infection. The subject in need of such prevention and / or treatment may have a chronic wound or an infected wound. By way of example only, a subject at risk of developing a chronic wound may have diabetes, chronic venous insufficiency, or peripheral arterial occlusive disease.

[0120] Embodiments of the compositions or methods of the invention that utilize anti-infective agents may also be useful in preventing or treating scarring that may be associated with disorders such as infection (eg, bacterial keratitis).

[0121] Analgesics suitable for incorporation as active agents in the compositions of the present invention may be selected from the group consisting of analgesics, anesthetics such as benzocaine, proparacaine, tetracaine, articaine, dibucaine, lidocaine, prilocaine, pramoxine, and dyclonine, or esters, amides, or ethers thereof; salicylates such as salicylic acid or acetylsalicylic acid; rubefacients such as menthol, capsaicin, and / or camphor, and nonsteroidal anti-inflammatory drugs (NSAIDs) such as ibuprofen.

[0122] The composition of the present invention comprising an analgesic agent can be used in a method for preventing and / or treating pain. Thus, such a composition can be administered to a subject in need of pain prevention and / or treatment. Preferably, the subject in need of such prevention and / or treatment may have or be at risk of a condition related to skin or musculoskeletal pain.

[0123] Anti-inflammatory agents suitable for incorporation into the compositions of the present invention as active agents can be selected from the group consisting of steroids, such as corticosteroids (e.g., prednisolone or dexamethasone); NSAIDs, such as ibuprofen, or COX-1 and / or COX-2 enzyme inhibitors; antihistamines, such as H1 receptor antagonists; interleukin-10; pirfenidone; immunomodulators; and heparin-like agents. Dextran or modified dextran sulfate and decorin also represent suitable agents that can be incorporated into the compositions of the present invention as anti-inflammatory agents. Those skilled in the art will understand that these molecules can exert either anti-inflammatory or pro-inflammatory effects in vivo, and will be aware that the scientific and clinical literature provides a wealth of information that allows for the selection of appropriate doses to exert the desired activity (anti-inflammatory or pro-inflammatory).

[0124] The composition of the present invention comprising an anti-inflammatory agent can be used in a method for preventing and / or treating inflammation.Therefore, such a composition can be administered to a subject who needs to prevent and / or treat inflammation.Preferably, the subject may have or be at risk of developing chronic inflammation or acute inflammation.By way of example only, chronic inflammation may be associated with rheumatoid arthritis or dermatitis.Acute inflammation may be caused by a wound.

[0125] Antiproliferative agents for incorporation into the compositions of the present invention as active agents can be selected from the group consisting of toll-like receptor 7 (TLR7) agonists, toll-like receptor 2 (TLR2) agonists, toll-like receptor 4 (TLR4) agonists, toll-like receptor 9 (TLR9) agonists; and antimetabolites. A suitable example of such a TLR7 agonist is imiquimod. A suitable example of such an antimetabolite is fluorouracil (5-FU).

[0126] The composition of the present invention comprising an antiproliferative agent can be used in a method for preventing and / or treating a proliferative disorder.Therefore, such a composition can be administered to a subject in need of prevention and / or treatment of a proliferative disorder.Preferably, the subject may have or be at risk of developing a skin proliferative disorder, such as psoriasis, cancer (e.g., melanoma or non-melanoma skin cancer), eczema, or ichthyosis.

[0127] Keratolytic agents suitable for incorporation as active agents in the compositions of the invention can be selected from the group consisting of acids, such as salicylic acid, alpha hydroxy acids, beta hydroxy acids, and / or lactic acid; enzymes, such as papain and / or bromelain; and retinoids, such as retinol and / or tretinoin. Compositions or methods of the invention utilizing a keratolytic agent (e.g., bromelain) can be used in debridement of wounds, such as burns.

[0128] Suitable extracellular matrix modifying agents for incorporation into the compositions of the present invention may be selected from the group consisting of proteinases (e.g., proteinase K), matrix metalloproteinases (MMPs); membrane-associated MMPs (MTMMPs); adamalysin (ADAM); ADAM with thrombolysin (ADAMTS); disintegrins; tissue inhibitors of metalloproteinases (TIMPs); serine proteases such as urokinase; tissue plasminogen activator; elastase; matriptase; and enzymes involved in matrix remodeling processes such as cathepsins, heparanase, and sulfatases.

[0129] Compositions or methods of the invention utilizing extracellular matrix modifying agents can be used in applications requiring ECM regulation and remodeling and / or modulation of cell-cell adhesion and cell-matrix interactions. By way of example, such applications can include the treatment of hypertrophic or keloid scars. Compositions or methods according to such embodiments can provide clinical benefit by promoting a beneficial balance of collagen ratios or by directly targeting the production of ECM components, such as collagen.

[0130] Suitable cell-cell junction modifiers for incorporation into the compositions of the present invention can be selected from the group consisting of adenosine triphosphate (ATP); cyclic adenosine monophosphate (cAMP); inositol triphosphate (IP3); glucose; glutathione; glutamate; and ions selected from sodium, potassium, and calcium ions. Preferably, such cell-cell junction modifiers can be antibodies or other peptides that affect components of cell-cell junctions, such as connexins. Examples of such proteins include cadherins and α- and β-catenins. Preferably, such agents can achieve microtubule inhibition. Tight junctions may be affected by inhibition by components such as occludin, claudins, and junctional adhesion molecule-1 (JAM-1).

[0131] Platelet-rich plasma (serum) can be incorporated into the compositions of the present invention.

[0132] The compositions or methods of the present invention utilizing cell junction altering agents can be used in the treatment of difficult-to-heal chronic wounds, such as ulcers.

[0133] A basement membrane-altering agent suitable for incorporation into the compositions of the present invention may be an agent against adhesions. Such an agent may be selected from the group consisting of blocking antibodies or competitive peptides that inhibit the activity of integrins, laminins, or components of focal adhesions (e.g., vinculin, talin, α-actinin, kindlin, etc.). Alternatively, a suitable basement membrane-altering agent may include a proteinase, such as proteinase K.

[0134] The compositions or methods of the invention utilizing basement membrane altering agents may also be used in the treatment of difficult-to-heal chronic wounds, such as ulcers.

[0135] For purposes of this disclosure, a biological lubricant will be considered to be an agent derived from a biological source that can act as a lubricant. In a preferred embodiment, the biological lubricant for incorporation into the hydrogel composition of the present invention may be serum. As presented below, serum has therapeutic utility in treating a number of eye disorders. Thus, the hydrogel composition of the present invention comprising serum may be suitable for administration to the eye as an eye drop.

[0136] The compositions or methods of the present invention utilizing a biological lubricant, such as serum, can be used in the prevention and / or treatment of conditions including those selected from the group consisting of dry eye syndrome and Sjogren's syndrome.

[0137] The compositions or methods of the present invention may utilize a pigment modifying agent. The pigment modifying agent for incorporation into the compositions of the present invention as an active agent may be selected from the group consisting of depigmenting agents and pigmentation enhancers.

[0138] The depigmenting agent suitable for incorporation into the composition of the present invention as an active agent can be selected from the group consisting of turmeric, melanin production inhibitors, and antioxidants. Suitable examples of melanin production inhibitors include hydroquinone, resorcinol, resveratrol, and azelaic acid. Suitable examples of antioxidants include vitamin C, vitamin E, glutathione, turmeric, and ferulic acid.

[0139] Suitable pigmentation enhancers for incorporation into the compositions of the present invention include substances that affect components of the melanin pathway. These can be selected from the group consisting of tyrosine (hydroxylated to L-3,4-dihydroxyphenylalanine (DOPA) by tyrosinase) and DOPA (oxidized to DOPAquinone, which, in the presence of a cysteine ​​group, produces pheomelanin). Eumelanin production requires the action of two additional enzymes: tyrosinase-related protein 1 (TRP1) and 2 (TRP2 / Dct), which reorganize DOPAchrome (produced from repeated spontaneous oxidation of DOPAquinone) to form DHI-2-carboxylic acid (DHICA). These enzymes or their substrates also represent suitable pigmentation-modifying agents.

[0140] The compositions or methods of the invention utilizing pigmentation-altering agents can be used in a wide range of clinical contexts related to unwanted hypo- or hyperpigmentation, including scarring, such as following surgical or pathological scarring (e.g., hypertrophic or keloid scarring).

[0141] The composition of the present invention containing a depigmenting agent can be used in a method for preventing and / or treating hyperpigmentation disorders. Therefore, such a composition can be administered to a subject in need of prevention and / or treatment of hyperpigmentation disorders. Preferably, the subject may have or be at risk of developing melasma, post-inflammatory hyperpigmentation, or Addison's disease.

[0142] In a preferred embodiment, the composition according to the invention may comprise an antifibrotic agent for use in combination with one or more agents selected from the group consisting of steroids and antibacterial agents. The antifibrotic agent, steroid and antibacterial agent may be formulated in separate compositions or as part of the same composition.

[0143] Preferably, compositions of the present invention may comprise decorin for use in combination with the anti-infective agent gentamicin and the anti-inflammatory agent prednisolone. Such compositions may comprise decorin, prednisolone, and gentamicin. Such compositions of the present invention are suitable for use in inhibiting scarring associated with bacterial keratitis, as illustrated by the data presented in the Examples.

[0144] In a preferred embodiment, the compositions of the present invention may include anti-inflammatory and analgesic agents. Such compositions may be particularly useful, for example, in the context of chronic inflammatory diseases such as dermatitis or rheumatoid arthritis, where it may be desirable to prevent and / or treat pain and inflammation.

[0145] In another example, the compositions of the present invention may include an analgesic and an anti-infective agent. Such compositions may be particularly useful in the context of skin wounds and may be desirable for preventing and / or treating pain and infection. Other suitable active agent combinations will be known to those skilled in the art.

[0146] The compositions of the present invention for medical use can incorporate a therapeutically effective amount of an active agent. Such a therapeutically effective amount will be capable of achieving a desired clinical outcome, either as a single administration or as part of a treatment regimen involving multiple administrations. Those skilled in the art will be aware of suitable protocols and procedures for calculating therapeutically effective amounts for various types of active agents.

[0147] Preferably, the active agent can be incorporated into the compositions of the present invention at a concentration of between 0.1 ng / mL and 10 mg / mL. For example, the active agent can be incorporated into the compositions of the present invention at a concentration of between 1 ng / mL and 5 mg / mL, between 10 ng / mL and 2.5 mg / mL, or between 20 ng / mL and 1 mg / mL, between about 0.1 μg / mL and 0.5 μg / mL, preferably about 0.24 μg / mL.

[0148] antifibrotic agents Anti-fibrotic agents are agents that are capable of causing inhibition of scarring in a subject or body site to which they are provided. Inhibition of scarring is discussed more generally below.

[0149] Many antifibrotic agents are known to those skilled in the art. Thus, those skilled in the art will be able to easily identify antifibrotic agents that can be advantageously incorporated into the compositions of the present invention for use in inhibiting scarring. Below is provided a non-exclusive list of examples of antifibrotic agents suitable for such use.

[0150] Suitable anti-fibrotic agents can be selected from the group consisting of anti-fibrotic extracellular matrix (ECM) components; anti-fibrotic growth factors (which for purposes of this disclosure should be considered to also encompass anti-fibrotic cytokines, chemokines, and the like); polymers, such as dextran or modified dextran sulfate; and inhibitors of fibrotic agents, such as function-blocking antibodies. It should be understood that the therapeutic effectiveness of such agents depends on the dose provided by the compositions of the present invention. Those skilled in the art will be aware of extensive literature and clinical resources that will enable them to select an appropriate dose of any of the listed agents to meet the required therapeutic objectives.

[0151] Dextran or modified dextran sulfate can exert both antifibrotic and profibrotic effects in vivo. In the context of antifibrotic uses of dextran or modified dextran sulfate, those skilled in the art will understand that a suitable dose for antifibrotic purposes may be between 0.1 and 10 mg / kg of subject body weight. In a preferred embodiment, dextran or modified dextran sulfate for use in the compositions of the present invention may have a molecular weight of 10 kDa or less.

[0152] Antibodies are useful for interfering with certain cellular activities by binding to cell signaling agents, thereby blocking the function caused by the activity of the agent. Examples of such activities that can be blocked include cell proliferation, cell migration, protease production, apoptosis, and anoikis. By way of example only, a suitable blocking antibody may be capable of binding to one or more of the following groups of cell signaling agents: ECM components, growth factors, cytokines, chemokines, or matrikines.

[0153] Decorin is an example of an anti-fibrotic ECM component that can be advantageously incorporated into the compositions of the present invention. The decorin can be human decorin. Preferably, the decorin can be human recombinant decorin. An example of a human recombinant decorin that can be incorporated into the compositions of the present invention is that manufactured and sold by Catalent Pharma Solutions, Inc. under the name "Galacorin™."

[0154] Decorin for incorporation into the compositions of the invention can be the full-length, naturally occurring version of this proteoglycan. Alternatively, the compositions of the invention can utilize anti-fibrotic fragments or anti-fibrotic variants of naturally occurring decorin.

[0155] Naturally occurring decorin is a proteoglycan. Proteoglycans (including both the core protein and glycosaminoglycan chains) or fragments thereof can be used in the hydrogel compositions of the present invention. However, the core protein alone (without the glycosaminoglycan chains) has been shown to be sufficient to inhibit scarring in the eye. Therefore, references herein to decorin (or fragments or variants thereof) can be interpreted as referring instead to the core protein without the glycosaminoglycan chains. It is believed that the core protein of decorin binds to fibrotic growth factors (e.g., TGF-β) and plays a role in blocking their biological functions.

[0156] Suitable anti-fibrotic fragments of decorin may comprise up to 50% of the full-length naturally occurring molecule, up to 75% of the full-length naturally occurring molecule, or up to 90% of the full-length naturally occurring molecule. Suitable anti-fibrotic fragments of decorin may comprise the TGF-β binding portion of decorin.

[0157] Anti-fibrotic variants of decorin will differ from naturally occurring proteoglycans by the presence of one or more mutations in the amino acid sequence of the core protein. These mutations may result in the addition, deletion, or substitution of one or more amino acid residues present in the core protein. By way of example only, suitable anti-fibrotic variants of decorin suitable for incorporation into compositions of the invention may have at least one, at least two, at least three, at least four, at least five, at least 10, at least 15, or at least 20 mutations compared to the amino acid sequence of the naturally occurring core protein.

[0158] Unless the context requires otherwise, reference herein to decorin with respect to the incorporation of this agent in compositions of the invention should also be considered as encompassing the use of antifibrotic fragments or antifibrotic variants of decorin.

[0159] In a preferred embodiment, decorin is the only ECM component present in the compositions of the invention.

[0160] Anti-fibrotic growth factors suitable for incorporation into the compositions of the present invention include those selected from the group consisting of transforming growth factor-β3, platelet-derived growth factor AA, insulin-like growth factor-1, epidermal growth factor, fibroblast growth factor (FGF) 2, FGF7, FGF10, FGF22, vascular endothelial growth factor A, keratinocyte growth factor, and hepatocyte growth factor.

[0161] Fibrotic agent inhibitors represent suitable anti-fibrotic agents that can be incorporated into the compositions of the present invention. Examples of such inhibitors include agents that bind to fibrotic agents and thereby block their activity. Examples of such inhibitors include function-blocking antibodies (discussed further above) or soluble fragments of cell receptors through which fibrotic agents induce cell signaling. Other examples of such inhibitors include agents that prevent the expression of fibrotic agents. Examples of these types of inhibitors include those selected from the group consisting of antisense oligonucleotides and interfering RNA sequences.

[0162] Compositions of the invention suitable for use in inhibiting scarring may incorporate a therapeutically effective amount of an anti-fibrotic agent. Such a therapeutically effective amount will be capable of inhibiting scarring either in a single dose or as part of a treatment comprising multiple doses. Details of how inhibition of scarring may be assessed, and how a therapeutically effective amount may therefore be calculated or recognised, are discussed above.

[0163] By way of example only, anti-fibrotic agents such as decorin may be incorporated into the compositions of the present invention at concentrations of between 0.1 ng / mL and 10 mg / mL, between 1 ng / mL and 5 mg / mL, between 10 ng / mL and 2.5 mg / mL, between 20 ng / mL and 1 mg / mL, between about 0.1 μg / mL and 0.5 μg / mL, preferably about 0.24 μg / mL.

[0164] Topical Administration and Topical Compositions The compositions of the present invention are suitable for topical administration to a subject. For the avoidance of doubt, in the context of the present disclosure, "topical administration" is considered to relate to the administration of a composition directly to the surface of the body or the surface of an organ. Compositions of the present invention suitable for such topical administration may be referred to as topical compositions of the present invention.

[0165] Preferably, the topical composition of the present invention can be administered to one or more body surfaces selected from the group consisting of the surface of the eye; skin; brain surface; and mucous membrane.For example, the topical composition of the present invention can be administered to a body surface during or after surgery.Preferably, the topical composition of the present invention can be administered to such surfaces associated with abdominal surgery (for example, to inhibit adhesion formation) or brain surgery (for example, to provide a desired therapeutic agent to the brain).

[0166] The topical compositions of the present invention may be for administration to a site of infection or injury on the surface of the body (including, but not limited to, abrasions, burns, and puncture wounds). For example, the compositions of the present invention may be for administration to a site of infection or injury on the surface of the eye (e.g., a site of bacterial keratitis), or to a site of infection or injury to the skin (e.g., a skin burn or abrasion).

[0167] It should be understood that the topical composition can be formulated in a conventional manner for use in such contexts. For example, a suitable topical composition can be formulated so as not to induce irritation or inflammation of the infected or injured area to which it is administered.

[0168] We present a novel eye drop system for the sustained delivery of a potent anti-scarring molecule (hrDecolin). The novelty of this eye drop lies in the method of structuring during manufacturing, creating a material that can transition between solid and liquid states, allowing for retention in a dynamic environment where it is slowly removed through blinking. In a mouse model of Pseudomonas keratitis, application of the eye drop resulted in a reduction of corneal opacity within 16 days. More remarkably, the addition of hrDecolin resulted in scar-free restoration and corneal integrity, as evidenced by complete re-epithelialization and reductions in αSMA, fibronectin, and laminin. This drug delivery system is an ideal non-invasive anti-fibrotic treatment for patients with bacterial keratitis and has the potential to save many eyesights without resorting to surgery in developing countries where corneal transplants are not available.

[0169] This paper reports a novel class of eye drop materials that enable long-term retention of therapeutic agents on the ocular surface while being gradually removed through the blinking process. The materials are formed through shearing of gellan-based hydrogels, a material currently used in diluted form to thicken eye drops (e.g., Timoptol) during the gelation process. The application of shear prevents the formation of a continuous polymer network and results in the formation of interacting particles that can exhibit spherical and ribbon-like morphologies. Following the shearing process, if the solution is at rest, these particles interact and form a continuous structure. However, when shear is applied (e.g., when extruded through an eyedropper), the continuous network of particles is disrupted and the material liquefies. Subsequent removal of the shear force results in immediate repair. The solid-liquid-solid transition that this material can undergo means that it conforms perfectly to the ocular surface and is gradually removed by the blinking movement of the eyelid. Importantly, gellan gum is optically transparent, allowing the material to continue to transmit light after application, minimizing disruption to the patient.

[0170] A fluid-gel eye drop loaded with decorin has been developed that can provide localized drug delivery and retention at the ocular surface. The material combines structured gellan gum with the proteoglycan decorin. Additionally, the FDA-approved polymer (FDA Reference Number 172.665) combined with clinical-grade hr-decorin, along with high optical clarity, can be rapidly introduced into the clinic. Therefore, this study investigated the effects of a fluid gel with or without hr-decorin on corneal opacity, wound healing, and fibrosis in a well-established mouse model of Pseudomonas keratitis, as a precursor to clinical application for the management of severe bacterial infection.

[0171] Fluid-gel eye drops as described herein have been shown to be beneficial in the prevention and / or treatment of glaucoma. Fluid-gel eye drops according to the present invention, particularly for use according to this aspect of the present invention, may comprise a shear-thinning hydrogel composition comprising gellan. As shown in results disclosed elsewhere herein, shear-thinning hydrogel compositions according to the present invention have been found to be capable of reducing intraocular pressure (a well-known experimental model for glaucoma) even when formulated without an active agent.

[0172] Fluid Gel Formulations and Properties Processing of the fluid gel involves passing a polymer solution, gellan, through a jacketed pin-stirrer, where the fluid gel experiences high levels of shear while being thermally forced through its sol-gel transition (Figure 1a). This limits the long-range ordering typically observed in quiescent gel formation and restricts the growth of gel nuclei into discrete particles. [34、35]The microstructure in eye drops prepared in this manner is demonstrated using two techniques: 1) optical microscopy, whereby the refractive index of the continuous phase is manipulated using polyethylene glycol, and 2) freeze-drying for imaging using scanning electron microscopy (SEM) (Figures 1a(i) and 1a(ii), respectively). Both microscopy techniques highlight the chain-like microstructure of the resulting gelled entity, where these large length-to-width ratios and subsequent large hydrodynamic radii result in the properties (viscosity and elastic structuring) of the resulting material.

[36] .

[0173] The unique properties of fluid gels are such that they exhibit quasi-solid properties at rest but can be made to flow when subjected to force, where increasing shear forces exerted on the system result in non-Newtonian shear thinning behavior typical of highly flocculated or concentrated polymer dispersions / solutions.

[37] (Figure 1b). Thus, at low shear, a large viscosity, over several orders of magnitude higher than typical water-based eye drops, is observed, which thins during application and subsequent blinking as a result of particle release and alignment during flow. [38、39] This makes the microgel suspension ideal for application through a dropper bottle, where it rapidly shear-thinns through a nozzle upon application to the eye (Figure 1c). Restoration of the three-dimensional structured matrix after application is crucial for achieving a high retention time on the ocular surface. Time-dependent shear removal on a timescale relative to the initial ramp was used to probe the hysteresis of the eye drop system and glean information on such structuring. The eye drop system exhibited a degree of thixotropy (Figure 1b), which resulted in the recovery of much of the original viscosity. The presence of weak gel-ribbon interactions was investigated using linear rheology, utilizing the development of elastic structures at strains within the linear viscoelastic region (Figure 1d). Initially, after shear, the fluid gel was observed to exhibit typical liquid-like behavior, with the loss modulus (G") exceeding the storage modulus (G'). This was followed by an increase in G' as a function of the formation of interactions between the gelled ribbons, achieving crossover, at which point the system began to behave as a solid gel.

[40] Thus, further structuring over time results in quasi-solid behavior, where a continuous network is formed between the gelled entities. The ability to shear thin upon application while being able to rapidly restructure after shear allows the eye drops to be applied to the ocular surface and act as a barrier. Using a single application of 5 μl of fluid gel eye drops, a uniform distribution of the gel was shown to cover the entire ocular surface, including the cornea, adjacent conjunctiva, and fornix (the space between the eyelid and the eyeball), in rodent eyes (Figure 1e).

[0174] In vitro activity of eye drops A gellan-based eye drop system was formulated for drug delivery with the candidate antifibrotic agent hr-decorin and used in the study. The release rate of hr-decorin from the eye drop system was nearly linear over time (Figure 2a). Turbidity was used as a measure of fibrillogenesis (the formation of large, unoriented collagen fibers) as a function of hr-decorin (Figures 2b and c). It is also clear that hr-decorin plays a pivotal role in the kinetics of fibrillogenesis, delaying the onset of fibrillogenesis and reaching equilibrium more quickly (Figure 2b). Above a critical concentration of 0.5 μg / ml, the active effect of hr-decorin in inhibiting fibrillogenesis was observed, highlighting its concentration dependency until a minimum turbidity (>10 μg / ml) was achieved, above which no further decrease occurred (Figure 2c). Furthermore, the assay demonstrated that the fluid gel carrier had no effect on fibrillogenesis, closely correlating with the collagen-only control.

[0175] In vivo efficacy of loaded and unloaded eye drops against corneal opacities Using a well-established model of bacterial keratitis

[41] Anesthetized mice (n = 6 per group) were inoculated with Pseudomonas aeruginosa (10 5 The eyes were challenged with 1000 CFU (10 ...

[0176] Following the sterilization phase, 2 days after the initial inoculation, a single 5 μl gellan eye drop was administered every 4 hours between 8:00 AM and 8:00 PM for an additional 13 days across the 1) gentamicin and prednisolone (GP); 2) gentamicin, prednisolone and fluid gel (GPFG); and 3) gentamicin, prednisolone and hrDecorin fluid gel (GPDecFG) treatment groups (Table 1).

[0177] Corneal images were taken at intervals throughout the 16-day experiment to measure changes in corneal opacity (Figure 3a). All mice were euthanized on day 16. The area of ​​opacity (measured independently by two clinical ophthalmologists blinded to treatment group) showed a more rapid decrease in size in eyes treated with fluid gel and hrDecorin fluid gel eye drops in addition to standard care compared to eyes treated with standard care (gentamicin and prednisolone) treatment alone. Thus, on day 9, eyes treated with standard care with hrDecorin fluid gel had a significantly larger area than eyes treated with gentamicin and prednisolone alone (3.5 ± 0.4 mm). 2 ) had a significantly (p<0.001) lower opacity area (1.9±0.3mm) compared with 2 On day 12, mice receiving hrDecorin fluid gel eye drops with standard care maintained significantly lower (p<0.01) opacity area compared to the gentamicin and prednisolone group and the fluid gel with standard care group (mean opacity area in group 1 = 3.5 ± 0.7 mm 2 , group 2=3.0±0.1mm 2 , group 3=2.1±0.2mm 2 Compare with Fig. 3b).

[0178] Effect of fluid gel eye drops with or without hrdecorin on corneal re-epithelialization Epithelial stratification / maturation, along with stromal thickness, were selected as outcome measures to assess corneal re-epithelialization and to observe stromal thickening resulting from edema and cellular infiltration (as a marker of infection). Pseudomonas infection severely disrupted corneal structure, with a mean increase in corneal thickness of 218.7 ± 24 μm at 2 days post-infection, compared with an untreated corneal thickness value of 129.3 ± 10.7 μm. Infected corneas at 2 days had a thinner epithelial layer compared with normal intact controls (19.2 ± 2.1 μm vs. 35.5 ± 1.7 μm; Figures 4a and b). Improved re-epithelialization was evident with the addition of fluid gel alone and eye drop treatment with hrDecolin over 13 days. Treatment with hrDecorin-loaded fluid gel eye drops resulted in an improved degree of epithelial stratification (26.1 ± 2.4 μm thick, made up of 3.6 ± 0.2 cell layers) compared with the epithelium in the gentamicin and prednisolone group (22.5 ± 2.1 μm thick, made up of 2.7 ± 0.2 cell layers) and the gentamicin, prednisolone, and fluid gel group (22.8 ± 1.3 μm thick, made up of 3.4 ± 0.1 cell layers). However, the differences between the various groups did not reach statistical significance (Figure 4b, c, and d).

[0179] Effects of fluid gels on myofibroblast and extracellular matrix levels The extent of fibrosis was assessed using αSMA immunoreactivity (IR) as the ratio of pixel intensity above the baseline (referred to here as threshold) obtained from intact corneas. Untreated intact corneas exhibited very low levels of αSMA immunoreactivity (IR) in the corneal stroma, indicating the near absence of myofibroblasts (Figure 5a). Two days after infection and one day after sterilization, infected corneas showed a 23% increase in stromal αSMA staining to a level of 26.5 ± 3.0% above threshold (normalized to intact corneas), indicating increased myofibroblast differentiation. Stroma IR αSMA levels remained elevated at day 16 in eyes treated with standard therapy alone, at 32.7 ± 6.1%. When eyes were treated with fluid gel eye drops with or without hr decorin, stromal αSMA IR levels were also significantly lower at 13.4±2.9% and 2.0±0.4%, respectively, on day 16, suggesting less activation of myofibroblasts in the corneal stroma. The hr decorin fluid gel was most effective at keeping αSMA IR levels low, resulting in values ​​similar to those in intact corneas, suggesting that the addition of hr decorin to the fluid gel has an additional beneficial effect on myofibroblast differentiation relative to the fluid gel alone (Figure 5a).

[0180] The levels of stromal ECM produced by myofibroblasts were examined using fibronectin and laminin IR (Figures 5b and c). Increased levels of stromal IR fibronectin were observed 2 days after infection and remained high at 16 days after gentamicin and prednisolone treatment (IR fibronectin 83.9 ± 5.5% and 75.3 ± 11.5% at days 0 and 16, respectively). Fluid gels with or without hr decorin significantly reduced fibronectin IR levels to 31.6 ± 5.8% and 13.9 ± 5.3%, respectively, indicating a borderline significant difference between the two eye drop treatment groups (p = 0.051). IR laminin levels (Figure 5c) show that infection increased laminin levels from 2.15 ± 0.6% in intact corneas to 16.3 ± 4.6% in the infected group at 2 days compared to intact corneas. IR laminin levels continued to increase to 42.5±8.2% by day 16 after gentamicin and prednisolone treatment. Similar to the gentamicin and prednisolone group, mean IR laminin levels remained high at day 16 after treatment with fluid gel, with IR laminin levels of 38.0±12.0%. Addition of hr decorin to fluid gel significantly reduced laminin levels compared to gentamicin and prednisolone treatment (12.4±5.5% vs. 42.3±8.2%), while fluid gel without hr decorin had no effect on this ECM parameter.

[0181] Effect of fluid gel on myofibroblast levels in vitro Human dermal fibroblasts were grown in 6-well plates at a density of 150,000 cells / well. Cells were allowed to attach for 24 hours and then serum-starved in HFDM-1 medium before treatment with the experimental compositions.

[0182] Experimental hydrogel compositions of the present invention were prepared with and without the anti-fibrotic agent decorin. These are shown in the graph of Figure 25 as "GEL+dec" and "GEL-dec," respectively. During the study, 1 ml of the experimental gel composition was added per well, followed by administration of TGF-β1 at 5 ng / ml (with the exception of "GEL+dec (2nd gel)," in which TGF-β1 was administered before administration of the gel, indicating that the order did not significantly alter the effect).

[0183] As can be seen from Figure 25, the addition of TGF-β1 stimulated the expression of α-sma, indicating the formation of myofibroblasts characteristic of scarring. The application of the hydrogel composition of the present invention reduced this α-sma expression. This was observed both in the presence and absence of the antifibrotic agent decorin, illustrating the ability of the hydrogel composition of the present invention to inhibit scarring even in the absence of additional antifibrotic active agents.

[0184] Consideration Precorneal tear film turnover (approximately 20% per minute)

[42] Because aqueous solutions (e.g., ocular ophthalmic solutions) result in rapid elimination of aqueous drugs and reduce the amount delivered to the target tissue site, improving ocular retention is key to increasing both biological efficacy and therapeutic response to topical therapies. Consequently, many ocular conditions are currently treated through invasive methods, including intensive topical therapies delivered throughout the day and night, or intraocular or intravitreal injections targeting intraocular pathology, which are disliked by many patients. In more severe cases where drugs are ineffective, surgery may be required to treat or remove the resulting corneal scar, increasing the risk of morbidity and the duration of patient discomfort following the procedure. Structured or "fluid-gels" formed from gellan offer an important advancement because they enable the sustained delivery of molecules such as hrdecorin, which can prevent scarring and eliminate the need for invasive surgical repair strategies. A key benefit of gellan fluid-gels is their ability to transition between solid and liquid states, passing through an applicator and solidifying on the surface of the cornea. This unique set of properties stems from the material's microstructure, which consists of ribbons and particles that weakly interact with each other at zero shear. These interactions are disrupted by the application of shear and reorganize following the removal of shear. In this way, the material is then gradually removed from the ocular surface by the natural blinking mechanism. The development of a weakly elastic structure when applied to the surface of the cornea results in the formation of a transparent and absorbent dressing, possessing the benefits of eye drops (in application) and hydrogel lenses (sustained release) without the drawbacks of either. Indeed, the fluid-gel alone appears to provide a microenvironment conducive to wound healing, accompanied by a reduction in markers of corneal opacity and scar formation, even without the addition of decorin. Importantly, as demonstrated by collagen fibrillogenesis data, the fluid-gel does not interfere with the biological activity of hrdecorin. Thus, the system provides a technology that is an excellent candidate for clinical settings, accompanied by improved drug compliance across numerous patient cohorts.

[0185] A murine model of Pseudomonas aeruginosa keratitis provides a robust, clinically relevant method to evaluate the anti-scarring potential of hrDecorin-loaded fluid gel versus the current standard of care (gentamicin and prednisolone) for Pseudomonas infections

[43] Once infection is established, P. aeruginosa invades corneal epithelial cells and disrupts the natural healing response, which involves the transformation of corneal fibroblasts into corneal myofibroblasts, resulting in a fibrogenic microenvironment.

[44] Topical administration of eye drops with or without hrDecolin resulted in a reduction in the level of corneal opacity 7 and 10 days after eye drop treatment, with the addition of hrDecolin demonstrating a clear additional benefit. The effect of fluid gel treatment alone was not as predicted by initial in vitro studies, indicating that this carrier appears to be inert. The therapeutic efficacy of fluid gel alone may be attributed to the formation of a permissive microenvironment in the injured cornea, and the occlusive effect of the gel ribbon (forming a barrier around the entangled wound) provides two key benefits. First, it provides a therapeutic dressing that prevents the biomechanical trauma caused by blinking on the ulcerated eye. Second, it sequesters the steroids and gentamicin within its structure, enhancing retention of the therapeutic substances on the ocular surface and thereby improving bioavailability, similar to artificial replacement of the ecosystem (PROSE™ device) but with the added benefit of being absorbable. Such a reduction in corneal opacity would benefit patients with regard to preserving their vision.

[45] .

[0186] A key aspect to the healing phase involves the restoration of a stratified, non-keratinized epithelium. Along with the tear film, the apical mucosa (composed of lipid, mucin, and aqueous layers) provides nutrition and lubrication to the ocular surface and is critical to the eye's first line of defense. Eyes treated with hrDecolin showed the most improved restoration to normal anatomy, with reduced stromal edema, thickness, and extracellular matrix deposition, accompanied by improved epithelial morphology. The reduction of fibrotic markers by hrDecolin has previously been shown across numerous animal models, modulating various growth factors (e.g., VEGF, IGF-1, EGF, PDGF) and their receptors, particularly TGFβ signaling via the SMAD2 and 3 pathways, and preventing corneal fibroblast differentiation. Additionally, modulation of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) results in fibrolysis and attenuated scar formation. [29、46~48] .

[0187] The inherent ability of hrDecolin to aid healing, particularly reducing scarring, is enhanced by the introduction of a fluid gel carrier, improving retention time on the ocular surface. The benefits of this fluid gel formulation have been clearly demonstrated in vivo, observed both physically with reduced corneal opacity and pharmacologically with reduced fibrosis markers. However, due to regulatory constraints, data generated during this study are limited to the 16-day time point. However, it would be interesting to investigate later time points in future studies.

[0188] The effect of the fluid gel alone on the injured corneal surface suggests an influence over endogenous growth factors, an effect enhanced by the addition of hr-decorin. The fluid-gel may aid corneal healing through several mechanisms: first, its unique viscoelastic properties allow it to act as a self-assembling liquid on the ocular surface, forming a semi-solid, therapeutic, occlusive dressing for stable healing; second, the helical domains formed during gelation of the fluid gel provide a pseudo-scaffold for endogenous decorin to bind and sequester key growth factors, such as TGF-β and / or exogenously delivered hr-decorin; and third, the fluid gel matrix, which is primarily water (99.1%), creates a gradient-driven diffusion of cytokines out of the wound site, restoring the natural equilibrium needed to prevent fibrosis again.

[0189] In conclusion, we demonstrated that a novel eye drop technology can be used to achieve localized, sustained delivery of antifibrotic drugs, such as hrDecolin, to the cornea in a clinically relevant mouse model of bacterial keratitis-associated fibrosis. The eye drop maintains hrDecolin in contact with the ocular surface for a sufficient length of time and with sufficient potency to significantly reduce corneal scarring. Furthermore, this study demonstrates that unfilled fluid gels can also have healing effects in their own right, likely through their inherent material microstructure and subsequent properties. Not only do the material properties of the eye drop enhance the retention time of the anti-scarring drug, but the readily available nature of the eye drop will be welcomed by patients and provide a simple treatment for preventing the scarring pathology that frequently occurs after corneal infection. The successful reduction of corneal opacity and markers commonly indicative of the scarring process has been demonstrated, making this technique an ideal treatment option for patients with bacterial keratitis, reducing the incidence of visually significant corneal opacities and potentially eliminating the need for corrective surgery compared to the current standard of care. Given that the availability of transplants and facilities for surgical procedures are often unavailable in developing countries, it is believed that this technique could help save the sight of many patients in the future.

[0190] Materials and Methods Study Design The purpose of this study was to investigate the use of a novel fluid gel for the delivery of decorin to the ocular surface to reduce corneal opacity and scarring following bacterial keratitis. The study used a mouse model of Pseudomonas keratitis (eyes were sterilized after infection) and compared it with the current standard of care. It was divided into three evaluation phases: (i) material properties for ease of eye drop application, (ii) in vitro evaluation of the bioactivity of formulated hr-decorin, and (iii) in vivo anti-scarring efficacy of the fluid gel with and without hr-decorin. Because the effect size was unknown, sample size (n = 6 per experimental group) was based on a resource equation. All analyses were performed by an observer blinded to the experimental grouping, and mice were randomly assigned to both treatment and control groups.

[0191] material Fabrication of fluid gel (FG) and hr-decorin fluid gel (DecFG) Preparation of fluid gel eye drops Fluid gels were prepared by first dissolving low-acyl gellan gum (Kelco gel CG LA, Azelis, UK) in deionized water. Gellan powder was added to deionized water at ambient temperature in the correct ratio to obtain a 1% (w / v) solution. The sol was heated to 70°C on a hot plate equipped with a magnetic stirrer under stirring until all the polymer was dissolved. Once dissolved, the gellan sol was added to the cup of a rotational rheometer (AR-G2, TA Instruments, UK) equipped with a cup and blade geometry (cup: 35 mm diameter, blade: 28 mm diameter). The system was then cooled to 40°C. hr decorin (Galacorin™; Catalent, USA) (4.76 mg / ml) in PBS and aqueous sodium chloride (0.2 M) were then added to give a final concentration of 0.9% (w / v) gellan, 0.24 mg / ml hr decorin, and 10 mM NaCl. Following this, the mixture was cooled under shear (450 / s) at a rate of 1°C / min to a final temperature of 20°C. Samples were then removed and stored at 4°C until further use. For fluid gels without hr decorin, the ratio was adjusted so that the final eye drops had a composition of 0.9% (w / v) gellan, 10 mM NaCl.

[0192] Characterization of fluid gel eye drop materials Microscopy: For transmission microscopy, samples were first diluted with polyethylene glycol 400 (PEG400) at a ratio of 1:4 (eye drops to PEG400). Following this, samples were analyzed using an Olympus FV3000. Images were processed using ImageJ (http: / / imagej.nih.gov / ij / ; provided in the public domain by the National Institutes of Health, Bethesda, MD, USA).

[0193] For scanning electron microscopy, samples were first prepared for lyophilization by diluting gellan in deionized water at a 1:9 ratio in the same manner as for transmission microscopy. The samples were then rapidly frozen using liquid nitrogen and placed in a freeze dryer overnight, leaving a powder. The dried samples were then adhered to carbon stubs and analyzed using SEM.

[0194] Rheology: Viscosity profiles were obtained at 20 °C using an AR-G2 (TA Instruments, UK) rheometer equipped with sandblasted parallel plates (40 mm, 1 mm gap height). A 2 min equilibration was used to ensure a constant test temperature. Following this, a time-dependent up-down ramp was applied ranging from 0.1 to 600 / s (sweep time 3 min). Recovery profiles were obtained using the same instrument at a single frequency. Samples were subjected to rejuvenation by shearing at 600 / s for 10 s. Following this, storage and loss (G', G"), respectively, were monitored at 1 Hz and 0.5% strain. The crossover point was used as the point at which the sample began to behave like a viscoelastic solid.

[0195] Release of hr-decorin from fluid gels The level of hr-decorin released from the gel was determined cumulatively by placing 1 ml of fluid gel containing hr-decorin in a 6-well plate. 2 ml of DMEM was then placed on the sample, and the plate was incubated at 37°C. At each time point, the medium was removed and replaced with fresh medium for hr-decorin measurement. Decorin release was quantified using a human decorin-specific ELISA (R&D Systems, Minneapolis, USA) according to the manufacturer's protocol.

[0196] In vitro hr decorin bioactivity assay Collagen fibrillogenesis: For dose-response curves, 75 μl of PBS was added to each well of a 96-well plate placed on ice. Various hr decorin doses were prepared by adding 400 μg / ml of hr decorin to the first well and then serially diluting (2-fold) across the plate. Following dilution, an additional 150 μl of PBS buffer was added to each well. Next, 75 μl of type I collagen (rat tail; Corning, UK) (800 μg / ml) was added to each well and incubated for 2 hours at 37°C. Absorbance readings were then taken using a 405 nm plate reader. Each assay consisted of duplicate blank controls and triplicate standard dilutions, followed by triplicate sample dilutions. Fibril formation kinetics was determined using a similar setup to the dose-response, but without serial dilutions, by incubating samples in the plate reader and taking data points every 2 minutes.

[0197] Pseudomonas keratitis model and in vivo stereomicroscopy The treatment dosing regimen for the in vivo Pseudomonas model is shown in Figure 6. Groups of untreated intact and infected corneas taken on day 2 were also included in the experimental design. Due to unknown effect sizes, a sample size of n=6 for each control or treatment group was determined based on the resource equation:

[49] Mice were randomly assigned to each treatment and control group and then infected with Pseudomonas. Each treatment procedure and sample size are described in further detail below. For in vivo studies, analyses were performed by investigators blinded to the experimental groups.

[0198] In vivo mouse model of Pseudomonas keratitis P. aeruginosa strain PAO1 was grown in high-salt LB (supplemented with 10 g tryptone, 5 g yeast extract, and 11.7 g NaCl, 10 mM MgCl, and 0.5 mM CaCl per liter) at 37°C for 18 hours. Subcultures were obtained at an optical density (OD) of 0.2 (OD 650 nm, approximately 1 × 10 8 P. aeruginosa was washed in PBS (x3), centrifuged at 300 rpm for 5 minutes, and diluted to 1 x 10 CFU / ml in PBS.5 CFU / 2.5 μl were resuspended. C57BL / 6 mice (Jackson Laboratory, CA, USA) were housed in pathogen-free conditions with free access to water and food in accordance with the ARRIVE guidelines, the ARVO statement for the use of animals in ophthalmic and vision research, and in compliance with guidelines set forth by the University of California, Irvine. For inoculation, mice were anesthetized, and one corneal epithelium was scraped with a 26G needle in a 3 × 1 mm parallel scratch, and 2.5 μl of Pseudomonas aeruginosa (1 × 10 5 CFU) (PAO1 strain) 64、65 Mice were allowed to rest and recover for 2 hours after inoculation to allow the infection to penetrate the eye. After 24 hours, conscious mice were treated with 5 μl of gentamicin (1.5%, QEHB Pharmacy, Birmingham, UK) every 2 hours for 12 hours to sterilize the infection. After another 12 hours, mice received eye drops (5 μl of each compound) every 4 hours between 8:00 AM and 8:00 PM for an additional 13 days, depending on the treatment group: (1) gentamicin + prednisolone (0.5%, QEHB Pharmacy), (2) gentamicin + prednisolone + fluid gel, or (3) gentamicin + prednisolone + fluid gel with hrDecolin. Mice were examined for corneal opacification, ulceration, and perforation. En-face 24-bit color photographs of the cornea were taken with a SPOT RTKE camera (Diagnostic Instruments) interfaced with a Leica MZF III stereomicroscope. Mice were euthanized by cervical dislocation under anesthesia on day 16, and eyes were enucleated and placed in 4% PFA in PBS for processing for immunohistochemistry.

[0199] Quantification of opacity Two blinded independent ophthalmologists analyzed all photographs in the same randomized order (the order was provided by an independent statistician) for the area of ​​opacification using ImageJ. The definitions of corneal opacification, sufficient, and insufficient images were agreed upon by the observers before the start of image analysis. Measurements were reported in mm 2 The area was recorded ± SEM. The randomized order required that there should be no temporal trends in the areas measured.

[0200] Tissue processing and immunohistochemical staining for re-epithelialization and ECM Eyes enucleated for IHC were postfixed overnight in 4% PFA in PBS at 4°C and then cryoprotected using increasing concentrations of sucrose in PBS (10%, 20%, and 30%; Sigma) for 24 hours at 4°C. Eyes were then embedded in optimal cutting temperature (OCT) embedding medium (Thermo Shandon, Runcorn, UK) in peel-away mold containers (Agar Scientific, Essex, UK) and later sectioned at 15 μm thickness in the parasagittal plane at −22°C using a cryostat microtome (Bright, Huntingdon, UK) and placed on Superfrost slides (Fisher Scientific, USA). Central sections (in the optic nerve plane) were used for all IHC studies and stored at −80°C. Frozen sections (optic nerve plane) were thawed for 30 min, washed 3 times with PBS for 5 min, and then permeabilized for 20 min with 0.1% Triton X-100 (Sigma). Nonspecific antibody binding sites in the tissue sections were blocked for 30 min with 0.5% BSA, 0.3% Tween-20 (all from Sigma), and 15% normal goat serum (Vector Laboratories, Peterborough, UK). Then, the sections were incubated overnight at 4°C in primary antibodies (αSMA, laminin, and fibronectin; 1:200; all from Sigma), washed again 3 times for 5 min, and incubated for 1 h at room temperature with secondary antibodies (goat anti-mouse Alexa Fluor 488 1:500, goat anti-mouse Alexa Fluor 594 1:500; Molecular Probes, Paisley, UK). Sections were then washed 3 x 5 min and mounted in Vectorshield mounting medium containing DAPI (Vector Laboratories). Control tissue sections incubated with secondary antibody alone were all negatively stained.

[0201] Imaging and quantification of immunohistochemical staining After IHC, sections were imaged at ×20 on a Zeiss Axioscanner fluorescence microscope (Axio Scan.Z1, Carl Zeiss Ltd.) using the same exposure time for each antibody. IHC staining was performed according to previously described methods. 61 , quantification was performed by measuring pixel intensity. Briefly, the region of interest used for ECM IR quantification was defined as a region of interest of the same defined size in the stroma for all eyes / treatments. Each stroma had a total of 30 individual intensity measurements (region of interest) taken to encompass the entire area. ECM deposition was quantified in these defined regions of interest, and the percentage of IR pixels exceeding a standardized background threshold from intact corneas was calculated using ImageJ. For each antibody, a threshold level of brightness in the stroma region was established using intact, untreated corneas to define the reference level for test group analysis. Images were assigned randomized filenames to ensure blinding of the assessors to the treatment groups.

[0202] statistical analysis All statistical analyses were performed using SPSS 20 (IBM, Chicago, IL, USA). Normal distribution tests were performed to determine the most appropriate statistical analysis for comparing treatments. Statistical significance was determined at P < 0.05. For opacity measurements, corneal width, epithelial thickness, αSMA, fibronectin, and laminin data were evaluated using ANOVA with Tukey's post-hoc test. For DAPI measurements of epithelial cell layer number, the Kruskal-Wallis test was used because the data were not normally distributed.

[0203] [Table 1]

[0204] Further technical information 1. List of biopolymers:

[0205] [Table 2]

[0206] 1. Material properties of "fluid gel" (microparticle suspension): Viscosity / flow behavior The optimal eye drop viscosity was determined by two main methods: rheological characterization of current commercially available eye drops / ointments and consultation with ophthalmic clinicians. Characterization of commercially available ophthalmic products highlights the large range of viscosities across both eye drops and ointments used to medicate conditions such as dry eye, with optimally long retention times required. Viscosity was collected and 1s -1 (selected as values ​​during the first stage of shear thinning to avoid instrumental artifacts) (Table 2 and Figure 6 (section A.1.)), highlighting the similar viscosities between the products as a function of polymer, which were mainly made based on paraffin, carbomer and biopolymer.

[0207] [Table 3]

[0208] For both paraffin- and carbomer-based ophthalmic products, warnings are provided in the instructions to inform patients that the drops may cause blurring and discomfort. Therefore, outer limits for the viscosity of the formulations were set based on values ​​obtained for these products. Maximum -200 Pa.s; and minimum 4 Pa.s

[0209] In all cases where all formulations were tested, these values ​​were not exceeded. Thus, all formulations prepared using gellan as the biopolymer used for gelation could be used within these limits. However, a more optimal formulation in terms of viscosity was narrowed down with the help of clinical advice.

[0210] A panel of different formulations was created and clinicians were asked to handle the products and rate them for a possible ophthalmic solution product. 5~50Pa.s Eye drops in the viscosity range are more easily applied, retain better, Approximately 10~20Pa.s It was found that the optimum fall was

[0211] Furthermore, the system should exhibit shear thinning behavior.

[0212] 1.1.1. Defined parameters

[0213] [Table 4]

[0214] Elasticity Elasticity at rest plays a major role in the use of products to retain and deliver active substances in a controlled manner. The ability of microgel suspensions to create a weak elastic network at rest is believed to cause high retention times for the products. Again, the limits are based on the characterization of commercially available eye drops and ointments (Figure 3 (section A.1.)). As seen for viscosity, similar correlations were observed between the various products, grouped into polymer types (Table 4).

[0215] [Table 5]

[0216] Again, similar to viscosity, for all formulations tested, the values ​​obtained for the current product were not exceeded, thus all formulations prepared with gellan as the biopolymer used for gelation could be used within these limits. Maximum -20000Pa; and minimum 1Pa

[0217] However, when analyzed by a clinician, this 1~250Pa The optimal formulation is 20~40Pa It ranged from.

[0218] 1.1.2. Defined parameters

[0219] [Table 6]

[0220] pH Due to the chemical composition of biopolymers and the various chemical moieties along their individual backbones, they have different natural pHs. The pH of the product that will come into contact with the ocular surface is important, as numerous chemical lesions form in the pH range <4 and >10, with normal physiology approaching 7.11 ± 1.5. Therefore, eye drops are formulated within this range (4-10), with some products reaching a pH as low as 3.5 (e.g., proparacaine hydrochloride solution). 1 Therefore, based on this data from the literature, the eye drop formulation 3.5~8.6 The pH should be within the range of

[0221] However, the delivery of many active substances, including proteins, requires that the formulation be neutral. In these cases, PBS (phosphate buffered saline) can be added to the eye drops to limit the pH to neutral acidity. Therefore, in the formulation, pH 6.5~7.5 The optimized formulation is 7.4 is.

[0222] 1.1.3. Defined parameters

[0223] [Table 7]

[0224] 2. "Fluid gel" (microparticle suspension) formulation: Biopolymer concentration (See experimental article A.1)

[0225] Ultimately, the material properties of the formulation are controlled by the initial polymer concentration in the product. Therefore, upper and lower limits of polymer concentration were established, and upper and lower material properties were used to evaluate the material formulation. Since all systems exhibit shear thinning behavior, the limits were set based on viscosity (1 s -1 ) and elastic behavior at rest. 0.5 to 2.5% (w / v) A maximum range of values ​​for the eye drop formulations was established, and values ​​within these were observed for the commercial products. This is consistent with clinician advice. 0.5 to 1.5% (w / v) The formulation is narrowed down and optimized to 0.9% (w / v) It consists of:

[0226] 2.1.1 Defined Parameters

[0227] [Table 8]

[0228] Crosslinker concentration (See experimental article A.2)

[0229] Data obtained from the characterization of gellan formulations showed that salt content does not affect the viscosity of the systems, but does have an effect on the elastic response of the gel at rest. Again, none of the formulated systems exceeded the upper and lower limits set by the commercial products; therefore, the upper and lower concentrations were: 5 to 40 mM It was defined as follows:

[0230] However, mechanical spectroscopy showed that at higher salt concentrations, a significant decrease in the elastic network occurs when deforming outside the linear viscoelastic region. This suggests that lower salt concentrations result in more plastic behavior, which may be more comfortable for patients. Therefore, the narrower limits for the formulation are: 5 to 20 mM The adjusted and optimized formulation is 10 mM.

[0231] PBS The addition of PBS can be used to manipulate the pH of the system. In these cases, 5% v / v (5% was determined as the amount added with the therapeutic decorin, so no further testing was performed in this area) was added, which will affect the level of salt in the system. The concentrations of monovalent ions in PBS were calculated and are summarized in Table 8.

[0232] [Table 9]

[0233] Therefore, the range for the crosslinker was modified (Table 9), and the lower limit was reduced so that the ionic content in PBS was sufficient to trigger the gelation process.

[0234] 2.1.3. Defined Parameters

[0235] [Table 10]

[0236] 3. "Fluid Gel" (microparticle suspension) processing parameters: Heat Treatment Thermal treatment during manufacturing is crucial for gel formation. Typically, thermal parameters fall into two categories: processing temperature and cooling rate.

[0237] 4.1.1. Processing temperature: The inlet and outlet are key to ensuring that the polymer is a sol prior to processing and exits below the gelation transition. Initially, due to the protein active substance actively denaturing at higher temperatures, the inlet temperature was set as close to the gelation temperature as possible. Therefore, the inlet temperature was set at 40°C. However, this is not necessary; the key aspect of the inlet temperature is to maintain it above the gelation temperature to prevent premature gelation and blockage. The function of the outlet temperature is to ensure that the polymer ordering / structuring is complete before storage. This prevents aggregation and the formation of heterogeneous suspensions during this stage. Therefore, for gellan, this temperature is defined as 20°C, allowing the polymer to go through the gelation process. The outlet temperature is therefore controlled by the mill jacket and set to provide sufficient cooling during processing. This can be varied to provide various cooling rates.

[0238] 4.1.2. Cooling rate (See experimental article A.3) It is known that the cooling rate during the sol-gel transition is very important with respect to the final material properties, as a higher cooling rate leads to rapid formation of structure and a weaker overall elastic modulus. This is observed for microgel suspensions, but only at higher polymer concentrations. For the optimal eye drop formulation, no change in material properties is observed. 0.1~6℃min -1 It was suggested that a large range of parameters could be used.

[0239] On the other hand, at 1.8% w / v polymer, there was more reliance on the required elastic structure.

[0240] 4.1.3. Defined Parameters

[0241] [Table 11]

[0242] 4.2 Shear rate (See experimental article A.3)

[0243] The shear rate during processing showed very similar results for the cooling rate, and the optimized polymer concentration was not affected by the processing shear. Again, higher concentrations showed a dependency. Therefore, for the optimized formulation, 50-2000 rpm (device limit) A very wide range of shear can be applied, 500~1500rpm In the optimized configuration, 1000 rpm (to prevent stress on processing equipment) is.

[0244] 4.2.1 Defined Parameters

[0245] [Table 12]

[0246] 5.Suspension parameters summary:

[0247] [Table 13]

[0248] Further experimental data A.1. Experimental - Gellan Concentration: Effect of Polymer Concentration on the Response of the Resulting Fluid Gel Material the purpose: · Understand how polymer concentration affects key material properties (viscosity and elasticity) following processing into microgel suspensions. Narrowing down the polymer concentration tolerances for suitable eye drop formulations.

[0249] Materials and Methods: material: Gellan (Kelco) NaCl (Fisher Chemicals, Lot No. 1665066)

[0250] Preparation of gellan microgel suspension (MS): Preparation of stock solutions: Preparation of NaCl solution NaCl (0.2 M) was prepared by adding dried crystals (1.16 g) to deionized water (100 ml) using a volumetric flask. The NaCl was then dissolved using an inverting technique to aid processing. Once fully dissolved, the solution was kept at ambient conditions until further use.

[0251] Preparation of gelansol: Gelan sols were prepared by dissolving powdered polymer in various ratios in water / NaCl solutions to achieve final concentrations equal to 0.5, 0.9, 1.35, 1.8, and 2.35% (w / v) after processing. Briefly, gellan powder was weighed out (2.5, 4.5, 6.75, 9.0, and 11.75 g) and added to 450 ml of deionized water. The mixture was heated to 95°C under stirring to dissolve the polymer. Once fully dissolved, 25 ml of NaCl stock solution (0.2 M) was added to the solution to achieve a concentration of 10 mM after processing. The sol was then allowed to reach thermal equilibrium at 95°C before processing.

[0252] Preparation of gellan MS: MS was prepared using a jacketed pin grinder set at 20°C. Gellan sol was pumped into the pin grinder at 3 ml / min using a peristaltic pump, entering the processing chamber at 40°C. Water was injected into the gellan stream (at a rate of 0.16 ml / min) prior to entry using a syringe and syringe pump, causing them to collide, diluting the gellan sol to final concentrations (0.5, 0.9, 1.35, 1.8, and 2.35% (w / v), 10 mM NaCl). The mixture was then cooled under shear (500 rpm or 1000 rpm) as it passed through the grinding unit. Upon exiting at 20°C, the gels were packaged and stored at 4°C until further testing.

[0253] Material analysis: Fluid measurement method: All samples were tested at 20 °C using a rheometer (TA, AR-G2) equipped with sandblasted parallel plates (40 mm diameter, 1 mm gap height). The results are shown in Figures 7 to 9.

[0254] Amplitude Sweep: Amplitude sweeps were obtained in strain-controlled mode over the range 0.1 to 100.0%. The sample was loaded into the instrument and the upper geometry was lowered. Once trimmed, the sample was equilibrated at 20°C before testing. Measurements were obtained logarithmically at 1 Hz.

[0255] Flow Profile: Viscosity profiles for the samples were obtained using a continuous ramp. The sample was loaded onto the instrument and the upper geometry was lowered. Once trimmed, the sample was allowed to equilibrate at 20°C before testing. Increasing shear was applied in rate-controlled mode from 0.1 to 600 s. -1 A 3 minute ramp between was applied to the sample and data points were acquired using logarithmic scale.

[0256] result: Small deformation rheology: See Figures 7-9 and the discussion above. Large Deformation Rheology: See Figures 10-12 and the discussion above.

[0257] Consideration: The effect of polymer concentration can be observed on both these elastic properties and viscosity, with both properties following the same trend, increasing until reaching a plateau at concentrations above 1.8% (w / v) (Figures 2 and 3). Such observations arise from the formation of microgelled particles; by applying shear during the gelation of the polymer system, confinement prevents the formation of a continuous network morphology. The primary consequence of this process is that gelled entities are dispersed in a non-gelling medium, similar to W1 / W2 emulsions. Thus, the rheology of suspensions also closely correlates with that of emulsions, with an increase in the droplet or particle phase volume (in this case) resulting in closer proximity and an increase in both the elastic properties (G') and viscosity of the system. In this case, increasing the polymer concentration leads to a greater number of particles until a maximum packing fraction is reached, beyond which no further changes in material properties are observed.

[0258] The elasticity (storage modulus, G') and viscosity of the various suspensions were compared with data collected for current eye drops / ointments across a range of materials: paraffin-, carbomer-, and biopolymer-based systems (Figures 3 and 6). All gellan systems were observed to exhibit G' and viscosities within the thresholds of current commercial ophthalmic products, suggesting that all systems would be suitable regardless of polymer concentration. However, for ease of application (from a single-use applicator) and comfort (blurred vision as described by the packaging), values ​​closest to carbomer- and biopolymer-based eye drops were optimal. Therefore, gellan concentrations ranging from 0.5 to 1.35% (w / v) were most suitable. Additionally, consultation with independent clinicians with an eye toward ophthalmic applications resulted in a concentration of 0.9% (w / v), which best mimicked the characteristics defined by the clinicians.

[0259] The yield behavior of a suspension is also very important, especially within a delivery system, because a rapidly yielding system results in rapid clearance. Conversely, if a system does not yield at all, the material will not be easily excreted from the body. The linear viscoelastic region (LVR) is a good indicator of the yield behavior of a suspension; as the system leaves this linear region, weak interparticle interactions begin to break down and the system begins to flow. The length of the LVR is observed as a function of polymer concentration and shows an inverse correlation with gellan content (Figure 1). Here, at lower polymer concentrations, the suspension can operate at higher strains before collapsing, providing a sealing barrier in dynamic regions of the body that will be slowly resorbed. Similar LVRs are observed in the 0.5–1.35% (w / v) range, suggesting that these will behave similarly.

[0260] The following yield shear thinning behavior is crucial for both application and drainage, allowing the suspension to flow easily upon liquefaction. Shear thinning was observed across all systems, regardless of polymer concentration (Figure 4). The high degree of shear thinning, which occurred through the breakdown of interparticle interactions and alignment during flow, allows the system to be easily applied through a nozzle (syringe, single-use applicator, etc.), with small pressures resulting in high levels of shear.

[0261] Conclusion: In summary, polymer concentration was shown to play a crucial role in the material properties of the resulting gellan microgel suspensions. Material characteristics, such as elasticity, as represented by the material's intrinsic G' value, and viscosity were found to increase as a function of polymer concentration until a plateau was formed at 1.8% (w / v). Effectively, this meant that, in close comparison with already commercially available products, all systems were suitable for application or injection in the ocular environment and exhibited the strong shear-thinning behavior required for extrusion through small orifices. Furthermore, by comparing with commercially available products and through discussions with independent clinicians, the polymer range between 0.5 and 1.35% (w / v) was narrowed down to 0.9% (w / v), which proved optimal for the final formulation.

[0262] A.2. Experimental - Crosslinker (NaCl) Concentration: Effect of Crosslinker Concentration on the Reaction of the Resulting Fluid Gel Material the purpose: · Understand how crosslinker concentration affects key material properties (viscosity and elasticity) following processing into microgel suspensions. Narrowing down the acceptable crosslinker concentrations for suitable eye drop formulations.

[0263] Materials and Methods: Gellan (Kelco) NaCl (Fisher Chemicals, Lot No. 1665066)

[0264] Preparation of gellan microgel suspension (MS): Preparation of stock solutions: Preparation of NaCl solution NaCl (0.1, 0.2, 0.4, and 0.8 M) was prepared by adding dried crystals (0.58, 1.16, 2.32, and 4.64 g) to deionized water (100 ml) using a volumetric flask. The NaCl was then dissolved using an inversion technique to aid processing. Once fully dissolved, the solution was kept at ambient conditions until further use.

[0265] Preparation of gelansol: Gellan solutions were prepared by dissolving powdered polymer in a water / NaCl solution to achieve final concentrations equal to 0.9% and 1.8% (w / v) after processing. Briefly, gellan powder was weighed out (4.5 g, 9.0 g) and added to 450 ml of deionized water. The mixture was heated to 95°C under stirring to dissolve the polymer. Once fully dissolved, 25 ml of NaCl stock solution (either 0.1, 0.2, 0.4, or 0.8 M) was added to the solution to achieve concentrations of 5, 10, 20, or 40 mM after processing. The sol was then allowed to reach thermal equilibrium at 95°C before processing.

[0266] Preparation of gellan MS: MS was prepared using a jacketed pin grinder set at 20°C. Gellan sol was pumped into the pin grinder using a peristaltic pump at 3 ml / min, entering the processing chamber at 40°C. Water was injected into the gellan stream (at a rate of 0.16 ml / min) prior to entry using a syringe and syringe pump, diluting the gellan sol to final concentrations (0.9% and 1.8% (w / v); 5, 10, 20, or 40 mM NaCl). The mixture was then cooled under shear (1000 rpm) as it passed through the grinding unit. Upon exiting at 20°C, the gels were packaged and stored at 4°C until further testing.

[0267] Material analysis: Fluid measurement method: All samples were tested at 20°C using a rheometer (TA, AR-G2) equipped with sandblasted parallel plates (40 mm diameter, 1 mm gap height).

[0268] Amplitude Sweep: Amplitude sweeps were obtained in strain-controlled mode over the range 0.1 to 100.0%. The sample was loaded into the instrument and the upper geometry was lowered. Once trimmed, the sample was equilibrated at 20°C before testing. Measurements were obtained logarithmically at 1 Hz.

[0269] Flow Profile: Viscosity profiles for the samples were obtained using a continuous ramp. The sample was loaded onto the instrument and the upper geometry was lowered. Once trimmed, the sample was allowed to equilibrate at 20°C before testing. Increasing shear was applied in rate-controlled mode from 0.1 to 600 s. -1 A 3 minute ramp between was applied to the sample and data points were acquired using logarithmic scale.

[0270] result: Small deformation rheology: see Figures 13-14. Large deformation rheology: see Figures 15-16.

[0271] Consideration: Mechanistically, salts play an important role in the gelation of many polymers, including gellan. The type of salt, particularly the valence (mono, di, tri, etc.), is crucial for the properties of the resulting gel; typically, increasing the valence increases the strength of the gel, as more crosslinks are formed between the polymers. However, in the case of gellan, divalent ions, such as Ca, 2+ leads to cloudiness (increased turbidity) of the resulting gel. + can be used to strengthen the interhelix binding sites and form a three-dimensional gel structure. Thus, the strength of the resulting gel is a function of the concentration of the added salt, also referred to as the crosslinker. The effect of crosslinker concentration on the resulting microgel suspension ("fluid gel") formed through shear gelation can be clearly seen in Figures 1 and 2. Here, a correlation between NaCl concentration and elastic (G') response can be observed, consistent with the known gelation mechanism (increased strength for higher crosslinker concentrations) for both polymer concentrations tested (Figure 2). Furthermore, mechanical spectra (Figure 1) highlight the change in the material's yield characteristics. At the highest salt concentration (40 mM), the strain dependence of the material was observed to increase, exhibiting a more rapid decrease in G' upon leaving the LVR (linear viscoelastic region). Such results closely match typical material behavior, where gels become stronger and more brittle. In these cases, as the system becomes more densely crosslinked, the material is expected to behave more like fracture as it reaches a critical strain, as opposed to deforming plastically. Higher concentrations are diffusible, but the enhanced strain dependence precludes their use in ophthalmic applications; increased plasticity upon deformation leads to smoother surfaces, predictably improving clarity and comfort.

[0272] The effect of salt concentration on the viscosity of the eye drops was also examined. Little change was observed across all systems (Figure 2), and all formulations exhibited significant shear-thinning behavior, with the overall viscosity ultimately dependent on the biopolymer concentration. However, with the 0.9% (w / v) gellan suspension, which had the highest salt concentration (40 mM), the overall viscosity of the suspension was lower. Such results are accompanied by increased error, which may result from some degree of syneresis (water expulsion), where the increased crosslinker density pulls the polymers closer together, resulting in insufficient polymer to structure the aqueous phase. Therefore, the stability of these systems may be compromised, resulting in inhomogeneous systems over time.

[0273] Conclusion: In summary, the addition of salt to biopolymer systems results in manipulations beyond the strength of the final product. Increasing salt concentration ultimately increases the number of crosslinks in the system and the elastic behavior of the final material. Additionally, while such effects did not appear to have a dramatic effect on viscosity, at lower polymer concentrations, excessive crosslinking could result in inhomogeneous suspensions and poor stability. Demonstrating elastic structure using strain sweeps allowed the yield behavior of the suspensions to be analyzed, highlighting the higher strain dependence in the 40 mM formulation. A decrease in plasticity would be expected to cause patient discomfort in ocular applications, and therefore an upper limit of 20 mM crosslinker is suggested.

[0274] A.3. Experimental - Cooling Rate: Effect of Cooling Rate Applied During Processing on the Production of Gellan Microgel Suspensions ("Fluid Gels") the purpose: · Understand the role that cooling rate plays on the material properties (viscosity and elasticity) of the resulting gellan microgel suspension. Narrow down the cooling rate suitable for processing eye drops to the allowable amount.

[0275] Materials and Methods: Gellan (Kelco) NaCl (Fisher Chemicals, Lot No. 1665066)

[0276] Preparation of gellan microgel suspension (MS): Preparation of stock solutions: Preparation of NaCl solution NaCl (0.2 M) was prepared by adding dried crystals (1.16 g) to deionized water (100 ml) using a volumetric flask. The NaCl was then dissolved using an inversion technique to aid processing. Once fully dissolved, the solution was kept at ambient conditions until further use.

[0277] Preparation of gelansol: Gellan solutions were prepared by dissolving powdered polymer in a water / NaCl solution to equal final concentrations of 0.9% and 1.8% (w / v) after processing. Briefly, gellan powder was weighed out (4.5 g, 9.0 g) and added to 475 ml of deionized water. The mixture was heated to 95°C under stirring to dissolve the polymer. Once fully dissolved, 25 ml of NaCl stock solution (0.2 M) was added to the gellan sol to obtain a final concentration of 10 mM. The sol was then allowed to reach thermal equilibrium at 95°C before processing.

[0278] Preparation of gellan MS: MS was prepared using a jacketed pin mill, whereby the jacket temperature and residence time in the mill were varied at 1, 3 and 6°C min. -1 As an example, the jacket was set at 5°C and 20 ml of -1 The temperature of the fluid is 46°C at the inlet and 16°C at the outlet, and the residence time at this velocity is 5 minutes. Therefore, the cooling rate is 6°C / min. -1 Upon exit, the gels were packaged and stored at 4°C until further testing.

[0279] Material analysis: Fluid measurement method: All samples were tested at 20°C using a rheometer (TA, AR-G2) equipped with sandblasted parallel plates (40 mm diameter, 1 mm gap height).

[0280] Amplitude Sweep: Amplitude sweeps were obtained in strain-controlled mode over the range 0.1 to 100.0%. The sample was loaded into the instrument and the upper geometry was lowered. Once trimmed, the sample was equilibrated at 20°C before testing. Measurements were obtained logarithmically at 1 Hz.

[0281] Flow Profile: Viscosity profiles for the samples were obtained using a continuous ramp. The sample was loaded onto the instrument and the upper geometry was lowered. Once trimmed, the sample was allowed to equilibrate at 20°C before testing. Increasing shear was applied in rate-controlled mode from 0.1 to 600 s. -1 A 3 minute ramp between was applied to the sample and data points were acquired using logarithmic scale.

[0282] result: Small deformation rheology: see Figures 17 and 18. Large deformation rheology: see Figures 19 and 20.

[0283] Consideration: Cooling plays a key role in the formation of gellan hydrogels, driving the polymer from a random coil to a helix transition. To assess the associated changes in material behavior, the effect of cooling rate on the formation of fluid gels was examined. At lower polymer concentrations (0.9% w / v), cooling rate was observed to have little effect on both the degree of elasticity and overall viscosity of the system. However, at higher concentrations (1.8% w / v), cooling rate had a much more pronounced effect on the elastic modulus (G') (Figure 2). At higher polymer concentrations, particles are likely held much closer together and thus are much more strongly affected by particle deformation. A slower cooling rate allows particles to form much more slowly, resulting in a more ordered and robust structure. While little effect was observed on viscosity, particles were characterized as "squeeze past" each other at the microscale, suggesting that they interact to a similar degree.

[0284] The data obtained suggests an increased degree of control over material properties at higher polymer concentrations. It is possible to engineer specific elastic properties into the system without changing the overall viscosity. It is important to be able to place semi-solid-like structures in situ that provide a barrier or long-term retention in delivery systems to various regions of the body. Furthermore, the ability to maintain the same viscosity means that the system is still injectable, even though it behaves more like a solid at rest.

[0285] Conclusion: The effect of cooling rate was found to be dependent on polymer concentration, and the optimized eye drop formulation was found to be independent of the applied cooling rate. However, at higher concentrations, the elastic structure can be fine-tuned without affecting the viscosity profile. Thus, the degree of solidity at rest can be manipulated, yet the system remains flowable (injectable) upon larger deformations.

[0286] A.4. Experimental - Mixing Rate Applied during Processing: Effect of Mixing Rate Applied During Processing on the Formation of Gellan Microgel Suspensions ("Fluid Gels") the purpose: · Understand the role that mixing speed during processing plays on the material properties (viscosity and elasticity) of the resulting gellan microgel suspension. Refine the mixing speed during processing to a suitable tolerance for eye drop formulations.

[0287] Materials and Methods: Gellan (Kelco) NaCl (Fisher Chemicals, Lot No. 1665066)

[0288] Preparation of gellan microgel suspension (MS): Preparation of stock solutions: Preparation of NaCl solution NaCl (0.2 M) was prepared by adding dried crystals (1.16 g) to deionized water (100 ml) using a volumetric flask. The NaCl was then dissolved using an inversion technique to aid processing. Once fully dissolved, the solution was kept at ambient conditions until further use.

[0289] Preparation of gelansol: Gellan solutions were prepared by dissolving powdered polymer in a water / NaCl solution to equal final concentrations of 0.9% and 1.8% (w / v) after processing. Briefly, gellan powder was weighed out (4.5 g, 9.0 g) and added to 450 ml of deionized water. The mixture was heated to 95°C under stirring to dissolve the polymer. Once fully dissolved, 25 ml of NaCl stock solution (0.2 M) was added to the solution to achieve a concentration of 10 mM after processing. The sol was then allowed to reach thermal equilibrium at 95°C before processing.

[0290] Preparation of gellan MS: MS was prepared using a jacketed pin grinder set at 20°C. Gellan sol was pumped into the pin grinder at 3 ml / min using a peristaltic pump, entering the processing chamber at 40°C. Water was injected into the gellan stream (at a rate of 0.16 ml / min) prior to entry using a syringe and syringe pump, diluting the gellan sol to final concentrations (0.9 and 1.8% (w / v), 10 mM NaCl). The mixture was then cooled under shear (100, 500, 1000, and 2000 rpm) as it passed through the grinding unit. Upon exiting at 20°C, the gel was packaged and stored at 4°C until further testing.

[0291] Material analysis: Fluid measurement method: All samples were tested at 20°C using a rheometer (TA, AR-G2) equipped with sandblasted parallel plates (40 mm diameter, 1 mm gap height).

[0292] Amplitude Sweep: Amplitude sweeps were obtained in strain-controlled mode over the range 0.1 to 100.0%. The sample was loaded into the instrument and the upper geometry was lowered. Once trimmed, the sample was equilibrated at 20°C before testing. Measurements were obtained logarithmically at 1 Hz.

[0293] Flow Profile: Viscosity profiles for the samples were obtained using a continuous ramp. The sample was loaded onto the instrument and the upper geometry was lowered. Once trimmed, the sample was allowed to equilibrate at 20°C before testing. Increasing shear was applied in rate-controlled mode from 0.1 to 600 s. -1 A 3 minute ramp between was applied to the sample and data points were acquired using logarithmic scale.

[0294] result: Small deformation rheology: see Figures 21 and 22. Large deformation rheology: see Figures 23 and 24.

[0295] Consideration: The degree of shear applied throughout the sol-gel transition of gellan biopolymer was tested at two concentrations: 0.9% (w / v) and 1.8% (w / v). At the lower polymer concentrations, both the elasticity and viscosity, as defined by G', were independent of the degree of shear experienced during the gelation profile. In all cases, the resulting materials exhibited shear-thinning properties over large deformations and solid-like behavior at rest, although the magnitude of such results remained unchanged (Figures 2 and 4). The same was observed for the viscosity profile of the 1.8% (w / v) system, where increased viscosity was observed compared to the 0.9% (w / v) system, but these were independent of the shear applied during processing. However, the elastic properties of the system at rest showed a dependence, with the final storage modulus (G') decreasing as shear increased. It is believed that increased mixing applied during the gelation process directly affects the microstructure of individual particles, and the increased level of confinement prevents the growth of more rigid particles. As a result, the particles are more deformable and G' is lower.

[0296] Conclusion: In summary, varying the mixing speed during processing does not play a significant role in the material properties of the resulting low polymer concentration microgel suspension. Therefore, a wide range of shear treatments can be applied without changing the final properties of the eye drop formulation. However, for the higher concentrations used for "cream-like" spreadable systems, shear treatment plays a more important role in the degree of solid-like behavior at rest. In these cases, the degree of elasticity can be manipulated for the intended use.

[0297] Further experimental information: Formation and properties of fluid gels the purpose: · To demonstrate the ability to prepare fluid gels from a variety of starting biopolymers (gellan, kappa-carrageenan, alginate, agar) using different gelation mechanisms at various concentrations. · To demonstrate the material properties of these fluid gels.

[0298] Materials and Methods: Preparation of fluid gel: The fluid gel was prepared as follows.

[0299] Gellan (thermal gelling): · Addition of gellan powder to water with 5% PBS to form a 0.5-2% polymer solution. The solution is heated above the gel point. Add cross-linker (sodium chloride (final concentration 10 mM)) The solution is cooled through the gel point (approximately 38°C) under constant shear.

[0300] Kappa-carrageenan (thermal gelling): · Addition of gellan powder to water with 5% PBS to form a 0.5-2% polymer solution. The solution is heated above the gel point. Add cross-linker (potassium chloride (final concentration 10 mM)) The solution is cooled through the gel point (approximately 40°C) under constant shear.

[0301] Alginate (ionotropic gelation): · Addition of alginate powder to water with 5% PBS to form a 0.5-1% polymer solution. · Fully hydrate the polymer (if heat assisted and cooled to room temperature). · Add the crosslinker (calcium chloride added (10 mM final concentration)) slowly using a syringe and needle with constant shear.

[0302] Agar (thermal gelation with hysteresis (melting and gelling points are not the same)): · Addition of agar powder to water with 5% PBS to form a 0.5-2% polymer solution. The solution is heated above the gel point (above 90°C). Add cross-linker (sodium chloride (10 mM final concentration)) The solution is cooled through the gel point (approximately 36°C) under constant shear.

[0303] Rheological testing: All samples were tested at 20°C using a rheometer equipped with serrated parallel plates (40 mm diameter, 1 mm gap height).

[0304] Amplitude Sweep: Amplitude sweeps were obtained over the range 0.1 to 500.0% in strain-controlled mode. Once mounted, the samples were equilibrated at 20°C before testing. Measurements were taken logarithmically at 1 Hz.

[0305] Frequency Sweep (Figure 27): A frequency sweep was performed at strains in the linear viscoelastic region of the amplitude sweep. Once mounted, the samples were equilibrated at 20°C before testing. The samples were tested logarithmically between 0.01 and 10 Hz.

[0306] Flow profile (Figure 28): Viscosity profiles for the samples were obtained using a continuous ramp. The sample was loaded into the instrument and allowed to equilibrate at 20°C before testing. Increasing shear, speed control mode, 0.1~600s -1 A 3 minute ramp between was applied to the sample and data points were acquired using logarithmic scale.

[0307] result: Figures 26-28 show amplitude sweep, frequency sweep and viscosity sweep data obtained for agar, gellan, kappa carrageenan and alginate.

[0308] Consideration: The data obtained showed the following: All systems were shown to exhibit mechanical properties typically associated with fluid gels: weak solid-like behavior at rest (frequency sweep); breakdown of solid behavior with applied strain, yielding (amplitude sweep); and shear-thinning behavior (viscosity profile). The data show that fluid gels can be made using a variety of different biopolymers, including gellan.

[0309] The data show that various gelation mechanisms can be used to fabricate fluid gels. a. thermally driven process (gellan, agar, K-carrageenan); b. Ionotropic gelation - gelation through cross-linking via ionic species (without heating) (alginate). Furthermore, for thermally driven processes, various ion species can be used (Na + and K. + ) indicates The mechanical response of the gels can be classified according to the material properties previously reported in patents (either within the outer limits or within a narrowed window described as more optimal).

[0310] Conclusion: In summary, this data shows that fluid gels can be fabricated from a variety of polymers. This is shown using a variety of biopolymers with different gelation mechanisms: thermal, hysteretic thermal, ionotropic, and free radical. This extensive example suggests a comprehensive method for fabricating fluid gels, such that any biopolymer solution that meets or exceeds the required gelation concentration, induced through its sol-gel transition (thermal, ionotropic, radical-induced, etc.), under appropriate shear to prevent the formation of a complete, continuous gel network, can be used to fabricate a fluid gel.

[0311] Further experimental information - Active substance release for various fluid gels the purpose: To demonstrate the ability to release a variety of active agents across a variety of different indications (antifibrotic, anti-infective, analgesic, anti-inflammatory, ECM modifying, basement membrane modifying, and profibrotic) from fluid gel matrices fabricated from a variety of starting polymers (particularly gellan and alginate).

[0312] Materials and Methods: Preparation of fluid gel: A fluid gel was prepared by:

[0313] Gellan (thermal gelling): · Addition of gellan powder to water with 5% PBS to form a 0.5-2% polymer solution. The solution is heated above the gel point. Add cross-linker (sodium chloride (final concentration 10 mM)) The solution is cooled through the gel point (approximately 38°C) under constant shear.

[0314] Alginate (ionotropic gelation): · Addition of alginate powder to water with 5% PBS to form a 0.5-1% polymer solution. · Fully hydrate the polymer (if heat assisted and cooled to room temperature). · Add the crosslinker (calcium chloride added (10 mM final concentration)) slowly using a syringe and needle with constant shear.

[0315] Preparation of the active substance: Aliquot of active substances: penicillin-streptomycin (0.1 ml), dexamethasone (50 mg), proteinase K (10 mg), ibuprofen (200 mg), dextran (300 mg), blue dextran (100 mg), vancomycin (50 mg), Galacorin™ (decorin) (2.4 mg / ml) Add the active substance to PBS to make a total volume of 1 ml. Mix thoroughly on a vortex mixer until dissolved

[0316] Preparation of gel loaded with active substance: Add 0.9 ml of gel to the Eppendorf. To each gel, add 0.1 ml of the active substance in PBS. Mix thoroughly using a vortex mixer. · Refrigerate for 24 hours before testing.

[0317] Determination of the standard curve: Standard concentrations of the active substance in PBS were prepared. Standards were pipetted into quartz cuvettes (path length 1 mm). UV / vis spectroscopy was used to measure the absorbance at wavelengths between 200 and 700 nm. The curve was plotted and used to determine the standard curve to be used for concentration determination. (For Galacorin™, determine decorin concentration using a pre-made ELISA kit according to the kit instructions.)

[0318] Release assay: · 0.5 ml of PBS was added to the wells of a 24-well plate. ·PBS was incubated at 37°C until equilibration. 0.1 ml of fluid gel containing the active substance was placed in the Transwell insert. The Transwell insert was placed into a well containing PBS. After the designated time, the Transwell insert was removed and placed in a well of fresh PBS. The release medium was then removed and analyzed using UV / vis spectroscopy. Concentrations were derived from the standard curve and cumulative release was plotted as a function of time.

[0319] result: FIG. 29 illustrates standard curves obtained for shear-thinning hydrogel compositions according to the present invention incorporating the following active agents: penicillin-streptomycin; dexamethasone; proteinase K; ibuprofen; dextran; and blue dextran.

[0320] FIG. 30 illustrates curves obtained for shear-thinning hydrogel compositions according to the present invention incorporating the following active agents: penicillin-streptomycin; dexamethasone; proteinase K; ibuprofen; dextran; and blue dextran.

[0321] Consideration: The data obtained in the above results showed the following: · Active substance loading and release could be achieved from all fluid gel matrices, regardless of biopolymer (gellan, alginate) or polymeric mechanism. Active substance loading and release could be achieved from all fluid gel matrices, regardless of the gelation mechanism: thermal, ionotropic, or radical-induced gelation. The fluid gels were able to release both small molecules (ibuprofen, dexamethasone, penicillin-streptomycin) and large molecules, polymers (dextran, blue dextran, proteinase K, Galacorin™ (decorin)). Fluid gels can be used for controlled delivery for the following indications: Anti-fibrotic – Galacroin™ (decorin), dextran Anti-infective - Vancomycin, Penicillin-Streptomycin Pain relief – Ibuprofen Anti-inflammatory - ibuprofen, dexamethasone ECM modification - Proteinase K Basement membrane modification - Proteinase K Pro-fibrosis - dextran

[0322] Conclusion: Shear-thinning hydrogel compositions (fluid gels) according to the present invention, made from a variety of polymers and using a variety of gelation techniques, can be used to deliver a wide range of therapeutic agents, both large and small molecules. This suggests that a wide range of therapeutic agents could be delivered in these cases. The suitability of a therapeutic agent for delivery by this method does not appear to be controlled by the size or type of molecule (protein or polysaccharide), but rather depends (in this study) on the agent being water-soluble. This represents an example of an active agent suitable for use in treating a wide range of indications.

[0323] Further experimental information: In vitro release and action of anti-infective molecules from gel compositions of the present invention the purpose: To demonstrate the effect of anti-infective therapeutic agents (exemplified by vancomycin and penicillin-streptomycin) on release from shear-thinning hydrogel compositions according to the present invention.

[0324] Materials and Methods: Preparation of fluid gel: A fluid gel was prepared by:

[0325] Gelan Addition of gellan powder to water with 5% PBS to form a 1% polymer solution. The solution is heated above the gel point. Add cross-linker (sodium chloride (10 mM final concentration)) The solution is cooled through the gel point under constant shear.

[0326] Alginate Addition of alginate powder to water with 5% PBS to form a 0.5% polymer solution. · Fully hydrate the polymer (if heat assisted and cooled to room temperature). · Add the crosslinker (calcium chloride added (10 mM final concentration)) slowly using a syringe and needle with constant shear.

[0327] Preparation of the active substance: Aliquots of active substances: penicillin-streptomycin (100 ml) and vancomycin (50 mg). Add the active substance to PBS to make a total volume of 1 ml. Mix thoroughly on a vortex mixer until dissolved.

[0328] Preparation of gel loaded with active substance: Add 0.9 ml of gel to the Eppendorf. To each gel, add 0.1 ml of the active substance in PBS. Mix thoroughly using a vortex mixer. · Refrigerate for 24 hours before testing.

[0329] Preparation of microorganisms: TSA plates are prepared by dissolving TSA in water, sterilizing by autoclaving, and placing into 90 mm Petri dishes. Cool the plate. ·Cultivate and inoculate microorganisms (E. coli and Staphylococcus aureus). - Allows microorganisms to form a "flora." Drilling, removing and preparing wells in gel.

[0330] Zone of Inhibition Assay: 0.25 ml of fluid gel containing the active substance is added to the wells of each plate. Add anti-infective in PBS to the control plates. Cover the plate and incubate for 24 hours for penicillin-streptomycin or up to 14 hours for vancomycin. -Measure the area where the microbial culture has been removed.

[0331] result: 31 shows photographs illustrating zone of inhibition results using shear-thinning hydrogel compositions according to the present invention comprising the polymers alginate or gellan in combination with an anti-infective (penicillin-streptomycin). These results demonstrate efficacy against E. coli and S. aureus. A summary of the results is also provided in the accompanying table.

[0332] 31 also includes a graph illustrating zone of inhibition results using a shear-thinning hydrogel composition according to the present invention comprising alginate in combination with an alternative anti-infective (vancomycin). Antimicrobial efficacy was tested against MRSA.

[0333] Consideration: The data shows: Anti-infective release and activity occurred across all systems, regardless of polymer type, gelation mechanism, or active agent.

[0334] Conclusion: Fluid gels made from a variety of polymers and using a variety of gelling techniques can be used to deliver anti-infective agents without compromising their activity. These results demonstrate the suitability of the shear-thinning gel compositions of the present invention for delivering a variety of anti-infective agents for use in therapies requiring such agents.

[0335] Further experimental information: In vitro action of proteinase K released from fluid gels the purpose: To demonstrate that the extracellular matrix remodeling agent, proteinase K, retains biological activity after release from a shear-thinning hydrogel composition according to the present invention.

[0336] Materials and Methods: Preparation of fluid gel: A fluid gel was prepared by:

[0337] Gelan Addition of gellan powder to water with 5% PBS to form a 1% polymer solution. The solution is heated above the gel point. Add cross-linker (sodium chloride (10 mM final concentration)) The solution is cooled through the gel point under constant shear.

[0338] Alginate Addition of alginate powder to water with 5% PBS to form a 0.5% polymer solution. · Fully hydrate the polymer (if heat assisted and cooled to room temperature). · Add the crosslinker (calcium chloride added (10 mM final concentration)) slowly using a syringe and needle with constant shear.

[0339] Preparation of the active substance: Aliquot of active substance: Proteinase K (10 mg) Add the active substance to PBS to make a total volume of 1 ml. Mix thoroughly on a vortex mixer until dissolved.

[0340] Preparation of gel loaded with active substance: Add 0.9 ml of gel to the Eppendorf. To each gel, add 0.1 ml of the active substance in PBS. Mix thoroughly using a vortex mixer. · Refrigerate for 24 hours before testing.

[0341] Matrix Disintegration Assay: 0.5 ml of fibrin gel (8.5 mg / ml) was formed in the wells of a 24-well plate. 0.5 ml of PBS was added to each well. 0.1 ml of the gel with the active substance was added to the Transwell insert and placed on top of the fibrin gel. The samples were incubated at 60°C to activate the proteinase K. Images were taken at various time points and compared to a control fibrin gel (PBS only) and a fibrin gel with no fluid gel carrier but with added proteinase K.

[0342] result: FIG. 32 shows photographs depicting the breakdown over time of an exemplary ECM molecule, fibrin (shown as a white gel in the photograph), under the action of the active agent, proteinase K, released from an alginate or gellan shear-thinning hydrogel composition according to the present invention.

[0343] Consideration: The data shows: Disintegration of the fibrin gel occurred in all cases except for the control group. Disintegration of the fibrin gel was faster in the proteinase K only group. The active substance remained potent after being released from the gel.

[0344] Conclusion: The shear-thinning hydrogel composition according to the present invention is capable of releasing an extracellular remodeling active agent (proteinase K) and retains its ability to modify the ECM, demonstrating the ability of the composition of the present invention to deliver such therapeutic molecules.

[0345] Further Experimental Information: In Vitro Effects of Galacorin™ (Decorin) Formulated in a Gellan Shear-Thinning Hydrogel Composition According to the Invention the purpose: To demonstrate the effect of an anti-fibrotic therapeutic agent (exemplified by the commercially available human recombinant decorin product, Galacorin™) on release from the shear-thinning hydrogel compositions of the present invention.

[0346] Materials and Methods: Preparation of fluid gel: A fluid gel was prepared by:

[0347] Gelan Addition of gellan powder to water with 5% PBS to form a 1% polymer solution. The solution is heated above the gel point. Add cross-linker (sodium chloride (10 mM final concentration)) Cool the solution to 40°C under constant shear and add Galacorin™ (final concentration of 240ug / ml) ·Continue cooling through the gel point under constant shear.

[0348] Collagen fibrillogenesis assay: · Prepare the diluent and reaction buffer by mixing sodium phosphate and sodium chloride and adjusting the pH to 7.4. · Samples (gellan fluid gel + Galacorin™, Galacorin only and gellan only) are added to a 96-well plate and serially diluted across the rows of the plate using dilution buffer. On ice, prepare collagen by mixing it with cold water to a concentration of 0.8 mg / ml. Add collagen to all wells. Add reaction buffer to all wells and mix. Incubate at 37°C for 2 hours. · Read using a plate reader at 405 nm.

[0349] result: Figure 33 illustrates the results of this study and is a graph comparing the absorbance at 405 nm (y-axis) for increasing concentrations of gellan fluid gel shear-thinning hydrogel compositions of the present invention incubated with collagen alone ("Collagen Only"), or decorin alone ("hrDecolin"), or with ("DecFG") or without ("FG") human recombinant decorin (Galacorin™).

[0350] Consideration: The data shows: Gellan fluid gel had no effect on collagen fibrillogenesis compared to the collagen-only control. Galacorin™ in or outside of the gellan fluid gel had the same effect on collagen fibrillogenesis.

[0351] Conclusion: Collagen fibrillogenesis can be used as an indicative assay for scarring. Thus, high absorbance as a result of poor collagen microstructure (corresponding to scarred tissue) indicates that the test composition has no effect on scarring. In contrast, a decrease in absorbance indicates a more ordered collagen microstructure, which corresponds to better in vivo healing. It has now been shown that Galacorin™ (decorin) loaded into a gellan fluid gel has the same effect as Galacorin™ added directly to collagen. Thus, this demonstrates in vitro that the gellan shear-thinning hydrogel composition according to the present invention can release a therapeutic antifibrotic agent and that it retains its activity upon release.

[0352] Further Experimental Information: In Vivo Effects of Galacorin™ (Decorin) from the Gellan Shear-Thinning Hydrogel Composition of the Present Invention the purpose: To demonstrate the efficacy of an anti-fibrotic therapeutic agent (Galacorin™) upon release from a fluid gel carrier in an in vivo mouse model of bacterial keratitis.

[0353] Materials and Methods: Preparation of fluid gel: A fluid gel was prepared by:

[0354] Gelan Addition of gellan powder to water with 5% PBS to form a 1% polymer solution. The solution is heated above the gel point. Add cross-linker (sodium chloride (10 mM final concentration)) Cool the solution to 40°C under constant shear and add Galacorin™ (final concentration of 240ug / ml) ·Continue cooling through the gel point under constant shear.

[0355] Mouse models: A description of the mouse model used in this study is presented in Figure 34. Briefly, it shows that the model progresses through three stages: the development of a bacterial keratitis model, the sterilization stage, and the healing stage. The endpoints used were in vivo stereomicroscopy (used to assess opacity on days 2, 3, 9, 12, and 16) and immunohistochemical analysis of tissue sections to examine the expression of ECM proteins and the extent of re-epithelialization.

[0356] Quantification of opacity: Two blinded, independent ophthalmologists analyzed all photographs in the same randomized order (the order was provided by an independent statistician). The area of ​​opacification was measured using Fiji, an open-source image processing package based on ImageJ. mm 2 Unit measurements were plotted using ggplot2 in R and loess smoothing was fitted to the time series for each rater.

[0357] Processing of tissue for re-epithelialization, αSMA, Lam, and FN, and IHC Eyes were fixed in 4% PFA in PBS. Eyes were then rapidly frozen in OCT and sectioned at 15 μm thickness in the parasagittal plane at −22°C. Sections were mounted on positively charged glass slides (Superfrost plus; Fisher Scientific, Pittsburgh, PA, USA). Central sections (optic nerve plane) were used for all IHC studies. Sections (optic nerve plane) were thawed for 30 min, washed with PBS, and subsequently permeabilized with 0.1% Triton X-100 (Sigma). Nonspecific antibody binding sites in the tissue sections were blocked with 0.5% BSA, 0.3% Tween-20, and 15% normal goat serum. Primary antibodies αSMA, laminin, and fibronectin (1:200 dilution) were added, followed by washing in PBS. Incubation with secondary antibodies (goat anti-mouse Alexa Flour 488 1:500, goat anti-mouse Alexa Flour 594 1:500) for 1 hour at room temperature was then attempted. Sections were then washed in PBS and mounted in Vectorshield mounting medium containing DAPI. Control tissue sections incubated with secondary antibody alone were all negatively stained (not shown).

[0358] IHC Imaging and Quantification: IHC staining was quantified by measuring pixel intensity. The region of interest used for quantification of ECM IR was defined as the region of interest that was the same defined size for all eyes / treatments in the stroma, with each stroma having a total of 30 individual intensity measurements (region of interest) taken to encompass the entire area of ​​the stroma. Extracellular matrix deposition was quantified in these defined regions of interest in the interstitium, and the percentage of immunofluorescent pixels above a standardized background threshold was calculated using ImageJ software. For each antibody, a threshold level of brightness in the stromal region was established using sections from intact, untreated eyes to define the reference level for test group analysis of pixel intensity.

[0359] result: The results of the study are illustrated in Figure 35, which presents graphs showing the area of ​​opacity associated with different treatments at different time points, the percentage of α-smooth muscle actin pixels above the threshold for the various control and treatment groups examined, the percentage of fibronectin pixels above the threshold for the various control and treatment groups examined, and the percentage of laminin pixels above the threshold for the various control and treatment groups examined.

[0360] Consideration: The data shows: The gellan shear thinning hydrogel (fluid gel) composition according to the present invention containing Galacorin™ reduced the area of ​​opacity over 16 days compared to standard of care (gentamicin + prednisolone only). The gellan shear-thinning hydrogel (fluid gel) composition of the present invention containing Galacorin™ significantly reduced all three tested markers of fibrosis compared to standard of care alone.

[0361] Conclusion: Application of anti-fibrotic agents in gellan shear-thinning hydrogel (fluid gel) compositions according to the present invention reduced scarring in vivo, as indicated by a reduction in the area of ​​opacity (scar formation) and a reduction in the expression of markers typically associated with fibrosis and scarring.

[0362] Further experimental information: Shear-thinning hydrogel compositions according to the present invention reduce intraocular pressure the purpose This study aimed to investigate the potential of a shear-thinning hydrogel composition without an active agent to reduce intraocular pressure in hypertensive rats (an animal model of glaucoma).

[0363] Materials and Methods Hypertension was induced in rats by intracameral injection of TGF-1 twice a week. The net change in intraocular pressure was determined in untreated hypertensive rats (n=9) and in rats receiving twice daily eye drop formulations of the shear-thinning hydrogel composition of the present invention made with gellan.

[0364] result The results are shown in Figure 36, where treated rats are shown as dashed lines and untreated controls are shown as solid black lines. The results were analyzed using a two-way analysis of variance with Sidak's multiple comparison test and showed that the composition of the present invention (p<0.05) significantly reduced intraocular pressure in intraocular hypertensive rats compared to controls by D28.

[0365] conclusion The results achieved demonstrate that the shear-thinning hydrogel formulations of the present invention are capable of reducing intraocular hypertension (indicating their ability to prevent or treat glaucoma). Surprisingly, this activity was observed even when the compositions were formulated without an active agent.

[0366] Throughout the description and claims of this specification, the words "comprise" and "contain" and variations thereof mean "including but not limited to" and are not intended to (and do not) exclude other parts, additions, components, numbers or steps. Throughout the description and claims of this specification, the singular includes the plural unless the context requires otherwise. In particular, where the indefinite article is used, the specification should be understood as contemplating both the plural and the singular unless the context requires otherwise.

[0367] It should be understood that any property, number, feature, compound, chemical moiety, or group described in connection with a particular aspect, embodiment, or example of the invention is applicable to any other aspect, embodiment, or example described herein, except where incompatible therewith. All features disclosed in this specification (including the accompanying claims, abstract, and drawings), and / or all steps of any method or process so disclosed, may be combined in any combination, except combinations in which at least some of such features and / or steps are mutually exclusive. The invention is not limited to the details of any preceding embodiment. The invention extends to any novel one or any novel combination of features disclosed in this specification (including the accompanying claims, abstract, and drawings), or any novel one or any novel combination of steps of any method or process so disclosed.

[0368] The reader's attention is directed to all papers and documents filed contemporaneously with or prior to this application in connection with this application and publicly accessible herewith, the contents of all such papers and documents being incorporated herein by reference.

Claims

1. (i) 0.1 to 5 wt % (e.g., 0.1 to 2.5 wt %) of a microgel particle-forming polymer; and (ii) 0.5 to 100 mM of a monovalent and / or polyvalent metal ion salt as a cross-linking agent; 1. A shear thinning hydrogel composition comprising: Dispersed in an aqueous vehicle; A shear-thinning hydrogel composition having a pH in the range of 3 to 8, the viscosity of which decreases when the gel is exposed to shear.

2. 10. The shear-thinning hydrogel composition of claim 1, which is free of collagen and / or fibrin.

3. 3. The shear-thinning hydrogel composition of claim 1, comprising 0.5 to 2.5 wt % of the microgel particle-forming polymer.

4. 4. The shear-thinning hydrogel composition of claim 1, comprising 0.8 to 1.8 wt % of the microgel particle-forming polymer.

5. 5. The shear-thinning hydrogel composition of any one of claims 1 to 4, comprising 0.8 to 1.0 wt% (e.g., 0.9 wt%) of the microgel particle-forming polymer.

6. 6. The shear-thinning hydrogel composition of claim 1, wherein the microgel particles are formed from one or more polysaccharide microgel particle-forming polymers.

7. 7. The shear-thinning hydrogel composition of claim 1, wherein the microgel particle-forming polymer is selected from one or more of the following group: gellan, alginate, carrageenan, agarose, chitosan, or gelatin.

8. 8. The shear-thinning hydrogel composition of any one of claims 1 to 7, wherein the microgel particles are transparent or translucent and formed from a microgel particle-forming polymer selected from one or more of the following: gellan, alginate, or carrageenan.

9. 9. The shear-thinning hydrogel composition of claim 1, wherein the microgel particle-forming polymer is gellan.

10. 10. The shear-thinning hydrogel composition of claim 1, wherein the microgel particle-forming polymer does not comprise decorin.

11. 11. The shear-thinning hydrogel composition of any one of claims 1 to 10, comprising 5 to 20 mM of a monovalent and / or polyvalent metal ion salt as a crosslinker.

12. 12. The shear-thinning hydrogel composition of any one of claims 1 to 11, comprising 5 to 15 mM of a monovalent and / or polyvalent metal ion salt as a crosslinker.

13. 13. The shear-thinning hydrogel composition of any one of claims 1 to 12, comprising 8 to 12 mM of a monovalent and / or polyvalent metal ion salt as a crosslinker.

14. 14. The shear-thinning hydrogel composition of claim 1, comprising 10 mM of a monovalent and / or polyvalent metal ion salt as a crosslinker.

15. 15. The shear-thinning hydrogel composition of any one of claims 1 to 14, wherein the microgel particle-forming polymer is gellan and comprises 0.5 to 40 mM, 5 to 15 mM, 8 to 12 mM, or 10 mM of a monovalent metal ion salt (e.g., NaCl) as a crosslinker.

16. The microgel particle-forming polymer is alginate and 0.5-40 mM, 5-15 mM, 8-12 mM, or 10 mM of a polyvalent metal ion (e.g., Ca 2+ 14. The shear-thinning hydrogel composition of claim 1, comprising a hydroxybenzoate salt as a crosslinker.

17. 17. The shear-thinning hydrogel composition of any one of claims 1 to 16, having a pH in the range of 6 to 8 or 6.5 to 8.

18. 18. The shear-thinning hydrogel composition of any one of claims 1 to 17, having a pH in the range of 7 to 7.5 (e.g., pH 7.4).

19. (i) has a viscosity of 1 Pa.s or greater (e.g., 1 Pa.s to 200 Pa.s) when exposed to zero shear, and the viscosity decreases (e.g., to less than 1 Pa.s) when the hydrogel composition is subjected to shear; (ii) has a viscosity of 2 Pa.s or greater (e.g., 2 Pa.s to 200 Pa.s) when exposed to zero shear, and the viscosity decreases (e.g., to less than 1 Pa.s) when the hydrogel composition is subjected to shear; (iii) The shear-thinning hydrogel composition of any one of claims 1 to 18, having a viscosity of 5 Pa s or greater (e.g., 5 Pa s to 200 Pa s) when exposed to zero shear, and wherein the viscosity decreases (e.g., to less than 1 Pa s) when the hydrogel composition is subjected to shear.

20. 20. The shear-thinning hydrogel composition of any one of claims 1 to 19, having an elastic modulus of 5 Pa to 200 Pa at zero shear.

21. 21. The shear-thinning hydrogel composition of any one of claims 1 to 20, further comprising one or more pharmacologically active agents.

22. 22. The shear-thinning hydrogel composition of claim 21, comprising one or more pharmacologically active agents selected from the group consisting of an anti-fibrotic agent (e.g., decorin); an anti-infective agent; an analgesic agent; an anti-inflammatory agent; an anti-proliferative agent; a keratolytic agent; an extracellular matrix modifying agent; an intercellular junction modifying agent; a basement membrane modifying agent; a biological lubricant, and a pigmentation modifying agent.

23. 23. The shear-thinning hydrogel composition of claim 21 or claim 22, comprising decorin at a concentration of between about 0.1 μg / mL and 0.5 μg / mL.

24. 24. A topical gel composition suitable for topical administration, the shear thinning gel composition of any one of claims 1 to 23.

25. 24. An ophthalmic gel composition suitable for administration to the eye, the shear thinning gel composition being the shear thinning gel composition of any one of claims 1 to 23.

26. 10. A method of making the shear thinning gel composition of claim 1, comprising: a) dissolving a microgel-forming polymer in an aqueous vehicle to form a polymer solution; b) mixing the microgel-forming polymer solution formed in step (a) with an aqueous solution of a monovalent or polyvalent metal ion salt at a temperature above the gelation temperature of the microgel particle-forming polymer; and c) cooling the mixture resulting from step b) to a temperature below the gelling temperature of the microgel particle-forming polymer to form the composition of any one of claims 1 to 24. A method comprising:

27. 27. The method of claim 26, wherein step (a) comprises heating and stirring the aqueous vehicle to promote dissolution of the microgel-forming polymer.

28. 28. The method of claim 27, wherein the aqueous vehicle is heated to a temperature above the gelation temperature of the microgel particle-forming polymer, which is 50°C.

29. 29. The method of any one of claims 26 to 28, wherein during step b), mixing of the microgel particle-forming polymer solution formed in step (a) with the aqueous solution of a monovalent or polyvalent metal ion salt occurs at elevated temperature and with shear mixing.

30. 30. The method of claim 29, wherein during step b), the mixing of the microgel-forming polymer solution formed in step (a) with the aqueous solution of the monovalent or polyvalent metal ion salt occurs at a temperature greater than 25°C.

31. 31. The method of any one of claims 26 to 30, wherein during step c) the mixture from step b) is cooled at a rate of 0.1 to 5°C / min with continuous mixing.

32. 32. The method of any one of claims 26 to 31, wherein during step c) the mixture from step b) is cooled at a rate of 0.5 to 2°C / min with continuous mixing.

33. 33. The method of any one of claims 26 to 32, wherein during step c) the mixture from step b) is cooled at a rate of 0.5 to 1.5°C / min (e.g. 1°C / min) with continuous mixing.

34. 34. The method of any one of claims 26 to 33, wherein during step c) the mixture is cooled to a temperature in the range of 0 to 25°C, 0 to 20°C, 0 to 10°C or 0 to 5°C.

35. 35. A shear-thinning gel composition obtainable by, obtained by or directly obtained by the method of any one of claims 26 to 34.

36. 36. The shear-thinning hydrogel composition of any one of claims 1 to 25 or claim 35 for use in therapy.

37. 27. The shear-thinning hydrogel composition for use according to claim 26, which is for topical administration.

38. 38. A shear-thinning hydrogel composition for use according to claim 35 or claim 37 in inhibiting scarring.

39. 39. The shear-thinning hydrogel composition for use according to claim 38, comprising an anti-fibrotic agent.

40. 40. The shear-thinning hydrogel composition for use according to claim 39, wherein the anti-fibrotic agent is selected from the group consisting of an anti-fibrotic extracellular matrix (ECM) component; an anti-fibrotic growth factor; and an inhibitor of a fibrotic agent.

41. 41. The shear-thinning hydrogel composition for use according to any one of claims 38 to 40, comprising the anti-fibrotic ECM component decorin.

42. 42. The shear-thinning hydrogel composition for use according to claim 41, wherein the decorin is present at a concentration of between about 0.1 μg / mL and 0.5 μg / mL.

43. 43. The shear-thinning hydrogel composition for use according to any one of claims 36 to 42, which is for administration to the ocular surface.

44. 44. The shear-thinning hydrogel composition for use according to claim 43, comprising a modified dextran sulfate.

45. 44. The shear-thinning hydrogel composition for use according to claim 43, wherein the shear-thinning hydrogel composition is for use in the treatment of bacterial keratitis.

46. 46. ​​The shear-thinning hydrogel composition for use according to claim 45, wherein the shear-thinning hydrogel composition is for use in combination with one or more agents selected from the group consisting of steroids and antibacterial agents.

47. 47. The shear-thinning hydrogel composition for use according to claim 46, wherein the shear-thinning hydrogel composition is for use in combination with one or more agents selected from the group consisting of prednisolone and gentamicin.

48. 48. A shear-thinning hydrogel composition for use according to claim 47, comprising decorin, prednisolone and gentamicin.

49. 44. The shear-thinning hydrogel composition for use according to claim 43, wherein the shear-thinning hydrogel composition is for use in the prevention and / or treatment of glaucoma.

50. 50. The shear-thinning hydrogel composition for use in the prevention and / or treatment of glaucoma according to claim 49, which does not contain an active agent.

51. 51. The shear-thinning hydrogel composition for use according to claim 50, wherein the particle-forming polymer comprises or consists of gellan.

52. 44. The shear-thinning hydrogel composition for use according to any one of claims 36 to 43, which is for administration to a wound.

53. 53. The shear-thinning hydrogel composition for use according to claim 52, for administration to a skin wound.

54. 54. The shear-thinning hydrogel composition for use according to claim 52 or claim 53, wherein the wound is selected from the group consisting of burns; incisions; excisions; abrasions; chronic wounds; and wounds resulting from the body's response to irritation.

55. 54. The shear-thinning hydrogel composition for use according to claim 52 or 53, wherein the wound is a surgical incision or surgical excision.

56. 55. The shear-thinning hydrogel composition for use according to claim 54, wherein the wound resulting from a bodily reaction to a stimulus is selected from the group consisting of: a systemic chemical and / or allergic reaction; and a genetically related disease, such as epidermolysis bullosa or Kindler syndrome.

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