Therapeutic hydrogel composition

Abstract

Disclosed is a shear-thinning hydrogel composition comprising 0.1-5 wt % (e.g., 0.1-2.5 wt %) of a microgel particle-forming polymer; and 0.5-100 mM of a monovalent and / or polyvalent metal ion salt as a crosslinker. The microgel particle-forming polymer is dispersed in an aqueous vehicle, and the hydrogel composition has a pH in the range of 3-8. The viscosity of the gel composition decreases when the gel is exposed to shear. Methods of making such compositions and medical uses of such compositions are also disclosed.

Description

Technical Field

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

Background Art

[0002] In 2018, the WHO reported that corneal opacity is a major global cause of blindness. Corneal infections caused by conditions such as bacterial keratitis result in tissue breakdown of collagen and the extracellular matrix and form scars. Treatment often requires recovery from 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 little success has been seen with the use of potent anti-scarring treatments. One limitation of current eyedrop treatments is low viscosity or weak gelling materials, which do not significantly enhance the drug retention time.

[0003] Corneal opacity is a major global cause of visual impairment, with an estimated 27.9 million people worldwide affected bilaterally or unilaterally. [1] . Such opacities typically result from changes in the complex and clear corneal tissue structure, which is extremely important for light refraction on the retina, and subsequent neuro-visual processing. Generally, corneal scarring is caused by eye infections from various 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], is an organism significantly caused by Pseudomonas aeruginosa. In the case of Gram-negative infections, such as Pseudomonas, the structural integrity of the cornea is impaired by multiple pathogenic factors, which then allows the microorganism to penetrate the epithelial cells, resulting in the activation of numerous inflammatory pathways. Subsequent inflammation, angiogenesis, cellular changes, and degradative stromal processes [5] result in the breakdown of complexly structured collagen fibrils [6] . Continuous inflammation leads to fibrosis, as well as remodeling with more extensive and disordered collagen fibrils and dysregulation of the stromal tissue matrix accompanied by loss of light transmission, impairment of light refraction, and loss of vision.

[0004] Typically, transforming growth factor beta (TGFβ) is mostly limited to the epithelium in a healthy cornea, while local trauma induces the production of cytokines, including TGFβ, in both the epithelium and stroma [7] . In an injured cornea, when physiological recovery or external drug treatment is insufficient to suppress the inflammatory response, the formation of disordered fibrils and deposition of dysregulated extracellular matrix (ECM) lead to a fibrotic reaction and persistent corneal scarring with visual impairment. Mechanistically, TGFβ activates corneal fibroblasts (corneal stromal cells), resulting in differentiation into myofibroblasts and promoting wound contraction via the excretion of ECM molecules containing collagen [7] .

[0005] Currently, the standard clinical treatment for patients suffering from bacterial keratitis initially focuses on sterilizing the affected eye by intensive topical administration of broad-spectrum antibiotics, followed by the addition of topical corticosteroids to reduce inflammation [8~9] . This includes intensive lubrication (to reduce the biomechanical trauma of the eyelids rubbing against the wound bed during blinking), and systemic agents in attempts to promote tissue remodeling (sub-antimicrobial dose of tetracycline for matrix metalloproteinase inhibition

[10] ) or supplement (vitamin C and free radical scavengers used as antioxidants)

[11] ) continue strategies to inhibit scar formation. Unfortunately, although effective for eye sterilization, in many cases a high degree of corneal clouding remains in the patient, and if the clouding obstructs the visual axis, it causes loss of vision. Surgical procedures for treating 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, if a visually significant central corneal scar is established, either excision of the scarred tissue and replacement with donor cornea. The reproducibility and repeatability of the clinical outcomes of amniotic membrane transplantation and corneal transplantation are associated with the risks of incompleteness and rejection [15~18] .

[0006] If the fibrotic response to injury and infection can be attenuated, it would be possible to maximize optical transparency, preserve visual function, and eliminate the need for surgical procedures and transplantation. Such an innovation would have the potential to prevent persistent vision loss in millions of subjects. As described above, fibrosis is caused by an increase in the level of TGFβ-1 activity, and therefore it may be possible to prevent fibrosis using TGFβ antagonists. Decorin is a naturally occurring, small leucine-rich anti-fibrotic proteoglycan that binds to collagen in the corneal stroma at high natural levels 22 and, when released, tightly regulates TGFβ activity by binding to growth factors and capturing them in the ECM

[19] . Decorin regulates innumerable growth factors including TGFβ {Zhang, 2007 #28} [20~24] and, by directly preventing the formation of collagen fibrils [25~28] , regulates cell proliferation, survival, and differentiation. Human recombinant (hr) decorin is currently 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 surface of the eye for in vivo treatment has not been reported. One possible reason for this is the relatively low viscosity of the eye drops, resulting in relatively rapid clearance from the corneal surface at an early stage (within a few minutes). [32、33] This could be the case, which would mean 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; vol. 91(1): pp. 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; Vol. 84 (No. 4): pp. 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; Vol. 79 (No. 8): pp. 777-786. [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 monovalent and / or polyvalent metal ion salts as crosslinking agents; A shear-reducing hydrogel composition comprising, Disperse in an aqueous vehicle; A shear-reducing hydrogel composition is provided, having a pH in the range of 3 to 8, and whose viscosity decreases when the hydrogel is subjected 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, an ophthalmic hydrogel composition suitable for application to the eye is provided, comprising, essentially, or consisting of a shear-thinning hydrogel composition as defined herein.

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

[0011] In a further embodiment, the present invention relates to a method for preparing a shear-thinning hydrogel composition as defined herein, a) A step of dissolving a microgel-forming polymer in an aqueous vehicle to form a polymer solution; b) A step of 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 exceeding the gelation temperature of the microgel particle-forming polymer; and c) A step of cooling the mixture obtained from step b) to a temperature below the gelation temperature of the microgel particle-forming polymer. This provides a method that includes [something].

[0012] A further aspect of the present invention is a method for preparing a shear-thinning hydrogel composition as defined herein, a) A step of dissolving a microgel-forming polymer in an aqueous vehicle containing 0.5 to 100 mM monovalent and / or polyvalent metal ion salt as a crosslinking agent 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) A step of mixing the microgel-forming polymer solution formed in step (a) at a temperature exceeding the gelation temperature of the microgel particle-forming polymer; and c) A step of cooling the mixture obtained from step b) to a temperature below the gelation temperature of the microgel particle-forming polymer under shear mixing. This provides a method that includes [something].

[0013] A further aspect of the present invention provides a shear-thinning hydrogel composition that can be obtained by, or is obtained by, or is obtained directly by, any of the preliminary methods defined herein.

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

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

[0016] A further aspect of the present invention provides a shear-thinning hydrogel composition, as defined herein, for inhibiting 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-reducing hydrogel composition, as defined herein, for administration to skin wounds.

[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 embodiment, the present invention provides compositions according to the present invention for use as pharmaceuticals. Examples of preferred medical uses of the compositions of the present invention are further described below. Preferably, the compositions of the present invention can be used as topical pharmaceuticals.

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

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

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

[0024] [Figure 1A]This diagram illustrates the processing and inherent material properties of a gellan-based fluid hydrogel eye drop. (a) This diagram schematically shows the production of the fluid gel, in which the initial sol is continuously processed under shear while being cooled, forming a “ribbon-like” gelled entity, as shown using (i) transmission microscopy and (ii) scanning electron microscopy. [Figure 1B] This figure shows the processing and inherent material properties of a gelan-based fluid hydrogel eye drop. It also shows the time-dependent viscosity profile obtained for the gelan eye drop, highlighting some degree of thixotropy. [Figure 1C] This diagram illustrates the processing and inherent material properties of a gelan-based fluid hydrogel eye drop. The diagram shows the fluid gel being dispensed from the eye dropper packaging (the gel is stained blue as seen in the photograph). [Figure 1D] This figure shows the processing and intrinsic material properties of a gelan-based fluid hydrogel eye drop. It also shows rheological data for small deformations obtained at a single frequency (1 Hz, strain 0.5%) as a function of time. The data demonstrates the development of an elastic network structure after shearing, resulting in a transition from liquid to solid-like behavior. [Figure 1E] This diagram illustrates the processing and inherent material properties of a gelan-based fluid hydrogel eye drop. The diagram also shows anterior segment OCT images of the ocular surface before (top image) and after (bottom image) fluid gel application. The images represent a uniform layer covering the entire ocular surface. [Figure 2] These are figures of in vitro assays representing the biological activity of formulated eye drops. (a) A figure of the cumulative release curve for eye drops filled with hr decorin over 4 hours (240 minutes). The fitted line follows the power function y=0.7x0.7 (R2=0.99). (b) A figure of turbidity data for collagen fibrillation for PBS control, collagen alone, and collagen + hr decorin. (c) A figure of turbidity data for collagen fibrillation using dose-response curves for collagen, collagen + hr decorin, collagen + fluid gel (FG) alone, and fluid gel filled with collagen + hr decorin (DecFG). [Figure 3]This diagram shows the measurement of corneal opacity area. (a) This diagram shows representative photographs taken on days 2, 3, 9, 12, and 16 after Psaudomonas infection and treatment, and (b) This graph shows the mean opacity area ± SEM (mm2) measured by two independent, blinded ophthalmologists from photographs taken from each individual mouse in each group (shown in panel a) (n=6; **p<0.01, ***p<0.001). [Figure 4A] This diagram illustrates corneal re-epithelialization. (a) Representative images of DAPI+ cell nuclei (blue) in the cornea used to approach the epithelium, illustrating epithelial thickness and lamination (number of cell layers) in an untreated, intact eye showing normal, non-keratinized, layered (approximately 5 layers) epithelium; an eye taken 2 days post-infection with thickened, edematous stroma with cell infiltration; and an eye taken 16 days after treatment showing re-epithelialization with 2-3 layers of lamination, with reduced interstitial edema in group 1 (gentamicin and prednisolone), increased lamination in group 2 (GPFG), and fully mature epithelium in group 3 (GPDecFG) (scale bar 100 μm). [Figure 4B] This figure shows corneal re-epithelialization. It also shows the quantification of corneal thickness ± SEM in an untreated, intact group (n=6) and eyes evaluated on days 2 and 16 from each treatment group (n=6 for each group). All quantifications were performed on blinded images unknown to the observer. [Figure 4C] This figure shows corneal re-epithelialization. It quantifies epithelial layer thickness ± SEM in an untreated, intact group (n=6) and in eyes evaluated on days 2 and 16 from each treatment group (n=6 for each group). All quantifications were performed on blinded images unknown to the observer. [Figure 4D] This figure shows corneal re-epithelialization. It quantifies cellular epithelial stratification layers ± SEM in eyes evaluated at 2 and 16 days from the untreated intact group (n=6) and from each treatment group (n=6 for each group). All quantifications were performed on blinded images unknown to the observer. [Figure 5] This is a diagram of the extracellular matrix level in the cornea. It is a representative image of immunohistochemical staining, with plots quantifying IR for (a) αSMA+ (green for staining myofibroblasts), (b) IR fibronectin+ (green for staining fibronectin in the ECM), and (c) laminin+ (red for staining laminin in the ECM), and DAPI+ used to stain cell nuclei (blue) in each case. Analysis was attempted with intact eyes, eyes taken 2 days after infection, and eyes obtained 16 days after infection, using various eye drop treatments: i) gentamicin and prednisolone (GP), ii) gentamicin, prednisolone and fluid gel (GPFG), and iii) gentamicin, prednisolone and hrdecolin fluid gel (GPDecFG). All tests were performed using n=6 treatment groups, and quantification was performed on blinded images unknown to the observer (scale bar = 100 μm). [Figure 6] This is a diagram of the in vivo experimental design. It is a diagram of the experimental design for an in vivo pseudomonas keratitis test comparing fluid gel eye drops with or without hr decorin with gentamicin and prednisolone eye drops alone. [Figure 7] The figures show the storage modulus (G') representing the elastic structure in a gelane microgel suspension as a function of the initial gelane polymer concentration, determined using amplitude sweep, (a) strain sweep figures obtained at 1 Hz (20°C) for various polymer concentrations prepared at a processing speed of 500 rpm, and (b) strain sweep figures obtained at 1 Hz (20°C) for various polymer concentrations at a processing speed of 1000 rpm. [Figure 8] This figure shows a comparison of storage modulus as a function of polymer concentration and processing rate. G' was obtained in the linear viscoelastic region (LVR) of the amplitude sweep shown in Figure 7. [Figure 9]This figure compares the storage moduli of commercially available eye drops / ointments for the treatment of dry eye. The data were obtained from amplitude sweeps attempted 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] These are flow rate profiles representing the ease of application of gelane microgel suspensions as a function of the initial gelane polymer concentration: (a) viscosity sweeps obtained between 0.1 and 600 s⁻¹ 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⁻¹ at 20°C for various polymer concentrations prepared at a processing speed of 1000 rpm. [Figure 11] This figure compares the viscosity of microgel suspensions at 1 s⁻¹ as a function of polymer concentration and processing rate. Instantaneous viscosity was obtained by measuring the value at 1 s⁻¹ using the sweep shown in Figure 10. [Figure 12] This figure compares the viscosity at 1s-1 for commercially available eye drops / ointments for the treatment of dry eye. The data were 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] Figures of the storage modulus (G') representing the elastic structure in a gelane microgel suspension as a function of added crosslinker, determined using amplitude sweeps. (a) Figures of strain sweeps obtained at 1 Hz (20°C) for various crosslinker concentrations for a 0.9% (w / v) system, and (b) Figures of strain sweeps obtained at 1 Hz (20°C) for various crosslinker concentrations for a 1.8% (w / v) polymer concentration. [Figure 14] This figure shows a comparison of storage moduli as functions of crosslinker and polymer concentration. G' was obtained in the linear viscoelastic region (LVR) of the amplitude sweep shown in Figure 7. [Figure 15]This diagram shows flow rate profiles representing the ease of application of gelane microgel suspensions as a function of crosslinker concentration. (Left) Viscosity sweeps obtained at 20°C and between 0.1 and 600 s⁻¹ for 0.9% (w / v) gelane systems prepared with various crosslinker concentrations. (Right) Viscosity sweeps obtained at 20°C and between 0.1 and 600 s for 1.8% (w / v) gelane systems prepared with various crosslinker concentrations. [Figure 16] This figure compares the viscosity of microgel suspensions at 1 s⁻¹ as a function of polymer and crosslinker concentrations. Instantaneous viscosity was obtained by measuring the value at 1 s⁻¹ using the sweep shown in Figure 3. [Figure 17] The figures show the storage modulus (G') representing the elastic structure in a gellan microgel suspension as a function of the cooling rate applied during processing, determined using amplitude sweeps: (a) a strain sweep obtained at 1 Hz (20°C) for various cooling rates for a 0.9% (w / v) system prepared at a processing rate of 1000 rpm; and (b) a strain sweep obtained at 1 Hz (20°C) for various cooling rates for a 1.8% (w / v) polymer concentration prepared at a processing rate of 1000 rpm. [Figure 18] This figure shows a comparison of storage modulus as a function of cooling rate and polymer concentration. G' was obtained in the linear viscoelastic region (LVR) of the amplitude sweep shown in Figure 7. [Figure 19] These are flow rate profiles representing the ease of application of gelane microgel suspensions as a function of the cooling rate applied during processing: (a) Viscosity sweep obtained at 20°C and between 0.1 and 600 s⁻¹ for 0.9% (w / v) gelane systems prepared at various cooling rates; (b) Viscosity sweep obtained at 20°C and between 0.1 and 600 s⁻¹ for 1.8% (w / v) gelane systems prepared at various cooling rates. [Figure 20] This figure compares the viscosity of microgel suspensions at 1 s⁻¹ as a function of polymer concentration and the cooling rate applied during processing. Instantaneous viscosity was obtained by measuring the value at 1 s⁻¹ using the sweep shown in Figure 9. [Figure 21] Figures of the storage modulus (G') representing the elastic structure in a gellan microgel suspension as a function of mechanical shear applied during processing, determined using amplitude sweeps, (a) figures of strain sweeps obtained at 1 Hz (20°C) for various processing rates for a 0.9% (w / v) system, and (b) figures of strain sweeps obtained at 1 Hz (20°C) for various processing rates for a 1.8% (w / v) polymer concentration. [Figure 22] This figure shows a comparison of storage modulus as a function of processing speed and polymer concentration. G' was obtained in the linear viscoelastic region (LVR) of the amplitude sweep shown in Figure 7. [Figure 23] These are flow rate profiles representing the ease of application of gelane microgel suspensions as a function of mechanical shear applied during processing: (a) Viscosity sweep obtained at 20°C and between 0.1 and 600 s⁻¹ for 0.9% (w / v) gelane systems prepared at various processing rates; (b) Viscosity sweep obtained at 20°C and between 0.1 and 600 s⁻¹ for 1.8% (w / v) gelane systems prepared at various processing rates. [Figure 24] This figure compares the viscosity of microgel suspensions at 1 s⁻¹ as a function of polymer concentration and processing rate during gelation. Instantaneous viscosity was obtained by measuring the value at 1 s⁻¹ using the sweep shown in Figure 9. [Figure 25] This figure shows that the shear-reducing hydrogel composition according to the present invention reduces the expression of scarring-related markers in cultured fibroblasts. Administration of TGF-β to cultured human dermal fibroblasts increases the expression of α-smooth muscle actin, a myofibroblast marker associated with scarring. The graph shows the effect of treatment with the experimental hydrogel composition on this expression. Hydrogel compositions with or without the antifibrotic agent decorin can reduce α-sma expression and exhibit the ability to inhibit scarring. [Figure 26] This figure shows the amplitude sweep data obtained for agar, gellan, kappa-carrageenan, and alginate. [Figure 27]This figure shows the frequency sweep data obtained for agar, gellan, kappa-carrageenan, and alginate. [Figure 28] This figure shows the viscosity sweep data obtained for agar, gellan, kappa-carrageenan, and alginate. [Figure 29] This figure illustrates the standard curves obtained for the 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] This figure illustrates the curves obtained for the shear-thinning hydrogel composition according to the present invention, incorporating the following active agents: penicillin-streptomycin; dexamethasone; proteinase K; ibuprofen; dextran; and blue dextran. [Figure 31] This figure shows photographs illustrating the results of an inhibition zone assay using a shear-thinning hydrogel composition according to the present invention, comprising a polymer alginate or gellan in combination with an anti-infective agent (penicillin-streptomycin). These results demonstrate efficacy against Escherichia coli (E. coli) and Staphylococcus aureus (S. Aureus). A summary of the results is also presented in the accompanying table. The figure also includes graphs illustrating the results of an inhibition zone assay using a shear-thinning hydrogel composition according to the present invention, comprising alginate in combination with an alternative anti-infective agent (vancomycin). The antimicrobial efficacy of vancomycin was tested against MRSA. [Figure 32] This figure shows a photograph illustrating the time-dependent disintegration of fibrin (shown as a white gel in the photograph) of an exemplary ECM molecule 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]This graph illustrates the results of this test and compares the absorbance (y-axis) at 405 nm for increasing concentrations of the gelan fluid gel shear-thinning hydrogel composition of the present invention, for collagen incubated with collagen alone ("collagen only") or with decorin alone ("hr decorin"), or with or without human recombinant decorin ("DecFG") ("FG"). [Figure 34] This figure shows a mouse model used to test the effects of the present invention's composition on experimental bacterial keratitis. [Figure 35] This graph shows the area of ​​turbidity associated with different treatments at different time points, the percentage of α-smooth muscle actin pixels exceeding the threshold for various control and treatment groups investigated, the percentage of fibronectin pixels exceeding the threshold for various control and treatment groups investigated, and the percentage of laminin pixels exceeding the threshold for various control and treatment groups investigated. [Figure 36] This figure shows the results of an intraocular pressure test in hypertensive rats, with treated rats shown by a dashed line and untreated controls shown by a solid black line. The results were analyzed using two-way ANOVA and the Sidak multiple comparison test, and it was shown that the composition of the present invention (p<0.05) significantly reduced intraocular pressure in hypertensive rats to D28 compared to the control. [Modes for carrying out the invention]

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

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

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

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

[0029] It should be understood that any reference to “treatment” or “treatment” includes the prevention and alleviation of established symptoms of a condition. “Treatment” or “treatment” of a condition, disorder, or state therefore includes: (1) preventing or delaying the appearance of any clinical symptoms of a condition, disorder, or state in a person who suffers from or is susceptible to the condition, disorder, or state but has not experienced or shown any clinical or subclinical symptoms of the condition, disorder, or state; (2) preventing the condition, disorder, or state, i.e., stopping, reducing, or delaying the onset of the disease or its recurrence (in the case of maintenance treatment) or at least one of its clinical or subclinical symptoms; or (3) alleviating or reducing the disease, i.e., causing the recovery of the condition, disorder, or state or at least one of its clinical or subclinical symptoms.

[0030] The "therapeutic dose" refers to the amount of a compound sufficient for such treatment to act on a disease when administered to a mammal. The therapeutic dose will vary depending on the compound, the disease and its severity, and the age, weight, etc., of the mammal being treated.

[0031] Throughout this description and claims, the words “including” and “containing” and their variations mean “including but not limited to” and are not intended to exclude (and do not exclude) other additions, components, numbers, or processes. Throughout this description and claims, singular forms include plural forms unless the context otherwise requires. In particular, where the indefinite article is used, this specification should be understood to intend both plural and singular forms unless the context otherwise requires.

[0032] The reader's attention is directed to all pages and documents filed concurrently with or prior to this specification in connection with this application and made publicly available together with this specification, the contents of all such pages and documents being incorporated herein by reference.

[0033] Hydrogel composition of the present invention In a further embodiment 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 monovalent and / or polyvalent metal ion salts as crosslinking agents; A shear-reducing hydrogel composition comprising, Disperse in an aqueous vehicle; A shear-reducing hydrogel composition is provided, having a pH in the range of 3 to 8, and whose viscosity decreases when the hydrogel is subjected to shear.

[0034] The hydrogel composition of the present invention is shear-thinning, meaning that the viscosity of the composition decreases when the hydrogel is subjected to shear. This property allows the viscosity of the hydrogel to decrease and flow when a shear force is applied, thereby allowing the hydrogel to be dispensed and administered, for example, from an eyedropper to a tube, by applying a shear force (for example, by constricting the end of an eyedropper or tube). Once administered, as the shear force applied to the hydrogel weakens, the viscosity of the hydrogel increases, forming a thicker gel that can remain at the administration site for a longer 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 a shear force for dispensing. At a viscosity of less than 1 Pa·s, the hydrogel composition will be able to flow. As a result, the static viscosity will typically be greater than 1 Pa·s, for example, 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 contain collagen and / or fibrin.

[0037] A microgel particle-forming polymer can be any polymer capable of forming microgel particles in an aqueous vehicle. Microgel particles formed by a microgel particle-forming polymer can have any preferred form (e.g., they can be linear filaments or regularly or irregularly shaped particles) and / or particle size. The formation of microgel particles promotes the desired shear-thinning characteristic, in contrast to a macrogel structure. While we do not wish to be constrained by any particular theory, it is assumed that in the absence of shear or at low levels of shear, the microgel particles would bind together, substantially hindering the overall flow of the hydrogel. However, when a shear force is applied, the interactions between adjacent microgel particles weaken, reducing viscosity and thereby allowing the hydrogel composition to flow. When the applied shear force is removed, the interactions between adjacent microgel particles then reform so that viscosity increases again, hindering the ability to flow easily.

[0038] Preferably, the hydrogel composition contains 0.5 to 5.0 wt% of a microgel particle-forming polymer. In one embodiment, the hydrogel composition contains 0.5 to 3.5 wt% of a microgel particle-forming polymer. In one embodiment, the hydrogel composition contains 0.5 to 2.5 wt% of a microgel particle-forming polymer. In one embodiment, the hydrogel composition contains 0.8 to 1.8 wt% of a microgel particle-forming polymer. In a further embodiment, the hydrogel composition contains 0.8 to 1.0 wt% (e.g., 0.9 wt%) of a 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 groups: 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 groups: agar, gellan, alginate, or carrageenan. In a particular embodiment, the microgel particle-forming polymer is selected from one or more of the following groups: gellan, alginate, or carrageenan. In a further 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. In a 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 gelan, alginate, and / or carrageenan. In a further embodiment, the hydrogel composition is transparent, and the microgel particle-forming polymer is selected from gelan, alginate, and / or carrageenan. In a particular embodiment, the hydrogel composition is transparent, and the microgel particle-forming polymer is gelan or alginate. In a further embodiment, the hydrogel composition is transparent, and the microgel particle-forming polymer is gelan.

[0043] Gellan (also known as 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 trademark name Kelco gel CG LA (Azelis, UK).

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

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

[0046] In a particular embodiment of the present invention, the microgel particle-forming polymer is gelan, 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 crosslinking agent.

[0047] In a further embodiment of the present invention, the microgel particle-forming polymer is an alginate, and the composition contains 0.5-40 mM, 5-15 mM, 8-12 mM, or 10 mM of a polyvalent metal ion salt (e.g., Ca 2+ Contains salt as a crosslinking 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 composition of the present invention has a static viscosity (i.e., viscosity at zero shear) of 1 Pa.s or more (e.g., 1 Pa.s to 200 Pa.s or 1 Pa.s to 100 Pa.s). More preferably, the static viscosity will be 2 Pa.s or more (e.g., 2 Pa.s to 200 Pa.s or 2 Pa.s to 100 Pa.s), 3 Pa.s or more (e.g., 3 Pa.s to 200 Pa.s or 3 Pa.s to 100 Pa.s), 4 Pa.s or more (e.g., 4 Pa.s to 200 Pa.s or 4 Pa.s to 100 Pa.s), or 5 Pa.s or more (e.g., 5 Pa.s to 200 Pa.s or 5 Pa.s to 100 Pa.s).

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

[0051] In one embodiment, the hydrogel composition has a static viscosity of 1 Pa.s or more (for example, 1 Pa.s to 200 Pa.s or 1 Pa.s to 100 Pa.s), and when subjected to shear force, the viscosity decreases to less than 1 Pa.s.

[0052] In another embodiment, the hydrogel composition has a static viscosity of 2 Pa.s or more (e.g., 2 Pa.s to 200 Pa.s or 2 Pa.s to 100 Pa.s), and when subjected to shear force, 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 static viscosity of 3 Pa.s or more (e.g., 3 Pa.s to 200 Pa.s or 3 Pa.s to 100 Pa.s), and when subjected to shear force, 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 static viscosity of 4 Pa.s or more (e.g., 4 Pa.s to 200 Pa.s or 4 Pa.s to 100 Pa.s), and when subjected to shear force, 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 static viscosity of 5 Pa.s or more (e.g., 5 Pa.s to 200 Pa.s or 5 Pa.s to 100 Pa.s), and when subjected to shear force, the viscosity decreases to less than 5 Pa.s (e.g., less than 1 Pa.s).

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

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

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

[0059] Specific Embodiments In a particular embodiment of the present invention, the shear-thinning hydrogel composition is: (I) 0.1-5.0 wt% of a microgel particle-forming polymer (e.g., gellan); 0.5-40 mM monovalent metal ion salt (e.g., NaCl) or polyvalent metal ion salt (e.g., Ca) as a crosslinking agent. 2+ ) It contains and has a pH of 3.5 to 8. (II) 0.1-5.0 wt% of a microgel particle-forming polymer (e.g., gellan); 0.5-40 mM monovalent metal ion salt (e.g., NaCl) or polyvalent metal ion salt (e.g., Ca) as a crosslinking agent. 2+ ) It contains and has a pH of 6 to 8. (III) 0.1-5.0 wt% of a microgel particle-forming polymer (e.g., gellan); 0.5-40 mM monovalent metal ion salt (e.g., NaCl) or polyvalent metal ion salt (e.g., Ca) as a crosslinking agent. 2+ ) It contains and has a pH of 6.5 to 7.5. (IV) 0.1-3.5 wt% of a microgel particle-forming polymer (e.g., gellan); 0.5-40 mM monovalent metal ion salt (e.g., NaCl) or polyvalent metal ion salt (e.g., Ca) as a crosslinking agent. 2+ ) It contains and has a pH of 3.5 to 8. (V) 0.1-3.5 wt% of a microgel particle-forming polymer (e.g., gellan); 0.5-40 mM monovalent metal ion salt (e.g., NaCl) or polyvalent metal ion salt (e.g., Ca) as a crosslinking agent. 2+ ) It contains and has a pH of 6 to 8. (VI) 0.1-3.5 wt% of a microgel particle-forming polymer (e.g., gellan); 0.5-40 mM monovalent metal ion salt (e.g., NaCl) or polyvalent metal ion salt (e.g., Ca) as a crosslinking agent. 2+ ) It contains and has a pH of 6.5 to 7.5. (1) 0.1-2.5 wt% of a microgel particle-forming polymer (e.g., gellan); 0.5-40 mM monovalent metal ion salt (e.g., NaCl) or polyvalent metal ion salt (e.g., Ca) as a crosslinking agent. 2+ ) It contains and has a pH of 3.5 to 8. (2) 0.1-2.5 wt% of a microgel particle-forming polymer (e.g., gellan); 0.5-40 mM monovalent metal ion salt (e.g., NaCl) or polyvalent metal ion salt (e.g., Ca) as a crosslinking agent.2+ ) It contains and has a pH of 6 to 8. (3) 0.1-2.5 wt% of a microgel particle-forming polymer (e.g., gellan); 0.5-40 mM monovalent metal ion salt (e.g., NaCl) or polyvalent metal ion salt (e.g., Ca) as a crosslinking agent. 2+ ) It contains and has a pH of 6.5 to 7.5. (4) 0.5-2.0 wt% of a microgel particle-forming polymer (e.g., gellan); 0.5-40 mM monovalent metal ion salt (e.g., NaCl) or polyvalent metal ion salt (e.g., Ca) as a crosslinking agent. 2+ ) It contains and has a pH of 3.5 to 8. (5) 0.8-1.8 wt% of a microgel particle-forming polymer (e.g., gellan); 0.5-40 mM monovalent metal ion salt (e.g., NaCl) or polyvalent metal ion salt (e.g., Ca) as a crosslinking agent. 2+ ) It contains and has a pH of 6 to 8. (6) 0.8-1.0 wt% of a microgel particle-forming polymer (e.g., gellan); 0.5-40 mM monovalent metal ion salt (e.g., NaCl) or polyvalent metal ion salt (e.g., Ca) as a crosslinking agent. 2+ ) It contains and has a pH of 6.5 to 7.5. (7) 0.5-2.5 wt% of a microgel particle-forming polymer (e.g., gellan); 0.5-40 mM monovalent metal ion salt (e.g., NaCl) or polyvalent metal ion salt (e.g., Ca) as a crosslinking agent. 2+ ) It contains and has a pH of 3.5 to 8. (8) 0.5-2.5 wt% of a microgel particle-forming polymer (e.g., gellan); 5-20 mM monovalent metal ion salts (e.g., NaCl) or polyvalent metal ion salts (e.g., Ca) can be used as crosslinking agents. 2+ ) It contains and has a pH of 6 to 8. (9) 0.5-2.5 wt% of a microgel particle-forming polymer (e.g., gellan); 5-15 mM monovalent metal ion salts (e.g., NaCl) or polyvalent metal ion salts (e.g., Ca) can be used as crosslinking agents. 2+ ) It contains and has a pH of 6 to 8. (10) 0.5-2.5 wt% of a microgel particle-forming polymer (e.g., gellan); As a crosslinking agent, use an 8-12 mM (e.g., 10 mM) monovalent metal ion salt (e.g., NaCl) or polyvalent metal ion salt (e.g., Ca 2+ ) It contains and has a pH of 6 to 8. (11) 0.5-2.5 wt% of a microgel particle-forming polymer (e.g., gellan); 0.5-40 mM monovalent metal ion salt (e.g., NaCl) or polyvalent metal ion salt (e.g., Ca) as a crosslinking agent. 2+ ) It contains and has a pH of 6 to 8. (12) 0.8-1.8 wt% of a microgel particle-forming polymer (e.g., gellan); 5-20 mM monovalent metal ion salts (e.g., NaCl) or polyvalent metal ion salts (e.g., Ca) can be used as crosslinking agents. 2+ ) It contains and has a pH of 6 to 8. (13) 0.8-1.0 wt% of a microgel particle-forming polymer (e.g., gellan); 5-15 mM monovalent metal ion salts (e.g., NaCl) or polyvalent metal ion salts (e.g., Ca) can be used as crosslinking agents. 2+ ) It contains and has a pH of 6 to 8. (14) 0.8-1.0 wt% of a microgel particle-forming polymer (e.g., gellan); As a crosslinking agent, use an 8-12 mM (e.g., 10 mM) monovalent metal ion salt (e.g., NaCl) or polyvalent metal ion salt (e.g., Ca 2+ ) Contains and has a pH of 6-8 These are some examples.

[0060] Therapeutic drugs 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 exist. 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; anti-proliferative agents; keratolytic agents; extracellular matrix modifiers; intercellular junction modifiers; basement membrane modifiers; and pigmentation modifiers. The antifibrotic agent may be decorin. In the context of the present invention, when decorin is incorporated into the hydrogel composition of the present invention, it should be understood that decorin may exist as an active agent incorporated into the hydrogel rather than as a component of the hydrogel itself.

[0061] The hydrogel composition may contain any suitable amount of the pharmacologically active agent. For example, the hydrogel composition may contain 0.01 to 50 wt% of the pharmacologically active agent.

[0062] In one embodiment, the hydrogel composition contains decorin in an optionally selected 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, with decorin optionally 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 the composition of the present invention, which contains an anti-infective agent, such as the antibiotic gentamicin, the anti-infective agent may be present in an amount of 1 to 5 mg / ml. For example, the anti-infective agent, such as gentamicin, may be present in an amount of 1 to 4 mg / ml, 1 to 3 mg / ml, or 1 to 2 mg / ml. The anti-infective agent, such as gentamicin, may be present in an amount of 2 to 4 mg / ml, or 2.5 to 3.5 mg / ml.

[0065] In one embodiment of the composition of the present invention, which contains 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. Preferably, 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 embodiment, the present invention provides an ophthalmic hydrogel composition suitable for administration to the eye, which is a shear-thinning hydrogel composition as defined herein.

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

[0068] The ophthalmic hydrogel composition of the present invention is suitable for application to the eye.

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

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

[0071] Method for preparing the hydrogel composition of the present invention The present invention relates to a method for preparing a shear-reducing hydrogel composition as defined herein, a) A step of dissolving a microgel particle-forming polymer in an aqueous vehicle to form a polymer solution; b) A step of 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 exceeding the gelation temperature of the microgel particle-forming polymer; and c) A step of cooling the mixture obtained from step b) to a temperature below the gelation temperature of the microgel particle-forming polymer under shear mixing. This provides a method that includes [something].

[0072] Preferably, step (a) is carried out by heating the microgel particle-forming polymer and aqueous vehicle to a temperature exceeding the gelation temperature for 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 should be understood that the amount of polymer to dissolve depends on the amount of polymer required in the hydrogel composition (i.e., this 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 thorough mixing. In one embodiment, mixing speeds of 300 to 900 rpm or 500 to 800 rpm can be used. Those skilled in the art will understand that the mixing speed and mixing apparatus can be varied to yield the desired level of shear / stirring.

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

[0076] It should be understood that the amount of monovalent or polyvalent metal ion salt solution added depends on the amount of metal ion salt required in the final hydrogel composition (i.e., this 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 gelation temperature of the microgel particle-forming polymer so that microgel particles are formed in the hydrogel composition. Preferably, the mixture from step b) is cooled gradually while being constantly mixed. In one embodiment, the mixture from step b) is cooled at a constant cooling rate by applying continuous stirring / shearing. Cooling under stirring / shearing 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 refrigeration.

[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 may be, for example, 50-2000 rpm, 300-900 rpm, or 400-500 (e.g., 450) rpm. Any suitable instrument can be used to bring the required agitation / shearing. In an accompanying embodiment, a rotary rheometer (AR-G2, TA Instruments, UK) with cup and blade geometry (cup: 35 mm diameter, blade: 28 mm diameter) is used to bring the required shearing.

[0079] Pharmacologically active drugs are: i) During process (a) ii) During process b); or iii) During step c), at a point when the mixture from step b) is at a temperature above the gelation temperature of the microgel particle-forming polymer. And it 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, and the mixture 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 in solution form to the mixture in either step b) or step c).

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

[0083] A further aspect of the present invention is a method for preparing a shear-thinning hydrogel composition as defined herein, a) A step of dissolving a microgel particle-forming polymer in an aqueous vehicle containing 0.5 to 100 mM monovalent and / or polyvalent metal ion salts as crosslinking agents; b) A step of mixing the microgel-forming polymer solution formed in step (a) at a temperature exceeding the gelation temperature of the microgel particle-forming polymer; and c) A step of cooling the mixture obtained from step b) to a temperature below the gelation temperature of the microgel particle-forming polymer. This provides a method that includes [something].

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

[0085] A further aspect of the present invention provides a shear-thinning gel composition that can be obtained by, or is obtained by, or is obtained directly by, any of the preliminary methods defined herein.

[0086] Medical use of the composition of the present invention, and method of treatment using the composition of the present invention. One aspect of the present invention provides compositions of the present invention for pharmaceutical use. The compositions of the present invention are suitable for medical use in inhibiting scarring (as presented in a further aspect of the present invention); and in preventing and / or treating infection; preventing and / or treating pain; preventing and / or treating inflammation; and preventing and / or treating proliferative disorders. Compositions used for such medical use may optionally contain active agents selected from the group consisting of antifibrotic agents; anti-infective agents; analgesics; anti-inflammatory agents; anti-proliferative agents; keratolytic agents; extracellular matrix modifiers; intercellular junction modifiers; basement membrane modifiers; biological lubricants; and pigmentation modifiers.

[0087] Without deviating from the foregoing, it has been found that compositions of the present invention that do not contain pharmacologically active agents can also be successfully used to inhibit scarring. Such uses are illustrated in the data presented herein.

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

[0089] In practice of such methods, the compositions of the present invention may be administered, as necessary, to subjects requiring inhibition of scarring; subjects requiring prevention and / or treatment of infection; subjects requiring prevention and / or treatment of pain; subjects requiring prevention and / or treatment of inflammation; subjects requiring prevention and / or treatment of proliferative disorders; subjects requiring prevention and / or treatment of hyperpigmentation; subjects requiring prevention and / or treatment of hypopigmentation; subjects requiring corneal lysis; subjects requiring modification of the extracellular matrix; subjects requiring modification of intercellular junctions; and subjects requiring modification of the basement membrane.

[0090] As described above, compositions used in such treatment methods may, as needed, include active agents selected from the group consisting of antifibrotic agents; anti-infective agents; analgesics; anti-inflammatory agents; anti-proliferative agents; keratolytic agents; extracellular matrix modifiers; intercellular junction modifiers; basement membrane modifiers; biological lubricants; and pigmentation modifiers.

[0091] A method for inhibiting scarring may involve administering a composition of the present invention that does not contain a pharmacologically active agent.

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

[0093] Inhibition of scarring In numerous clinical contexts, scarring is recognized as having detrimental effects. For example, ocular scarring may be associated with an increased risk of vision loss and blindness, while skin scarring may be associated with reduced mobility, discomfort, and aesthetic impairment (which can cause psychological distress).

[0094] Scarring can lead to complications and therefore reduce the effectiveness of surgical procedures. For example, scarring that occurs after the surgical insertion of a stent (e.g., in the treatment of glaucoma) can completely or partially occlude the passage within the stent, thus rendering the surgery ineffective.

[0095] It should be understood that "inhibition of scarring" encompasses both partial and complete inhibition of scarring. Preferred values ​​related to the range in which scarring can 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 parts. For example, the compositions of the present invention can be used in inhibiting scarring in the eyes; scarring in the skin; scarring in muscles or tendons; scarring in nerves; fibrosis of internal organs, such as the liver or lungs; or the formation of adhesions, such as surgical adhesions or omental adhesions.

[0097] Scarring in the eye that can be inhibited by the medical use of the compositions of the present invention includes corneal scarring, retinal scarring, ocular surface scarring, and scarring of or around the optic nerve. While the compositions of the present invention are suitable for topical use, it should be understood that topically administered drugs may have an effect on internal structures. Therefore, compositions administered to the surface of the eye may be effective in inhibiting intraocular scarring.

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

[0099] Keratitis can also result from injury or autoimmune diseases, including rheumatoid arthritis or Sjögren's syndrome. 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 the medical use of the compositions of the present invention may also include scarring associated with surgery, such as surgery for the treatment of glaucoma (e.g., by stent insertion), and surgical procedures, such as LASIK or LASEK surgery, as well as scarring associated with injuries caused by accidents.

[0101] Preferably, the compositions of the present invention for use in inhibiting scarring may contain gellan. Surprisingly, compositions of the present invention containing gellan can effectively inhibit scarring even in the absence of pharmacologically active agents, such as active antifibrotic agents. Nevertheless, the incorporation of antifibrotic agents into the compositions of the present invention exhibits beneficial properties in inhibiting scarring. Simply as an example, decorin represents an example of such antifibrotic agents that are suitable for incorporation into the compositions of the present invention for use in inhibiting scarring.

[0102] Those skilled in the art will recognize numerous suitable methodologies that enable the identification and quantification of scarring. These methodologies can also be used to identify the inhibition of scarring. Therefore, they can be used to describe the effective medical use of the compositions of the present invention, to identify therapeutically effective doses of antifibrotic agents, and in the identification and / or selection of antifibrotic agents to be incorporated into the compositions of the present invention.

[0103] Those skilled in the art will notice that there are numerous parameters that can be used to evaluate the inhibition of scarring in the eye. Examples of these will be discussed further in the examples. Some of these, such as the induction of myofibroblasts or ECM components, are common to body parts 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. Therefore, 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 reduction in the area of ​​the opaque cornea.

[0105] The ability of the composition of the present invention, which contains the antifibrotic agent decorin, to reduce corneal opacity and to maintain such reduction over time is illustrated in the data presented in the examples.

[0106] The compositions of the present invention can be used in inhibiting 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 a bodily reaction to irritation. Examples of this latter classification include systemic chemical and / or allergic reactions that cause severe blistering and shedding of the skin, as well as gene-related disorders that result in impaired skin structure and homeostasis. These reactions or disorders can result in blistering, peeling, and a dramatically increased risk and severity of wounds (even from relatively minor contact). Examples of such disorders include epidermolysis bullosa (e.g., simple epidermolysis bullosa, junctional epidermolysis bullosa, or dystrophic epidermolysis bullosa) and Kindler syndrome. The compositions or methods of the present invention are suitable for use in inhibiting scarring in subjects with such disorders.

[0107] Other parameters indicating scarring may be common to many different tissues. For example, scarring in many body parts can be indicated by an increased presence of myofibroblasts. Such an increase can be represented by increased α-smooth muscle actin expression. Therefore, inhibition of scarring can be indicated by a decrease in the number of myofibroblasts compared to a suitable control. This type of decrease in the number of myofibroblasts can be represented 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 α-smooth muscle actin (α-sma). Myofibroblasts may have numerous adverse effects on scar formation, including causing contraction in the healed area. The compositions of the present invention can inhibit α-sma expression when evaluated in vitro and in vivo.

[0109] As will be further discussed in the examples, the compositions of the present invention (with or without the antifibrotic agent decorin) can inhibit myofibroblast differentiation in vivo in an experimental model of bacterial keratitis. Compositions, particularly those incorporating decorin, can also maintain this reduced differentiation over time.

[0110] Myofibroblast differentiation can be increased in response to the action of TGF-β1, a fibrotic growth factor that induces α-sma expression. The examples present details of an in vitro test (in human dermal fibroblasts), which demonstrates the ability of the composition of the present invention to block this increase in α-sma expression. This demonstrates that the beneficial inhibition of scarring achieved by the composition of the present invention is not limited to the eye. Furthermore, the inhibition of scarring is observed even in the absence of active antifibrotic agents, suggesting that it is 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 may increase in scarring, and the structure of the ECM may differ from that observed in undamaged reference tissue. The data presented in the examples illustrate that treatment with the compositions of the present invention produces tissue in which the composition of ECM components more closely resembles that of undamaged tissue, thus illustrating the usefulness of these compositions in inhibiting scarring.

[0112] The compositions of the present invention are suitable for use in inhibiting scarring at surgical incision sites, which may otherwise occur with the healing of such surgical wounds.

[0113] An antifibrotic agent suitable for incorporation into the composition of the present invention may be capable of achieving at least 5% inhibition of fibrosis compared to a suitable control agent. For example, a suitable antifibrotic agent 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. An antifibrotic agent suitable for incorporation into the composition of the present invention may be capable of achieving substantially complete inhibition of scarring compared to a suitable control agent.

[0114] Similarly, the medical use of the compositions of the present invention, or methods of treatment using such compositions to inhibit scarring, can achieve at least 5% inhibition compared to a preferred control. For example, such medical use or methods of treatment may 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 preferred control. The medical use or methods of treatment of the present invention can achieve substantially complete inhibition of scarring compared to a preferred control.

[0115] The selection of suitable controls will be readily determined by those skilled in the art. For example, suitable controls for evaluating the ability of the compositions of the present invention to inhibit scarring in the eye can be provided by recognized standard treatments or their experimental alternatives.

[0116] Active agents suitable for incorporation into the composition of the present invention The compositions of the present invention intended for use in medical use or in methods of treatment may contain further active agents. Suitable active agents can be selected with respect to the intended medical use. However, as an example, suitable active agents may be selected from the group consisting of antifibrotic agents; anti-infective agents; analgesics; anti-inflammatory agents; anti-proliferative agents; keratolytic agents; extracellular matrix modifiers; intercellular junction modifiers; basement membrane modifiers; biological lubricants and pigmentation modifiers. To avoid doubt, the compositions of the present invention may preferably contain more than one active agent. Where a composition contains more than one active agent, the active agents may be more than one active agent within a particular class of active agents (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 composition of the present invention are discussed in more detail below.

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

[0119] Compositions of the present invention containing an anti-infective agent can be used in methods for the prevention and / or treatment of infection. Therefore, it should be understood that such compositions can be administered to subjects requiring the prevention and / or treatment of infection. Subjects requiring such prevention and / or treatment may have chronic wounds or infected wounds. For example, subjects at risk of developing chronic wounds may have diabetes, chronic venous insufficiency, or peripheral artery occlusive disease.

[0120] Embodiments of the compositions or methods of the present invention utilizing anti-infective agents may also be useful for the prevention or treatment of scarring that may be associated with disorders, such as infections (e.g., bacterial keratitis).

[0121] Suitable analgesics for incorporation into the compositions of the present invention as active agents can be selected from the group consisting of analgesics, anesthetics, such as benzocaine, propalacaine, tetracaine, artikaine, dibucaine, lidocaine, prilocaine, pramoxin, and diclonin, or their esters, amides, or ethers; salicylates, such as salicylic acid or acetylsalicylic acid; redness-inducing agents, such as menthol, capsaicin, and / or camphor; and nonsteroidal anti-inflammatory drugs (NSAIDs), such as ibuprofen.

[0122] The compositions of the present invention, comprising an analgesic, can be used in methods for the prevention and / or treatment of pain. Therefore, such compositions can be administered to subjects in need of pain prevention and / or treatment. Preferably, subjects in need of such prevention and / or treatment may be individuals who have or are at risk of conditions related to skin or musculoskeletal pain.

[0123] Suitable anti-inflammatory agents for incorporation into the compositions of the present invention as active agents can be selected from the group consisting of steroids, e.g., corticosteroids (e.g., prednisolone or dexamethasone); NSAIDs, e.g., ibuprofen, or COX-1 and / or COX-2 enzyme inhibitors; antihistamines, e.g., 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, but will also find that the scientific and clinical literature provides a wealth of information that enables the selection of appropriate doses to exert the desired activity (which is anti-inflammatory or pro-inflammatory).

[0124] Compositions of the present invention containing an anti-inflammatory agent can be used in methods for the prevention and / or treatment of inflammation. Therefore, such compositions can be administered to subjects requiring the prevention and / or treatment of inflammation. Preferably, subjects may be individuals who have or are at risk of developing chronic or acute inflammation. For example, chronic inflammation may be associated with rheumatoid arthritis or dermatitis. Acute inflammation may result from wounds.

[0125] The antiproliferative agent for incorporation into the composition of the present invention as the active agent 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 preferred example of such a TLR7 agonist is imiquimod. A preferred example of such antimetabolite is fluorouracil (5-FU).

[0126] Compositions of the present invention containing an antiproliferative agent can be used in methods for the prevention and / or treatment of proliferative disorders. Accordingly, such compositions can be administered to subjects in need of prevention and / or treatment of proliferative disorders. Preferably, subjects may be individuals who have or are at risk of developing skin proliferative disorders, such as psoriasis, cancer (e.g., melanoma or non-melanoma skin cancer), eczema, or ichthyosis.

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

[0128] Suitable extracellular matrix modifiers for incorporation into the compositions of the present invention can be selected from the group consisting of proteinases (e.g., proteinase K), matrix metalloproteinases (MMPs); membrane-bound MMPs (MTMMPs); adamarylsin (ADAM); thrombolysin-containing ADAMs (ADAMTS); disintegrins; tissue inhibitors of metalloproteinases (TIMPs); serine proteases, such as urokinases; tissue plasminogen activators; elastases; matryptases; and enzymes involved in matrix remodeling processes, such as cathepsins, heparanases, and sulfatases.

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

[0130] Suitable intercellular 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 intercellular junction modifiers may be antibodies or other peptides that affect components of intercellular junctions, such as connexins. Examples of such proteins include cadherins and α and β-catenins. Preferably, such agents can achieve microtubule inhibition. Tight junctions will be affected by inhibition by components such as occludins, claudins, and junction adhesion molecule-1 (JAM-1).

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

[0132] The compositions or methods of the present invention that utilize intercellular junction modifying agents can be used in the treatment of intractable chronic wounds, such as ulcers.

[0133] Suitable basement membrane modifiers for incorporation into the compositions of the present invention may be agents against adhesion. Such agents can be selected from the group consisting of blocking antibodies or competitive peptides that inhibit the activity of integrins, laminins, or adhesion site components (e.g., vinculin, talin, α-actinin, kindlin, etc.). Alternatively, suitable basement membrane modifiers may include proteinases, such as proteinase K.

[0134] The compositions or methods of the present invention that utilize basement membrane modifiers can also be used in the treatment of refractory chronic wounds, such as ulcers.

[0135] For the purposes of this disclosure, a biological lubricant is considered to be a drug derived from a biological source that is capable of acting as a lubricant. In one preferred embodiment, the biological lubricant for incorporation into the hydrogel composition of the present invention may be serum. As shown below, serum has therapeutic utility in the treatment of numerous eye disorders. Therefore, the hydrogel composition of the present invention containing serum may be suitable for ophthalmic use as an eye drop.

[0136] Compositions or methods of the present invention that utilize biological lubricants, such as serum, can be used in the prevention and / or treatment of conditions selected from the group consisting of dry eye syndrome and Sjögren's syndrome.

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

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

[0139] Suitable pigmentation promoters for incorporation into the compositions of the present invention include substances that affect the 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 produces pheomelanin in the presence of a cysteine ​​group). Eumelanin production requires the action of two further enzymes: tyrosinase-related proteins 1 (TRP1) and 2 (TRP2 / Dct), which rearrange DOPAchromium (produced from the spontaneous repeated oxidation of DOPAquinone) to form DHI-2-carboxylic acid (DHICA). These enzymes or their substrates also represent suitable pigmentation modifiers.

[0140] The compositions or methods of the present invention utilizing pigmentation modifiers can be used in a wide range of clinical contexts related to undesirable hypopigmentation or hyperpigmentation. These include scarring, such as surgical or pathological scarring (e.g., hypertrophic or keloid scarring).

[0141] The compositions of the present invention, comprising a depigmenting agent, can be used in methods for the prevention and / or treatment of hyperpigmentation disorders. Therefore, such compositions can be administered to subjects requiring the prevention and / or treatment of hyperpigmentation disorders. Preferably, subjects may be individuals with or at risk of developing melasma, post-inflammatory hyperpigmentation, or Addison's disease.

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

[0143] Preferably, the compositions of the present invention may contain decorin for use in combination with the anti-infective agent gentamicin and the anti-inflammatory agent prednisolone. Such compositions may contain 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 one preferred embodiment, the composition of the present invention may comprise an anti-inflammatory agent and an analgesic agent. Such a composition may be particularly useful, for example, in the context of chronic inflammatory diseases, such as dermatitis or rheumatoid arthritis, and may be desirable for preventing and / or treating pain and inflammation.

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

[0146] The compositions of the present invention for medical use can incorporate an active agent in a therapeutically effective dose. Such a therapeutically effective dose may enable the achievement of the desired clinical outcome, either as a single dose or as part of a treatment involving multiple doses. Those skilled in the art will readily recognize suitable protocols and procedures for calculating therapeutically effective doses of various types of active agents.

[0147] Preferably, the active agent can be incorporated into the composition of the present invention at a concentration between 0.1 ng / mL and 10 mg / mL. For example, the active agent can be incorporated into the composition of the present invention at a concentration 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 approximately 0.1 μg / mL and 0.5 μg / mL, preferably at approximately 0.24 μg / mL.

[0148] Antifibrotic agents Antifibrotic agents are drugs that can inhibit scarring in the target area or body part to which they are applied. Inhibition of scarring will be discussed more generally below.

[0149] Numerous antifibrotic agents are known to those skilled in the art. Therefore, it will be readily possible for those skilled in the art to identify antifibrotic agents that can be beneficially incorporated into the compositions of the present invention for use in inhibiting scarring. A list of non-exclusive examples of antifibrotic agents suitable for such use is provided below.

[0150] Suitable antifibrotic agents can be selected from the group consisting of antifibrotic extracellular matrix (ECM) components; antifibrotic growth factors (for the purposes of this disclosure, this should be considered to include antifibrotic cytokines, chemokines, etc.); polymers, such as dextran or modified dextran sulfate; and inhibitors of fibrotic agents, such as functional blocking antibodies. It should be understood that the therapeutic efficacy of such agents depends on the dose provided by the composition of the present invention. Those skilled in the art will find a wide range of literature and clinical resources that enable the selection of suitable doses of any of the listed agents to satisfy the required therapeutic objectives.

[0151] Dextran or modified dextran sulfate can exert both anti-fibrotic and pro-fibrotic effects in vivo. In the context of the anti-fibrotic use of dextran or modified dextran sulfate, those skilled in the art will understand that a suitable dose for anti-fibrotic purposes may be between 0.1 and 10 mg / kg of body weight of the subject. In a preferred embodiment, the dextran or modified dextran sulfate for use in the composition 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 and thereby blocking the function induced by the drug's activity. Examples of such activities that can be blocked include cell proliferation, cell migration, protease production, apoptosis, and anokis. As merely an example, suitable blocking antibodies may be capable of binding to one or more of the following groups of cell signaling agents: ECM components, growth factors, cytokines, chemokines, or matricines.

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

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

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

[0156] A suitable anti-fibrotic fragment of decorin may contain up to 50% of the naturally occurring molecule at full length, up to 75% of the naturally occurring molecule at full length, or up to 90% of the naturally occurring molecule at full length. A suitable anti-fibrotic fragment of decorin may contain the TGF-β binding moiety of decorin.

[0157] Antifibrillating variants of decorin will differ from naturally occurring proteoglycans in 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. As merely an example, suitable antifibrillating variants of decorin suitable for incorporation into the compositions of the present invention may have at least one, at least two, at least three, at least four, at least five, at least ten, at least fifteen, or at least 20 mutations compared to the amino acid sequence of the naturally occurring core protein.

[0158] Unless the context requires otherwise, any reference to decorin herein relating to the incorporation of this agent in the compositions of the present invention should also be understood to encompass the use of an antifibrotic fragment or antifibrotic variant of decorin.

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

[0160] Suitable anti-fibrotic growth factors 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] Inhibitors of fibrotic agents 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 functional blocking antibodies (discussed further above) or soluble fragments of cell receptors that thereby induce cellular signaling by fibrotic agents. 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] The compositions of the present invention, suitable for use in inhibiting scarring, can incorporate an antifibrotic agent in a therapeutically effective dose. Such a therapeutically effective dose may inhibit scarring either as a single dose or as part of a treatment involving multiple doses. Details of methods for evaluating the inhibition of scarring, and for calculating or recognizing the therapeutically effective dose, are discussed above.

[0163] For example, an antifibrotic agent, such as decorin, can be incorporated into the composition of the present invention at concentrations 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 approximately 0.1 μg / mL and 0.5 μg / mL, preferably at approximately 0.24 μg / mL.

[0164] Topical administration and topical compositions The compositions of the present invention are suitable for topical administration to a subject. To avoid doubt, in the context of this disclosure, “topical administration” is understood to refer to the direct administration of the composition to the surface of a body or 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 compositions of the present invention may be for administration to one or more body surfaces selected from the group consisting of the surface of the eye; skin; surface of the brain; and mucous membranes. For example, the topical compositions of the present invention may be administered to a body surface during or after surgery. Preferably, the topical compositions of the present invention may be administered to such surfaces in connection with abdominal surgery (e.g., to inhibit adhesion formation) or brain surgery (e.g., to provide the brain with a desired therapeutic agent).

[0166] The topical compositions of the present invention may be for administration to sites 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 sites of infection or injury on the surface of the eye (e.g., sites of bacterial keratitis), or to sites of infection or injury to the skin (e.g., burns or abrasions of the skin).

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

[0168] We provide a novel eye drop system for the sustained-release delivery of a potent anti-scarring molecule (hr-decolin). The novelty of this eye drop lies in the method of structuring during manufacturing, which creates a material that can transition between solid and liquid states and retain in a dynamic environment, 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 notably, the addition of hr-decolin resulted in complete re-epithelialization, as well as scar-free restoration and corneal integrity, as indicated by the reduction of 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 much vision without resorting to surgery in developing countries where corneal transplants are not available.

[0169] This report presents a novel class of eye drop materials that allows for the long-term retention of therapeutic agents on the surface of the eye while being gradually removed through the blinking process. The material is formed through shearing of a gellan-based hydrogel, a material currently used in diluted forms to concentrate eye drops (e.g., Timoptol) during the gelling process. The application of shearing prevents the formation of a continuous polymer network structure 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 shearing is applied (e.g., when extruded through an eye dropper), the continuous network structure of the particles is disrupted and the material liquefies. The subsequent removal of the shearing force results in immediate restoration. The solid-liquid-solid transition that this material can undergo means that it adheres perfectly to the surface of the eye and is gradually removed by the blinking motion of the eyelids. Importantly, gellan gum is light-transmitting, so the material can continue to transmit light after application, minimizing disturbance to the patient.

[0170] A fluid-gel eye drop has been developed that is filled with decorin, allowing for localized drug delivery and retention on the surface of the eye. The material combines structured gellan gum with the proteoglycan decorin. In addition, an FDA-approved polymer (FDA reference number 172.665) bound to clinical-grade hr-decolin, along with high optical clarity, can be rapidly introduced into clinical practice. Therefore, this study investigated the effects of fluid gels with and without hr-decolin 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 infections.

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

[0172] Fluid gel formulations and properties The processing of the fluid gel involves passing the polymer solution, gellan, through a jacketed stirring pin (pin-stirrer), where the fluid gel experiences a high level of shear while being subjected to (thermally) forces through its sol-gel transition (Figure 1a). This limits the long-range ordering usually observed in the formation of static gels and restricts the growth of gel nuclei into separate particles. [34、35]The microstructure in the eye drops prepared by this method is shown using two techniques: 1) optical microscopy, by which the refractive index of the continuous phase is manipulated using polyethylene glycol, and 2) freeze-drying for imaging using scanning electron microscopy (SEM) (Figure 1a(i) and 1a(ii), respectively). Both microscopy techniques highlight the chain-like microstructure of the resulting gelled entity, where their large length-to-width ratio and subsequent large hydrodynamic radius give rise to the properties (viscosity and elastic structuration) of the resulting material

[36] .

[0173] The unique properties of the fluid gel are such that it exhibits pseudo-solid properties at rest but can be made to flow when a force is applied. Here, an increase in the shear force exerted on the system results in non-Newtonian shear-thinning behavior typical of highly aggregated or concentrated polymer dispersions / solutions

[37] (Figure 1b). Thus, at low shear, a much higher viscosity is observed, exceeding several orders of magnitude compared to typical water-based eye drops, and it shear-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, rapidly shear-thinning through the nozzle upon application to the eye (Figure 1c). Restoration of the three-dimensional structural matrix after application is essential to obtain a high retention time on the eye surface. Removal of time-dependent shear on a time scale compared to the initial ramp was used to probe the hysteresis of the eye drops and collect information regarding such structuration. The eye drop system exhibited some thixotropy (Figure 1b), by which the majority of the original viscosity was recovered. The presence of weak interactions between the gel-ribbons was investigated using linear rheology, exploiting the generation of elastic structures at strains within the linear viscoelastic regime (Figure 1d). Initially, after shear, the fluid gel was observed to exhibit typical liquid-like behavior where the loss elastic modulus (G") exceeded the storage elastic modulus (G'). Subsequently, a crossover was achieved due to an increase in G' as a function of the formation of interactions between the gelled ribbons, at which point the system began to behave as a solid gel

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

[0174] In vitro activity of the eye drops The gellan-based eye drop system was formulated and used for drug delivery with the candidate anti-fibrotic agent hr decorin. The release rate of hr decorin from the eye drop system was approximately linear over time (Figure 2a). Turbidity was used as a measure of fibril formation (formation of large unoriented collagen fibers) shown as a function of hr decorin (Figures 2b and c). It was also clear that hr decorin plays an essential role in the kinetics of fibril formation, delaying the onset of fibril formation and reaching equilibrium faster (Figure 2b). A concentration dependence was emphasized until a minimum turbidity (>10 μg / ml) was achieved, above which the active effect of hr decorin in fibril formation inhibition was observed and no further decrease occurred beyond that concentration when exceeding the essential concentration of 0.5 μg / ml (Figure 2c). Furthermore, the assay showed that the fluid gel carrier had no effect on fibril formation and correlated closely with the collagen-only control.

[0175] In vivo efficacy of filled / unfilled eye drops against corneal opacity Using a well-established model of bacterial keratitis

[41] , anesthetized mice (n = 6 per group) were loaded with Pseudomonas aeruginosa (10 5 CFU) on the surface of the damaged cornea. A treatment protocol was developed to treat the infection based on the standard treatment for patients with bacterial keratitis. Twelve hours after incubation with Pseudomonas aeruginosa to establish corneal infection, the eyes were treated with a regimen of gentamicin (1.5%) for 1 hour for 12 hours to sterilize the infection (confirmed by swab culture).

[0176] Following the sterilization stage, two days after the initial inoculation, a single 5 μl dose of gellan eye drops was administered every four hours between 8 a.m. and 8 p.m. for a further 13 days to the treatment groups: 1) gentamicin and prednisolone (GP); 2) gentamicin, prednisolone and fluid gel (GPFG); and 3) gentamicin, prednisolone and hrdecolin fluid gel (GPDecFG) (Table 1).

[0177] Corneal images were taken at intervals throughout the 16-day experiment, and changes in corneal opacity were measured (Figure 3a). All mice were euthanized on day 16. The area of ​​opacity (independently measured by two blinded clinical ophthalmologists for each treatment group) showed earlier size reduction in eyes treated with the fluid gel and hr decorin fluid gel eye drops in addition to standard treatment, compared to eyes treated with standard treatment (gentamicin and prednisolone) alone. Therefore, on day 9, eyes treated with standard treatment using hr decorin fluid gel showed earlier size reduction than eyes treated with gentamicin and prednisolone alone (3.5 ± 0.4 mm). 2 Compared to (1.9 ± 0.3 mm²), the turbidity area was significantly lower (p < 0.001). 2 On day 12, mice treated with hr decorin fluid gel eye drops with standard treatment maintained a significantly lower (p<0.01) turbidity area compared to the gentamicin and prednisolone groups, as well as the fluid gel group with standard treatment (mean turbidity area in group 1 = 3.5 ± 0.7 mm²). 2 , group 2=3.0±0.1mm 2 , group 3=2.1±0.2mm 2 (Compared to Figure 3b)

[0178] The effect of fluid gel eye drops containing or not containing hr decorin on corneal re-epithelialization. Epithelial lamination / maturation, along with interstitial thickness, was selected as an outcome measure to assess corneal re-epithelialization and to observe interstitial thickening due to edema and cell infiltration (as a marker of infection). Pseudomonas infection severely disrupted corneal structure, with a mean increase in corneal thickness of 218.7 ± 24 μm on post-infection day 2 compared to an untreated corneal thickness of 129.3 ± 10.7 μm. Infected corneas on day 2 had a thinner epithelial layer compared to normal, intact controls (19.2 ± 2.1 μm vs. 35.5 ± 1.7 μm; Figures 4a and b). Treatment with fluid gel alone and eye drops containing hr decorin over 13 days clearly improved re-epithelialization. Treatment with hr-decolin-filled fluid gel eye drops resulted in improved degree of lamination in the epithelial layer (26.1±2.4 μm thickness, made from a 3.6±0.2 cell layer) compared to the gentamicin and prednisolone group (22.5±2.1 μm thickness with a 2.7±0.2 cell layer) and the gentamicin, prednisolone, and fluid gel group (22.8±1.3 μm thickness with a 3.4±0.1 cell layer). However, the differences between the various groups did not reach statistical significance (Figures 4b, c, and d).

[0179] Effects of fluid gels on myofibroblasts and extracellular matrix levels Immunoreactivity (IR) was used to assess the degree of fibrosis as the ratio of pixel intensities exceeding the baseline (referred to here as the threshold) obtained from intact corneas. In untreated, intact corneas, very low levels of αSMA immunoreactivity (IR) were observed in the corneal interstitium, indicating the near absence of myofibroblasts (Figure 5a). Two days after infection and one day after sterilization, infected corneas showed a 23% increase in interstitium αSMA staining to a level exceeding the threshold of 26.5 ± 3.0% (normalized from intact corneas), indicating increased myofibroblast differentiation. Interstitium IR αSMA levels were still elevated at 32.7 ± 6.1% on day 16 in eyes treated with standard treatment alone. Whether the eye was treated with a fluid gel eye drop containing or without hr decorin, the interstitial αSMA IR levels were significantly lower on day 16, at 13.4±2.9% and 2.0±0.4%, respectively, suggesting lower activation of myofibroblasts in the corneal interstitium. The hr decorin fluid gel was most effective in maintaining low αSMA IR levels, resulting in values ​​similar to those of intact corneas, suggesting that the addition of hr decorin to the fluid gel has an additional beneficial effect on myofibroblast differentiation compared to the fluid gel alone (Figure 5a).

[0180] ECM levels in the interstitium generated by myofibroblasts were tested using fibronectin and laminin IR (Figures 5b and c). An increase in the amount of interstitium IR fibronectin was observed on day 2 post-infection and remained high on day 16 after gentamicin and prednisolone treatment (IR fibronectin 83.9±5.5% and 75.3±11.5% on day 0 and day 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, showing a significant difference on the borderline between the two eye drop treatment groups (p=0.051). The level of IR laminin (Figure 5c) indicates that infection increased laminin levels from 2.15±0.6% in intact cornea to 16.3±4.6% in the infected group on day 2, compared to intact cornea. IR laminin levels continued to rise to 42.5±8.2% up to day 16 after gentamicin and prednisolone treatment. Similar to the gentamicin and prednisolone group, the mean IR laminin level remained high at 38.0±12.0% on day 16 after treatment with the fluid gel. Addition of hr decorin to the fluid gel significantly reduced laminin levels compared to gentamicin and prednisolone treatment (12.4±5.5% vs. 42.3±8.2%), while the fluid gel without hr decorin had no effect on this ECM parameter.

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

[0182] The experimental hydrogel compositions of the present invention were prepared with or without the antifibrotic agent decorin. These are shown as "GEL+dec" and "GEL-dec" in the graph of Figure 25, respectively. During the test, 1 ml of the experimental gel composition was added to each well, and then administered with TGF-β1 at 5 ng / ml (with the exception of "GEL+dec (second gel)," where TGF-β1 was introduced before the gel administration, indicating that the order did not significantly alter the effect).

[0183] As can be seen in Figure 25, the addition of TGF-β1 stimulated α-sma expression, leading to the formation of myofibroblasts characteristic of scarring. Supply of the hydrogel composition of the present invention reduced this α-sma expression. This was observed both in and out of the antifibrotic agent decorin, and explains the ability of the hydrogel composition of the present invention to inhibit scarring even in the absence of further antifibrotic active agents.

[0184] Consideration Corneal prelacical membrane turnover (approximately 20% per minute)

[42] Because the rapid elimination of aqueous drugs reduces the potency delivered to the target tissue site, improved retention in the eye is crucial for increasing both biological efficiency and the therapeutic response to topical therapy. Therefore, many ocular conditions are currently treated through invasive methods disliked by many patients, including intensive topical therapy delivered day and night, or intraocular or intravitreal injections targeting intraocular pathologies. In more severe cases where drugs are ineffective, surgery may be required to treat or remove the resulting corneal scarring, increasing the risk of morbidity and the duration of patient discomfort following the treatment. Structured or "fluid-gels" formed from gelan represent a significant advance because they enable sustained-release delivery of molecules such as hr-decolin, which can prevent scarring and eliminate the need for invasive surgical repair strategies. The primary benefit of gelan fluid-gels is their ability to transition between solid and liquid states, such as passing through an applicator and solidifying on the surface of the cornea. This unique set of properties stems from the microstructure of the material, 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 reorganized following the removal of the 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 corneal surface results in the formation of a transparent and absorbable bandage, possessing the benefits of eye drops (in application) and hydrogel lenses (sustained release) without the drawbacks of either. In fact, the fluid-gel alone appears to create a microenvironment that aids wound healing, with a reduction in markers of corneal opacity and scar formation, even without the addition of decorin. Importantly, as indicated by collagen fibrillation data, the fluid-gel does not interfere with the biological activity of hr decorin. Thus, the system provides an excellent candidate technology for clinical situations, accompanied by improved drug adherence across countless patient cohorts.

[0185] A mouse model of Pseudomonas keratitis provides a robust and clinically relevant method for evaluating the antiscarring capacity of hr decorin-filled fluid gel against current standard treatments for Pseudomonas infection (gentamicin and prednisolone).

[43] Once infection is established, Pseudomonas aeruginosa invades corneal epithelial cells, disrupting the natural healing response that involves the conversion of corneal fibroblasts into corneal myofibroblasts, and creating a fibrous microenvironment.

[44] Topical administration of eye drops with or without hr-decolin resulted in a reduction in corneal opacity levels 7 and 10 days after eye drop treatment, and the addition of hr-decolin showed a clear further benefit. The effect of treatment with fluid gel alone was not as predicted in the initial in vitro study, indicating that this carrier appears inactive. The therapeutic efficacy of fluid gel alone may be due to the formation of an acceptable microenvironment in the damaged cornea, and the sealing effect of the gel ribbon (forming a barrier around the entangled wound) yields two key effects: firstly, a therapeutic bandage that prevents biomechanical trauma caused by blinking over the ulcerated eye; and secondly, encapsulating steroids and gentamicin within the structure, enhancing the retention of therapeutic substances on the ocular surface, and thereby improving bioavailability, with the added benefit of being similar to but absorbable, an artificial replacement of the ecosystem (PROSE® device). Such a reduction in corneal opacity would be beneficial to the patient in terms of preserving vision.

[45] .

[0186] A key aspect of the healing process involves the restoration of the layered, non-keratinized epithelium. Along with the lacrimal membrane, the apical mucosa (composed of lipid, mucin, and aqueous layers) provides nourishment and lubrication to the ocular surface and is crucial for the eye's frontline defense. Eyes treated with hr-decolin showed the most improved restoration to normal anatomical structure, with reduced interstitial edema, thickness, and extracellular matrix deposition, and improved epithelial morphology. The reduction of fibrosis markers by hr-decolin has been previously demonstrated across numerous animal models, regulating various growth factors (e.g., VEGF, IGF-1, EGF, PDGF) and their receptors, particularly TGFβ signaling via the SMAD2 and SMAD3 pathways, and preventing corneal fibroblast differentiation. In addition, regulation of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) leads to fibrolysis and reduced scar formation. [29、46~48] .

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

[0188] The effect of the fluid gel alone on the damaged corneal surface suggests an influence over endogenous growth factors, and this effect is enhanced by the addition of hr-decolin. The fluid gel can assist in corneal healing through several mechanisms: firstly, the fluid gel's unique viscoelastic properties act as a self-organizing liquid on the ocular surface, forming a semi-solid therapeutic occlusive dressing for stable healing; secondly, the helix domains formed during the gelation of the fluid gel provide a pseudo-scaffold for the binding of endogenous decorin, sealing key growth factors, such as TGF-β and / or exogenously delivered hr-decolin; and thirdly, the fluid gel matrix, primarily water (99.1%), creates the diffusion of cytokines outside the wound site, induced by the gradient, resulting in the restoration of the natural equilibrium necessary to prevent fibrosis again.

[0189] In conclusion, the novel eye drop technology has been shown to be usable to provide local, sustained-release delivery of antifibrotic agents such as hr-decolin to the cornea in a clinically relevant mouse model of fibrosis associated with bacterial keratitis. The eye drops allow hr-decolin to remain in contact with the surface of the eye for a sufficiently long time and with sufficient potency, significantly reducing corneal scarring. Furthermore, this study shows that unfilled fluid gels also possess healing effects, suggested to arise from their inherent material microstructure and subsequent properties. The material properties of the eye drops not only enhance the retention time of the anti-scarring agent, but the easy-to-use nature of the eye drops will be welcomed by patients and will provide a simple treatment for preventing scarring pathology that frequently occurs after corneal infection. The technique has demonstrated a successful reduction in corneal opacity and a decrease in markers generally indicating the scarring process. Compared to current standard treatments, this technique offers an ideal treatment option for patients with bacterial keratitis, potentially reducing the incidence of visually significant corneal opacity and eliminating the need for corrective surgical intervention. Given the limited availability of transplants and surgical facilities in developing countries, this technique could potentially help save the vision of a large number of patients in the future.

[0190] Materials and methods Test design The objective of this study was to investigate the use of a novel fluid gel for the delivery of hr-decolin to the ocular surface to reduce corneal opacity and scarring after bacterial keratitis. The study was divided into three evaluation stages, using a mouse model of pseudomonas keratitis (eyes sterilized after infection) and comparing it with current standard treatment: (i) material properties relating to the ease of application of eye drops, (ii) in vitro evaluation of the bioactivity of formulated hr-decolin, and (iii) in vivo anti-scarring efficacy of fluid gels with / without hr-decolin. Since the effect size was unknown, the sample size (n=6 per experimental group) was based on the resource equation. All analyses were performed by blinded observers for experimental grouping, and mice were randomly assigned to both the treatment and control groups.

[0191] material Manufacturing of fluid gels (FG) and hrdecolin fluid gels (DecFG) Preparation of fluid gel eye drops The fluid gel was first prepared by 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 cup and blade geometry (cup: diameter 35 mm, blade: diameter 28 mm). The system was then cooled to 40 °C. hr decorin in PBS (Galacorin™; Catalent, USA) (4.76 mg / ml) and an aqueous sodium chloride solution (0.2 M) were then added to obtain final concentrations of 0.9% (w / v) gellan, 0.24 mg / ml hr decorin and 10 mM NaCl. Subsequently, the mixture was cooled at a rate of 1 °C / min under shear (450 / s) to a final temperature of 20 °C. The samples were then removed and stored at 4 °C until further use. For the fluid gel without hr decorin, the ratio was adjusted so that the final eye drop had a composition of 0.9% (w / v) gellan and 10 mM NaCl.

[0192] Characterization of the materials of the fluid gel eye drops Microscopy: For transmission microscopy, samples were first diluted using polyethylene glycol 400 (PEG400) in a ratio of 1:4 (eye drop to PEG400). Subsequently, the samples were analyzed using an Olympus FV3000. The 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, the sample was first prepared for freeze-drying by diluting gelan in deionized water in a 1:9 ratio, using the same method as for transmission microscopy. The sample was then rapidly frozen using liquid nitrogen and left in a freeze-dryer overnight to leave a powder. The dried sample was then attached to a carbon stub and analyzed using SEM.

[0194] Rheology: Viscosity profiles were obtained at 20°C using an AR-G2 (TA Instruments, UK) rheometer equipped with a sandblasted parallel plate (40 mm, gap height 1 mm). A 2-minute equilibrium period was used to ensure a constant test temperature. Subsequently, time-dependent up and down ramps were applied in the range of 0.1–600 / s (sweep time 3 minutes). Recovery profiles were obtained at a single frequency using the same instrument. The samples were rejuvenated by shearing at 600 / s for 10 seconds. Subsequently, storage and loss (G' and 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 hrdecolin from fluid gel The level of hr decorin released from the gel was cumulatively determined by placing 1 ml of fluid gel containing hr decorin into a 6-well plate. Next, 2 ml of DMEM was placed on top of 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-decolin bioactivity assay Collagen fibril formation: For dose-response curves, 75 μl of PBS was added to each well of a 96-well plate placed on ice. Various hr-decolin doses were prepared by adding 400 μg / ml of hr-decolin to the first well, followed by serial dilution (2-fold dilution) of the entire 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 at 37°C for 2 hours. Absorbance readings were then taken using a 405 nm plate reader. Each assay consisted of two blank controls, three standard dilutions, followed by three sample dilutions. The kinetics of fibril formation were determined by incubating the sample in a plate reader and taking data points every 2 minutes, using a configuration similar to the dose-response without serial dilution.

[0197] Pseudomonas keratitis model and in vivo stereomicroscopy method The treatment regimen for the in vivo Pseudomonas model is shown in Figure 6. The experimental plan also included untreated, intact corneas and infected corneas taken on day 2. Because the effect size is unknown, the n=6 sample size 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 subsequently infected with Pseudomonas. The treatment procedures and sample sizes are described in further detail below. For in vivo studies, analysis was performed by blinded investigators for each experimental group.

[0198] In vivo mouse model of Pseudomonas keratitis Pseudomonas aeruginosa strain PAO1 was cultured at 37°C for 18 hours in high-salt LB (10 g tryptone, 5 g yeast extract, and 11.7 g NaCl, 10 mM MgCl2, and 0.5 mM CaCl2 per liter). Subcultures were obtained at an optical density (OD) of 0.2 (OD 650 nm, approximately 1 × 10⁻⁶). 8 (CFU / ml). Wash Pseudomonas aeruginosa in PBS (x3), centrifuge at 300 rpm for 5 minutes, and add 1 x 10⁶ to PBS.5 The bacteria were resuspended in 2.5 μl of CFU. C57BL / 6 mice (Jackson Laboratory, CA, USA) were housed in a pathogen-free state, following the ARRIVE guidelines, the ARVO statement for the use of animals in ophthalmic and vision research, and the guidelines presented by the University of California, Irvine. They were allowed free access to water and food. For inoculation, the mice were anesthetized, and one corneal epithelium was scraped using a 26G needle with parallel scratches measuring 3 × 1 mm, and 2.5 μl of Pseudomonas aeruginosa (1 × 10⁶) was added. 5 Inoculated with CFU (PAO1 strain) 64、65 The mice were kept at rest for 2 hours after inoculation to allow the infection to penetrate the eye and recover. 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, the mice were administered eye drops (5 μl of each compound) every 4 hours between 8 a.m. and 8 p.m. for a further 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 containing hr decorin. The mice were examined for corneal opacity formation, ulceration, and perforation. En-face 24-bit color images of the cornea were taken using a SPOT RTKE camera (Diagnostic Instruments) connected to a Leica MZF III stereomicroscope. Mice were euthanized on day 16 by cervical dislocation under anesthesia, their eyes were removed, and they were placed in 4% PFA in PBS for immunohistochemical staining.

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

[0200] Tissue treatment and immunohistochemical staining for re-epithelialization and ECM. Eyes extracted for IHC were post-fixed by immersion in 4% PFA in PBS at 4°C overnight, and then cryoprotected for 24 hours each at 4°C using increasing concentrations of sucrose in PBS (10%, 20%, and 30%; Sigma). The eyes were then embedded in embedding medium (Thermo Shandon, Runcorn, UK) at the optimal cutting temperature (OCT) in a peel-away mold container (Agar Scientific, Essex, UK), and subsequently sectioned to a thickness of 15 μm 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 tests and stored at -80°C. Frozen tissue sections (optic nerve plane) were thawed for 30 minutes, then washed with PBS for 3 x 5 minutes, followed by permeabilization with 0.1% Triton X-100 (Sigma) for 20 minutes. Nonspecific antibody binding sites in the tissue sections were blocked for 30 minutes using 0.5% BSA, 0.3% Tween-20 (all from Sigma) and 15% normal goat serum (Vector Laboratories, Peterborough, UK) in PBS. Subsequently, the sections were incubated overnight at 4°C in primary antibodies (αSMA, laminin, and fibronectin; 1:200; all from Sigma), washed again for 3 x 5 minutes, and incubated with secondary antibodies (goat anti-mouse Alexa Fluor 488 1:500, goat anti-mouse Alexa Fluor 594 1:500, Molecular Probes, Paisley, UK) at room temperature for 1 hour. The sections were then washed for 3 × 5 minutes and mounted in Vectorshield mounting medium (Vector Laboratories) containing DAPI. All control tissue sections incubated with secondary antibody alone were negatively stained.

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

[0202] statistical analysis All statistical analyses were performed using SPSS 20 (IBM, Chicago, IL, USA). A normal distribution test was performed to determine the most appropriate statistical analysis for comparing treatments. Statistical significance was defined as P<0.05. For turbidity measurements, corneal width, epithelial thickness, αSMA, fibronectin, and laminin data were evaluated using ANOVA along with Tukey's post-hoc test. For DAPI measurements of epithelial cell layer count, 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" (particulate suspension): Viscosity / Flow Behavior The optimal viscosity of eye drops was determined through two main methods: rheological characterization of currently available eye drops / ointments, and consultation with ophthalmic clinicians. Characterization of commercially available ophthalmic products highlights a wide range of viscosities across both eye drops and ophthalmic ointments used for drug application in conditions such as dry eye, where optimal long retention time is required. Viscosity was collected, and 1s -1 The similar viscosities between products were compared (selected as the initial value of shear viscosity reduction to avoid artificial results for the apparatus) (Table 2 (Table 4) and Figure 6 (Section A.1)), highlighting the similar viscosities between products as a function of polymers mainly based on paraffin, carbomer, and biopolymer.

[0207] [Table 3]

[0208] For ophthalmic products based on both paraffin and carbomer, a warning is included in the instructions to inform patients that blurred vision and discomfort may occur due to the eye drops. Therefore, the outer viscosity limits 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. Therefore, all formulations prepared using gellan as the biopolymer used for gelling could be used within these limits. However, the most 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 the eye drop products they deemed appropriate. From this data, 5-50 Pa.s Eye drops within this viscosity range are easier to apply and retain well. Approximately 10~20Pa.s It was found to involve an optimal drop.

[0211] Furthermore, the system should exhibit shear-reducing viscosity behavior.

[0212] 1.1.1. Defined Parameters

[0213] [Table 4]

[0214] elasticity Resting elasticity plays a significant role in the use of products that are retained in a controlled manner and deliver active ingredients. The ability of microgel suspensions to create a weakly elastic network structure at rest is thought to result in high retention times for products. Again, the limits are based on the characterization of commercially available eye drops and ointments (Figure 3 (Section A.1)). Similar correlations were observed among various products, as seen for viscosity, and products were grouped within polymer types (Table 4 (Table 6)).

[0215] [Table 5]

[0216] Again, similar to viscosity, none of the tested formulations exceeded the values ​​obtained for the current product. Therefore, all formulations prepared using gellan as the biopolymer used for gelling could be used within these limits. Maximum -20000 Pa; minimum 1 Pa

[0217] However, when analyzed by clinicians, this 1-250 Pa The options were narrowed down, and the optimal formulation is 20-40 Pa It extended to that range.

[0218] 1.1.2. Defined Parameters

[0219] [Table 6]

[0220] pH Due to the chemical composition of biopolymers and the various chemical parts along their individual skeletons, they have a wide range of natural pH values. Since numerous chemical damages occur in the pH < 4 and pH > 10 ranges, and normal physiological function is close to 7.11 ± 1.5, the pH of products that come into contact with the ocular surface is important. Therefore, eye drops are formulated within this range (4-10), with some products having a pH of around 3.5 (propalacaine hydrochloride solution). 1 Therefore, based on this data from the literature, eye drop formulations are 3.5~8.6 It should have a pH within the specified range.

[0221] However, the delivery of numerous active substances, including proteins, requires the formulation to be neutral. In these cases, PBS (phosphate-buffered saline) can be added to the eye drops to limit the pH to a neutral acidity. Therefore, the pH in the formulation 6.5~7.5 The formulations have been narrowed down and optimized. 7.4 That is the case.

[0222] 1.1.3. Defined Parameters

[0223] [Table 7]

[0224] 2. "Fluid gel" (particulate suspension) formulations: Biopolymer concentration (Please refer to Experiment Article A.1)

[0225] Ultimately, the material properties of the formulation are controlled by the concentration of the initial polymer in the product. Therefore, upper and lower limits of polymer concentration were indicated and used to evaluate the upper and lower material properties of the material formulation. Since all systems exhibit shear-thinning behavior, the limit is viscosity (1 s). -1 ) was based solely on meeting the criteria for both the elastic behavior at rest and the elastic behavior at rest. Therefore, 0.5~2.5% (w / v) The maximum range was set for eye drop formulations, and values ​​within this range were accepted for commercially available products. This is to be applicable to clinician advice. 0.5~1.5% (w / v) The narrowed-down and optimized formulation is 0.9% (w / v) It consists of.

[0226] 2.1.1 Defined Parameters

[0227] [Table 8]

[0228] Crosslinker Concentration (Please refer to Experiment Article A.2)

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

[0230] However, the mechanical spectrum showed that at higher salt concentrations, a significant decrease in the elastic network structure occurred when deformed outside the linear viscoelastic region. This suggests that lower salt concentrations result in more plastic behavior, which would likely be more comfortable for the patient. Therefore, the narrowed limits for the formulation are: 5-20 mM The adjusted and optimized formulation is 10 mM.

[0231] 2.1.2.PBS The addition of PBS can be used to manipulate the pH of the system. In these cases, 5% v / v (5% is determined to be the amount added along with therapeutic decorin, and therefore no further testing has been performed in this region) is added, which will affect the salt levels in the system. The concentrations of monovalent ions in PBS were calculated and summarized in Table 8 (Table 10).

[0232] [Table 9]

[0233] Therefore, the range for crosslinkers was modified (Table 9 (Table 11)), and the lower limit was reduced so that the ion content in PBS was sufficient to induce the gelation process.

[0234] 2.1.3. Defined Parameters

[0235] [Table 10]

[0236] 3. Processing parameters for "fluid gel" (particulate suspension): heat treatment Heat treatment during manufacturing is crucial for gel formation. Typically, thermal parameters are divided into two categories: processing temperature and cooling rate.

[0237] 4.1.1. Processing temperature: The inlet and outlet temperatures are crucial to ensuring the polymer is in a sol state before processing and exits at a temperature below the gelation transition temperature. Initially, due to the protein-active substances that denature actively at higher temperatures, the inlet temperature was set as close to the gelation temperature as possible. Therefore, the inlet temperature was set to 40°C. However, this is not necessary; the crucial 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 ordering / structuring of the polymer is completed before storage. This prevents aggregation and the formation of heterogeneous suspensions during the process. 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 pulverizer jacket and set to ensure sufficient cooling during processing. This can be varied to result in various cooling rates.

[0238] 4.1.2. Cooling rate (Please refer to Experiment Article A.3) It is well 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 structure formation and a weaker overall elastic modulus. This is observed for microgel suspensions, however, only at higher polymer concentrations. For optimal eye drop formulations, no change in material properties is observed. 0.1~6℃min -1 This suggested that a wide range of parameters could be used.

[0239] On the other hand, for 1.8% w / v polymers, the elasticity depended more on the required structure.

[0240] 4.1.3. Defined Parameters

[0241] [Table 11]

[0242] 4.2 Shear rate (Please refer to Experiment Article A.3)

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

[0244] 4.2.1 Defined Parameters

[0245] [Table 12]

[0246] 5. Overview of suspension parameters:

[0247] [Table 13]

[0248] Further experimental data A.1. Experiment - Gellan concentration: Effect of polymer concentration on the reaction of the resulting fluid gel material the purpose: To understand how polymer concentration affects key material properties (viscosity and elasticity) following treatment of a microgel suspension. • Narrow down the acceptable range of polymer concentrations for suitable eye drop formulations.

[0249] Materials and methods: material: • Gerlan (Kelco) • NaCl (Fisher Chemicals, Lot No.: 1665066)

[0250] Preparation of gelan microgel suspension (MS): Preparation of the stock solution: Preparation of NaCl solution NaCl (0.2M) was prepared by adding dry crystals (1.16g) to deionized water (100ml) using a volumetric flask. The NaCl was then dissolved using the inverting technique to assist the process. 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 water / NaCl solutions in various ratios such that the final concentrations after processing were equal to 0.5, 0.9, 1.35, 1.8, and 2.35% (w / v). 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 with stirring to dissolve the polymer. Once fully dissolved, 25 ml of stock NaCl solution (0.2 M) was added to the solution, and a concentration of 10 mM was obtained after processing. The sols were then brought to thermal equilibrium at 95°C and subsequently processed.

[0252] Preparation of Gelan MS: MS was prepared using a jacketed pin mill set to 20°C. Gelansol was injected into the pin mill at 3 ml / min using a peristaltic pump so that it entered the processing chamber at 40°C. Before entry using a syringe and syringe pump, water was injected into the gellan stream so that they collided (at a rate of 0.16 ml / min) to dilute the gellansol to its final concentration (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 milling unit. Upon exit, the gel was packaged at 20°C 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 Corporation, AR-G2) equipped with a sandblasted parallel plate (40 mm in diameter, 1 mm gap height). The results are shown in Figures 7-9.

[0254] Amplitude sweep: Amplitude sweeps were obtained in strain control mode over a range of 0.1–100.0%. The sample was mounted on the instrument, and the upper geometric shape was lowered. After trimming, the sample was brought to equilibrium at 20°C before testing. Measurements were obtained at 1 Hz using logarithmic scales.

[0255] Flow profile: Viscosity profiles for the samples were obtained using a continuous ramp. The samples were mounted in the instrument, and the upper geometric shape was lowered. After trimming, the samples were brought to equilibrium at 20°C before testing. Increasing shear was applied in a speed-controlled mode for 0.1–600 s. -1 The sample was applied over a 3-minute ramp, and data points were obtained using logarithmic methods.

[0256] result: Rheology of small deformations: Please refer to Figures 7-9 and the above discussion. Rheology of large deformities: Please refer to Figures 10-12 and the above discussion.

[0257] Consideration: The effect of polymer concentration can be observed in both these elastic properties and viscosity, both of which show the same trend and increase until reaching a plateau at concentrations above 1.8% (w / v) (Figures 2 and 3). Such observations arise from the formation of microgelled particles, and by applying shear during the gelation of the polymer system, confinement prevents the formation of a continuous network structure. The main result of this treatment is that the gelled entities are dispersed in a non-gelling medium, similar to the W1 / W2 emulsion. Thus, the rheology of the suspension is closely correlated with the rheology of the emulsion, and an increase in the phase volume of droplets or particles leads to (in this case) closer proximity and an increase in both the elastic properties (G') and viscosity of the system. In this case, increasing the polymer concentration results in a larger number of particles until the maximum packing density is achieved. Beyond this, no further changes are observed in the material properties.

[0258] The elasticity (storage modulus, G') and viscosity of various suspensions were compared with data collected for current eye drops / ointments across various material-based systems: paraffin, carbomer, and biopolymer (Figures 3 and 6). All gelane systems were observed to exhibit G' and viscosity within the thresholds of current commercially available ophthalmic products, suggesting that all systems would be suitable regardless of polymer concentration. However, for ease of application (from single-use applicators) and comfort (blurred vision as described on the packaging), values ​​closest to those of carbomer and biopolymer-based eye drops were optimal. Therefore, gelane concentrations in the range of 0.5–1.35% (w / v) were most suitable. In addition, consultation with independent clinicians considering ophthalmic application resulted in a concentration of 0.9% (w / v), which best mimicked the clinician-defined properties.

[0259] The yielding behavior of a suspension is also very important, especially within retention mechanisms for delivery, because rapidly yielding systems result in quick clearance. Conversely, if a system does not yield at all, the material will not be easily expelled from the body. The linear viscoelastic region (LVR) is a good indicator of the yielding behavior of a suspension; as the system moves away from this linear region, weak interparticle interactions begin to break down and the system flows. 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 be manipulated with higher strain before breakdown, providing a sealing barrier in the dynamic region of the body where it will be slowly reabsorbed. Similar LVRs are observed in the range of 0.5–1.35% (w / v), suggesting that they will behave similarly.

[0260] The following yield shear viscosity reduction behavior is significant for both application and discharge, allowing the suspension to flow easily upon liquefaction. Shear viscosity reduction was observed across all systems regardless of polymer concentration (Figure 4). The high degree of shear viscosity reduction resulting from the breakdown of interparticle interactions and alignment during flow allows the system to be easily applied through nozzles (syringes, single-use applicators, etc.), with low pressure resulting in a high level of shear.

[0261] Conclusion: In summary, it was shown that polymer concentration plays a key role in the material properties of the resulting gelane microgel suspension. Material characteristics, such as elasticity (expressed by the material's inherent G' value) and viscosity, were found to be functions of polymer concentration, increasing until a plateau was formed at 1.8% (w / v). Effectively, this meant that, in close comparison with 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 from small openings. Furthermore, through comparison with commercially available products and discussions with independent clinicians, the polymer range between 0.5–1.35% (w / v) was narrowed down to 0.9% (w / v), which was deemed optimal for the final formulation.

[0262] A.2. Experiment - Crosslinker (NaCl) Concentration: Effect of Crosslinker Concentration on the Reaction of the Resulting Fluid Gel Material the purpose: To understand how crosslinker concentration affects key material properties (viscosity and elasticity) following treatment of microgel suspensions. • Narrow down the acceptable limits for crosslinker concentrations in suitable eye drop formulations.

[0263] Materials and methods: • Gerlan (Kelco) • NaCl (Fisher Chemicals, Lot No.: 1665066)

[0264] Preparation of gelan microgel suspension (MS): Preparation of the stock solution: Preparation of NaCl solution NaCl (0.1, 0.2, 0.4, and 0.8 M) was prepared by adding dry crystals (0.58, 1.16, 2.32, and 4.64 g) to 100 ml of deionized water using a volumetric flask. The NaCl was then dissolved using the inversion technique to assist the process. 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 water / NaCl solutions such that the final concentrations after processing were equal to 0.9% and 1.8% (w / v). Briefly, gelellan powder was weighed out (4.5g and 9.0g) and added to 450ml of deionized water. The mixture was heated to 95°C with stirring to dissolve the polymer. Once fully dissolved, 25ml of stock NaCl solution (either 0.1, 0.2, 0.4, or 0.8M) was added to the solution, and concentrations of 5, 10, 20, or 40 mM were obtained after processing. The sol was then brought to thermal equilibrium at 95°C and subsequently processed.

[0266] Preparation of Gelan MS: MS was prepared using a jacketed pin mill set to 20°C. Gelansol was injected into the pin mill at 3 ml / min using a peristaltic pump so that it entered the processing chamber at 40°C. Before entry using a syringe and syringe pump, water was injected into the gellansol stream so that they collided (at a rate of 0.16 ml / min) to dilute the gellansol to its final concentration (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 milling unit. Upon exit, the gel was packaged at 20°C 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 Corporation, AR-G2) equipped with a sandblasted parallel plate (40 mm in diameter, 1 mm gap height).

[0268] Amplitude sweep: Amplitude sweeps were obtained in strain control mode over a range of 0.1–100.0%. The sample was mounted on the instrument, and the upper geometric shape was lowered. After trimming, the sample was brought to equilibrium at 20°C before testing. Measurements were obtained at 1 Hz using logarithmic scales.

[0269] Flow profile: Viscosity profiles for the samples were obtained using a continuous ramp. The samples were mounted in the instrument, and the upper geometric shape was lowered. After trimming, the samples were brought to equilibrium at 20°C before testing. Increasing shear was applied in a speed-controlled mode for 0.1–600 s. -1 The sample was applied over a 3-minute ramp, and data points were obtained using logarithmic methods.

[0270] result: Rheology of small deformations: Please refer to Figures 13-14. Rheology of large deformities: Please refer to Figures 15-16.

[0271] Consideration: Mechanistically, salts play a crucial role in the gelation of many polymers, including gellan. The type of salt, particularly its valence (mono, di, tri, etc.), is key to the properties of the resulting gel. Typically, increasing valence increases gel strength because more crosslinks are formed between polymers. However, in the case of gellan, divalent ions, such as Ca, are important. 2+ This results in cloudiness (increased turbidity) of the resulting gel. Therefore, monovalent ions, such as Na, are harmful. + This can be used to strengthen the bonding sites between helices and form a three-dimensional gel structure. Therefore, the strength of the resulting gel is a function of the concentration of the added salt, also called the crosslinker. The effect of the crosslinker concentration on the resulting microgel suspension ("fluid gel") formed through formation and sheared gelation is clearly visible in Figures 1 and 2. Here, a correlation can be observed between the NaCl concentration and the elastic (G') reaction, which is consistent with the known gelation mechanism for both polymer concentrations tested (increased strength for higher crosslinker concentrations) (Figure 2). Furthermore, the mechanical spectrum (Figure 1) highlights the change in the material's yield properties. At the highest salt concentration (40 mM), it was observed that the material's strain dependence increased, showing a more rapid decrease in G' as it left the LVR (linear viscoelastic region). Such results closely match typical material reactions, where the gel becomes stronger and more brittle. In these cases, it is thought that the material behaves more towards fracturing as it reaches critical strain, in contrast to plastically deforming as the system becomes more densely crosslinked. While higher concentrations are diffusible, their enhanced strain dependence prevents their use in eye-like applications, and the increased plasticity during deformation results in a smoother surface, improving clarity and comfort as expected.

[0272] The effect of salt concentration on the viscosity of the eye drops was also tested. Little change was observed across all systems (Figure 2), all formulations exhibited significant shear-thinning behavior, and the overall viscosity ultimately depended on the biopolymer concentration. However, with the highest salt concentration (40 mM) of 0.9% (w / v) gelan, the overall viscosity of the suspension was lower. Such results are accompanied by increased errors that may arise from some syneresis (water leaching), and the increased density of crosslinkers pulls the polymers closer together, resulting in insufficient polymer to structure the aqueous phase. Therefore, the stability of these systems may be compromised, leading to heterogeneous systems over time.

[0273] Conclusion: In summary, the addition of salt to the biopolymer system results in operations that exceed the strength of the final product. Increasing salt concentrations ultimately increase the number of crosslinks in the system and the elastic behavior of the final material. In addition, such effects did not appear to have a dramatic change in viscosity, but at lower polymer concentrations, excessive crosslinking could lead to heterogeneous suspensions and poor stability. Demonstrating the elastic structure using strain sweeps allowed for the yield behavior of the suspensions analyzed, highlighting a higher strain dependence at the 40 mM formulation. The decrease in plasticity is predicted to cause discomfort to patients when applied to the eyes, and therefore, the upper limit for crosslinkers is suggested to be 20 mM.

[0274] A.3. Experiment - Cooling Rate: Effect of cooling rate applied during the process of manufacturing gellan microgel suspension ("fluid gel") the purpose: • Understand the role that the cooling rate plays in the material properties (viscosity and elasticity) of the resulting gelan microgel suspension. • Narrow down the cooling rate to an acceptable level suitable for processing eye drops.

[0275] Materials and methods: • Gerlan (Kelco) • NaCl (Fisher Chemicals, Lot No.: 1665066)

[0276] Preparation of gelan microgel suspension (MS): Preparation of the stock solution: Preparation of NaCl solution NaCl (0.2M) was prepared by adding dry crystals (1.16g) to deionized water (100ml) using a volumetric flask. The NaCl was then dissolved using the inversion technique to assist the process. 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 water / NaCl solutions such that the final concentrations after processing were equal to 0.9% and 1.8% (w / v). Briefly, gelane powder was weighed out (4.5g and 9.0g) and added to 475ml of deionized water. The mixture was heated to 95°C with stirring to dissolve the polymer. Once fully dissolved, 25ml of stock NaCl solution (0.2M) was added to the gelane sol to obtain a final concentration of 10mM. The sol was then brought to thermal equilibrium at 95°C and subsequently processed.

[0278] Preparation of Gelan MS: MS was prepared using a jacketed pin grinder, thereby changing the jacket temperature and residence time in the grinder by 1, 3, and 6°C. -1 The following cooling rate was obtained. As an example, the jacket was set to 5°C, and 20 ml -1 Assuming a flow velocity of , the fluid temperature is 46°C at the inlet and 16°C at the outlet, the residence time at this velocity is 5 minutes, and therefore the cooling rate is 6°C. -1 It was equivalent to [the other result]. Upon release, the gel was 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 Corporation, AR-G2) equipped with a sandblasted parallel plate (40 mm in diameter, 1 mm gap height).

[0280] Amplitude sweep: Amplitude sweeps were obtained in strain control mode over a range of 0.1–100.0%. The sample was mounted on the instrument, and the upper geometric shape was lowered. After trimming, the sample was brought to equilibrium at 20°C before testing. Measurements were obtained at 1 Hz using logarithmic scales.

[0281] Flow profile: Viscosity profiles for the samples were obtained using a continuous ramp. The samples were mounted in the instrument, and the upper geometric shape was lowered. After trimming, the samples were brought to equilibrium at 20°C before testing. Increasing shear was applied in a speed-controlled mode for 0.1–600 s. -1 The sample was applied over a 3-minute ramp, and data points were obtained using logarithmic methods.

[0282] result: Rheology of small deformations: See Figures 17 and 18. Rheology of large deformities: See Figures 19 and 20.

[0283] Consideration: Cooling plays a crucial role in the formation of gelan hydrogels, causing the polymer to transition from a random coil to a helix. To evaluate the changes in the associated material reactions, the effect of the cooling rate on the formation of the fluid gel was tested. At lower polymer concentrations (0.9% (w / v)), the cooling rate was observed to have almost no effect on both the degree of elasticity and the overall viscosity in the system. However, at higher concentrations (1.8% (w / v)), the cooling rate had a much more pronounced effect on the modulus of elasticity (G') (Figure 2). At higher polymer concentrations, the particles are held much closer together, and thus are thought to be much more strongly influenced by particle deformation. Slower cooling rates allow the particles to form much more slowly, resulting in a more ordered and robust structure. While almost no effect was observed on viscosity, this suggests that the particles interact to a similar degree, and the particles were characterized as "squeeze pasting" each other at the microscale.

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

[0285] Conclusion: The effect of the cooling rate was found to depend on the 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 can be manipulated when the system is at rest, but it remains fluid (injectable) under greater deformation.

[0286] A.4. Experiment - Mixing rate applied to the process: Effect of the mixing rate applied during the process for the formation of gellan microgel suspension ("fluid gel"). the purpose: • Understand the role that the mixing rate during processing plays in the material properties (viscosity and elasticity) of the resulting gelan microgel suspension. • Reduce the mixing rate during processing to a suitable tolerance level for eye drop formulations.

[0287] Materials and methods: • Gerlan (Kelco) • NaCl (Fisher Chemicals, Lot No.: 1665066)

[0288] Preparation of gelan microgel suspension (MS): Preparation of the stock solution: Preparation of NaCl solution NaCl (0.2M) was prepared by adding dry crystals (1.16g) to deionized water (100ml) using a volumetric flask. The NaCl was then dissolved using the inversion technique to assist the process. 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 water / NaCl solutions such that the final concentrations after processing were equal to 0.9% and 1.8% (w / v). Briefly, gelane powder was weighed out (4.5g and 9.0g) and added to 450ml of deionized water. The mixture was heated to 95°C with stirring to dissolve the polymer. Once fully dissolved, 25ml of stock NaCl solution (0.2M) was added to the solution, and a concentration of 10mM was obtained after processing. The sol was then brought to thermal equilibrium at 95°C and subsequently processed.

[0290] Preparation of Gelan MS: MS was prepared using a jacketed pin mill set to 20°C. Gelansol was injected into the pin mill at 3 ml / min using a peristaltic pump so that it entered the processing chamber at 40°C. Before entry using a syringe and syringe pump, water was injected into the gellansol stream at a rate of 0.16 ml / min so that they collided, diluting the gellansol to its final concentration (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 milling unit. Upon exit, the gel was packaged at 20°C 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 Corporation, AR-G2) equipped with a sandblasted parallel plate (40 mm in diameter, 1 mm gap height).

[0292] Amplitude sweep: Amplitude sweeps were obtained in strain control mode over a range of 0.1–100.0%. The sample was mounted on the instrument, and the upper geometric shape was lowered. After trimming, the sample was brought to equilibrium at 20°C before testing. Measurements were obtained at 1 Hz using logarithmic scales.

[0293] Flow profile: Viscosity profiles for the samples were obtained using a continuous ramp. The samples were mounted in the instrument, and the upper geometric shape was lowered. After trimming, the samples were brought to equilibrium at 20°C before testing. Increasing shear was applied in a speed-controlled mode for 0.1–600 s. -1 The sample was applied over a 3-minute ramp, and data points were obtained using logarithmic methods.

[0294] result: Rheology of small deformations: See Figures 21 and 22. Rheology of large deformities: See Figures 23 and 24.

[0295] Consideration: The degree of shear applied during the sol-gel transition of gelane biopolymers was tested at two concentrations, 0.9% (w / v) and 1.8% (w / v). At lower polymer concentrations, both elasticity and viscosity, as defined by G', were independent of the degree of shear experienced during the gelation profile. In all cases, the resulting material exhibited shear reduction over large deformations and solid-like behavior at rest, but the scale 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 the process. However, the elastic properties of the system at rest showed a dependency, with the final storage modulus (G') decreasing as the shear increased. It is thought that the 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, changing the mixing rate during processing does not play a significant role in the material properties of the resulting low polymer concentration microgel suspension. Therefore, broad shearing treatments can be applied without altering the final properties of the eye drop formulation. However, for higher concentrations used in "cream-like" diffusible systems, shearing 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 various initiating 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 gels: The fluid gel was prepared as follows.

[0299] Gerlan (thermal gelling): • Addition of gellan powder to water with 5% PBS to form a 0.5-2% polymer solution. • Heat the solution above its gelation point. • Add crosslinker (sodium chloride (final concentration of 10 mM)). The solution is cooled while being constantly sheared, passing through the gelation point (approximately 38°C).

[0300] Kappa-carrageenan (thermogel): • Addition of gellan powder to water with 5% PBS to form a 0.5-2% polymer solution. • Heat the solution above its gelation point. • Add crosslinker (potassium chloride (final concentration of 10 mM)). The solution is cooled while being constantly sheared, passing through the gelation point (approximately 40°C).

[0301] Alginate (ionotropic gelation): • Addition of alginate powder to water with 5% PBS to form a 0.5-1% polymer solution. • The polymer is thoroughly hydrated (assuming heat is used and it is cooled to room temperature). • Add the crosslinking agent (calcium chloride added (final concentration of 10 mM)) slowly using a syringe and needle, maintaining a constant shearing motion.

[0302] Agar (thermal gelation accompanied by 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. • Heat the solution above its gelation point (above 90°C). • Add crosslinker (with added sodium chloride (final concentration of 10 mM)). The solution is cooled while being constantly sheared, passing through the gelation point (approximately 36°C).

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

[0304] Amplitude sweep: • Amplitude sweep was obtained in the strain control mode over a range of 0.1 to 500.0%. • After installation, the sample was kept at equilibrium at 20°C before testing. The measurements were obtained at 1 Hz using a logarithmic scale.

[0305] Frequency sweep (Figure 27): Frequency sweeps were performed using the strain in the linear viscoelastic region of the amplitude sweep. • After installation, the sample was kept at equilibrium at 20°C before testing. The samples were tested using a logarithmic scale 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 brought to equilibrium at 20°C before the test. • Increasing shear force, in speed control mode, 0.1~600s -1 The sample was applied over a 3-minute ramp, and data points were obtained using logarithmic methods.

[0307] result: Figures 26 to 28 show the 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 exhibited mechanical properties typically associated with fluid gels: weak solid-like behavior at rest (frequency sweep); collapse of solid behavior under strain and yield (amplitude sweep); and shear-induced viscosity reduction behavior (viscosity profile). The data shows that fluid gels can be fabricated using a variety of different biopolymers, including gellan.

[0309] The data demonstrates that various gelation mechanisms can be used to fabricate fluid gels. a. Thermally driven process (Gellinger agar, K-carrageenan); b. Ionotropic gelation - gelation via crosslinking through ionic species (without heating) (alginate). Furthermore, in the case of heat-driven processing, various ion species can be used (Na + and K + This indicates that. The mechanical response of the gel can be classified according to the material properties previously reported in the patent (either within the outer limit or within the narrowed window which is stated to be more optimal).

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

[0311] Further experimental information - release of active substances in various fluid gels the purpose: To demonstrate the ability to release a variety of active substances from fluid gel matrices fabricated from various initiating polymers (particularly gellan and alginate) for a wide range of different applications (anti-fibrotic agents, anti-infective agents, analgesics, anti-inflammatory agents, ECM modifiers, basement membrane modifiers, and pro-fibrosis agents).

[0312] Materials and methods: Preparation of fluid gels: The fluid gel was prepared as follows:

[0313] Gerlan (thermal gelling): • Addition of gellan powder to water with 5% PBS to form a 0.5-2% polymer solution. • Heat the solution above its gelation point. • Add crosslinker (sodium chloride (final concentration of 10 mM)). The solution is cooled while being constantly sheared, passing through the gelation point (approximately 38°C).

[0314] Alginate (ionotropic gelation): • Addition of alginate powder to water with 5% PBS to form a 0.5-1% polymer solution. • The polymer is thoroughly hydrated (assuming heat is used and it is cooled to room temperature). • Add the crosslinking agent (calcium chloride added (final concentration of 10 mM)) slowly using a syringe and needle, maintaining a constant shearing motion.

[0315] Preparation of active substances: • Aliquot of active ingredients: 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 (trademark) (Decolin) (2.4 mg / ml) • Add the active substance to PBS to prepare a total volume of 1 ml. Mix thoroughly on a vortex mixer until dissolved.

[0316] Preparation of gels filled with active substances: Add 0.9 ml of gel to the Eppendorf. • Add the active substance in 0.1 ml of PBS to each gel. Mix thoroughly using a vortex mixer. • Refrigerate for 24 hours before the exam.

[0317] Determination of the standard curve: Standard concentrations of the active substance in PBS were prepared. The standard was transferred to a quartz cuvette (optical path length 1 mM) using a pipette. • Absorbance was measured at wavelengths between 200 and 700 nm using UV / vis spectroscopy. • The curve was plotted and used to determine the standard curve used for determining the concentration. (For Galacorin (trademark), use a commercially available ELISA kit according to the kit's instructions to determine the decorin concentration.)

[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 equilibrium was reached. • A 0.1 ml fluid gel containing the active substance was placed in a Transwell insert. The Transwell insert was placed in a well containing PBS. After the specified time, the Transwell insert was removed and placed in the wells of fresh PBS. The emission medium was then removed and analyzed using UV / vis spectroscopy. • The concentration was derived from the standard curve, and the cumulative emission was plotted as a function of time.

[0319] result: Figure 29 illustrates the standard curves obtained for the 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] Figure 30 illustrates the curves obtained for the 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 from the above results were as follows: • The filling and release of active substances could be achieved from all fluid gel matrices, regardless of whether they were biopolymers (gellan, alginate) or polymer mechanisms. • The filling and release of active substances could be achieved from all fluid gel matrices, regardless of the gelation mechanism: thermal gelation, ionotropic gelation, or radical-induced gelation. The fluid gel was able to release both small molecules (ibuprofen, dexamethasone, penicillin-streptomycin) and macromolecules and macromolecules (dextran, blue dextran, proteinase K, Galacorin® (Decolin)). • Fluid gels can be used for controlled delivery for the following applications: • Antifibrosis - Galacroin (trademark) (Decolin), Dextran • Anti-infectives: vancomycin, penicillin-streptomycin • Pain reliever - ibuprofen • Anti-inflammatory drugs: ibuprofen, dexamethasone • ECM modification - Proteinase K • Basement membrane modification - Proteinase K • Promotes fibrosis - dextran

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

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

[0324] Materials and methods: Preparation of fluid gels: The fluid gel was prepared as follows:

[0325] Jeran • Addition of gellan powder to water with 5% PBS to form a 1% polymer solution. • Heat the solution above its gelation point. • Add crosslinker (with added sodium chloride (final concentration of 10 mM)). The solution is cooled while being constantly sheared, passing through the gelation point.

[0326] Alginate • Addition of alginate powder to water with 5% PBS to form a 0.5% polymer solution. • The polymer is thoroughly hydrated (assuming heat is used and it is cooled to room temperature). • Add the crosslinking agent (calcium chloride added (final concentration of 10 mM)) slowly using a syringe and needle, maintaining a constant shearing motion.

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

[0328] Preparation of gels filled with active substances: Add 0.9 ml of gel to the Eppendorf. • Add the active substance in 0.1 ml of PBS to each gel. Mix thoroughly using a vortex mixer. • Refrigerate for 24 hours before the exam.

[0329] Preparation of microorganisms: • TSA plates are prepared by dissolving TSA in water, sterilizing it by autoclaving, and placing it in a 90mm Petri dish. • Cool the plate. • Cultivate microorganisms (E. coli and Staphylococcus aureus) and sow the seeds. • Allow microorganisms to form a "microbial community". • Make holes in the gel, remove the material, and prepare the wells.

[0330] Block zone assay: Add 0.25 ml of the fluid gel containing the active substance to the wells of each plate. • Add the anti-infectant in PBS to the control plate. Cover the plate and incubate for 24 hours for penicillin-streptomycin and up to 14 hours for vancomycin. • Measure the area from which the microbial culture was removed.

[0331] result: Figure 31 shows photographs illustrating the inhibition zone results using the shear-thinning hydrogel composition according to the present invention, which contains a polymer alginate or gellan in combination with an anti-infective agent (penicillin-streptomycin). These results demonstrate efficacy against Escherichia coli and Staphylococcus aureus. A summary of the results is also presented in the accompanying table.

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

[0333] Consideration: The data shows the following: • The release and activity of anti-infective agents occurred in all systems, regardless of the type of polymer, gelation mechanism, or active substance.

[0334] Conclusion: Fluid gels prepared from various polymers using various gelation techniques can be used to deliver antiinfective agents without impairing their activity. These results illustrate the suitability of the shear-reducing gel compositions of the present invention for delivering various antiinfective agents for use in therapies requiring such agents.

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

[0336] Materials and methods: Preparation of fluid gels: The fluid gel was prepared as follows:

[0337] Jeran • Addition of gellan powder to water with 5% PBS to form a 1% polymer solution. • Heat the solution above its gelation point. • Add crosslinker (with added sodium chloride (final concentration of 10 mM)). The solution is cooled while being constantly sheared, passing through the gelation point.

[0338] Alginate • Addition of alginate powder to water with 5% PBS to form a 0.5% polymer solution. • The polymer is thoroughly hydrated (assuming heat is used and it is cooled to room temperature). • Add the crosslinking agent (calcium chloride added (final concentration of 10 mM)) slowly using a syringe and needle, maintaining a constant shearing motion.

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

[0340] Preparation of gels filled with active substances: Add 0.9 ml of gel to the Eppendorf. • Add the active substance in 0.1 ml of PBS to each gel. Mix thoroughly using a vortex mixer. • Refrigerate for 24 hours before the exam.

[0341] Matrix breakdown 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 gel containing the active substance was added to a Transwell insert and placed on top of the fibrin gel. The sample was incubated at 60°C to activate proteinase K. Images were taken at various points in time and compared with a control fibrin gel (PBS only) and a fibrin gel with proteinase K added without a fluid gel support.

[0342] result: Figure 32 shows a photograph illustrating the time-dependent decay of fibrin (shown as a white gel in the photograph) of an exemplary ECM molecule 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 the following: • Fibrin gel collapse occurred in all cases except for the control group. • Fibrin gel disintegration was faster in the group receiving only proteinase K. The active substance remained potent even after being released from the gel.

[0344] Conclusion: The shear-reducing hydrogel composition according to the present invention is capable of releasing extracellular remodeling agents (proteinase K) and retains its ability to modify the extracellular membrane (ECM). This demonstrates the ability of the composition of the present invention to deliver this type of therapeutic molecule.

[0345] Further experimental information: In vitro action of Galacorin® (Decorin) formulated in the gelan shear-thinning hydrogel composition according to the present invention. the purpose: To demonstrate the efficacy of an antifibrotic therapeutic agent (exemplified by commercially available human recombinant decorin products, Galacorin®) against the release from the shear-thinning hydrogel composition of the present invention.

[0346] Materials and methods: Preparation of fluid gels: The fluid gel was prepared as follows:

[0347] Jeran • Addition of gellan powder to water with 5% PBS to form a 1% polymer solution. • Heat the solution above its gelation point. • Add crosslinker (with added sodium chloride (final concentration of 10 mM)). • Cool the solution to 40°C under constant shear and add Galacorin® (final concentration of 240 ug / ml). • While maintaining a constant shearing force, the gelation point is passed and cooling continues.

[0348] Collagen fibrillation assay: The diluent and reaction buffer are prepared by mixing sodium phosphate and sodium chloride and adjusting the pH to 7.4. • Add the samples (Gelatin fluid gel + Galacorin®, Galacorin only, and Gelatin only) to a 96-well plate and serially dilute them across the rows of the plate using dilution buffer. • Prepare the collagen by mixing it with cold water on ice until it reaches a concentration of 0.8 mg / ml. Add collagen to all wells. Add the reaction buffer to all wells and mix. Incubate at 37°C for 2 hours. • Read using a plate reader at 405nm.

[0349] result: Figure 33 illustrates the results of this test and is a graph comparing the absorbance at 405 nm (y-axis) for increasing concentrations of the gelan fluid gel shear-thinning hydrogel composition of the present invention, for collagen incubated with collagen alone ("collagen only"), or with or without decorin alone ("hr decorin"), or with or without human recombinant decorin ("DecFG") ("FG").

[0350] Consideration: The data shows the following: • Gelan fluid gel had no effect on collagen fibrillation compared to a control containing collagen alone. Galacorin™ present in or outside of the gelan fluid gel had the same effect on collagen fibrillation.

[0351] Conclusion: Collagen fibrillation can be used as an assay to indicate scarring. Therefore, high absorbance resulting from 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 is shown here that Galacorin® (Decorin), filled into a gelan fluid gel, has the same effect as Galacorin® directly added to collagen. Therefore, this demonstrates in vitro that the gelan shear-reducing 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 action of Galacorin® (Decorin) from the gelan shear-reducing hydrogel composition of the present invention. the purpose: To demonstrate the efficacy of an anti-fibrotic agent (Galacorin®) against release from a fluid gel carrier in an in vivo mouse model of bacterial keratitis.

[0353] Materials and methods: Preparation of fluid gels: The fluid gel was prepared as follows:

[0354] Jeran • Addition of gellan powder to water with 5% PBS to form a 1% polymer solution. • Heat the solution above its gelation point. • Add crosslinker (with added sodium chloride (final concentration of 10 mM)). • Cool the solution to 40°C under constant shear and add Galacorin® (final concentration of 240 ug / ml). • While maintaining a constant shearing force, the gelation point is passed and cooling continues.

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

[0356] Quantification of turbidity: Two blinded, independent ophthalmologists analyzed all photographs in the same randomized order (the order was provided by an independent statistician). The area of ​​turbidity formation was measured using Fiji, an open-source image processing package based on imageJ. ·mm 2 The unit measurements were plotted using ggplot2 in R, and looses smoothing was applied to the time series for each evaluator.

[0357] Tissue treatment and IHC for re-epithelialization, αSMA, Lam, and FN The eyes were fixed with 4% PFA in PBS. The eye was then rapidly frozen in OCT and sectioned at -22°C in the parasagittal plane to a thickness of 15 μm. The sections were placed on positively charged glass slides (Superfrost plus; Fisher Scientific, Pittsburgh, PA, USA). • Central sections (optic nerve plane) were used in all IHC tests. The sections (optic nerve plane) were thawed for 30 minutes, then washed with PBS, and subsequently subjected to permeabilization using 0.1% Triton X-100 (Sigma). Nonspecific antibody binding sites in tissue sections were blocked with 0.5% BSA, 0.3% Tween-20, and 15% normal goat serum. The primary antibody αSMA, laminin, and fibronectin (1:200 dilution) were added, followed by washing in PBS. Next, we attempted incubation at room temperature for 1 hour with secondary antibodies (goat anti-mouse Alexa Flour 488 1:500, goat anti-mouse Alexa Flour 594 1:500). The sections were then washed in PBS and mounted in Vectorshield mounting medium containing DAPI. • All control tissue fragments incubated with secondary antibodies alone were negatively stained (not shown).

[0358] IHC imaging and quantification: IHC staining was quantified by measuring pixel intensity. The area of ​​interest used for quantifying ECM IR was defined as an area of ​​interest of the same specified size for all eyes / procedures in the interstitium, and each interstitium had a total of 30 individual intensity measurements (areas of interest) taken to encompass the entire area of ​​the interstitium. Extracellular matrix deposition was quantified within these defined target regions in the stroma, and the percentage of immunofluorescence pixels exceeding a standardized background threshold was calculated using ImageJ software. For each antibody, a threshold level of brightness in the interstitial region was established using intact, untreated eye sections to define a reference level for test group analysis of pixel intensity.

[0359] result: The results of the study are explained in Figure 35, where graphs are presented showing the area of ​​turbidity associated with different treatments at different time points, the percentage of α-smooth muscle actin pixels exceeding the threshold for various control and treatment groups investigated, the percentage of fibronectin pixels exceeding the threshold for various control and treatment groups investigated, and the percentage of laminin pixels exceeding the threshold for various control and treatment groups investigated.

[0360] Consideration: The data shows the following: The gelan shear-reducing hydrogel (fluid gel) composition according to the present invention, containing Galacorin™, reduced the area of ​​turbidity over 16 days compared to standard treatment (gentamicin + prednisolone only). The gelan shear-reducing hydrogel (fluid gel) composition according to the present invention, containing Galacorin™, significantly reduced markers for all three tested fibrosis conditions compared to standard treatment alone.

[0361] Conclusion: The application of an antifibrotic agent in the gelan shear-reducing hydrogel (fluid gel) composition according to the present invention reduced scarring in vivo. This reduction in scarring was indicated by a decrease in the area of ​​turbidity (scar formation) and a decrease in the expression of markers typically associated with fibrosis and scarring.

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

[0363] Materials and methods Hypertension was induced in rats by intracavitary injection of TGF-1 twice a week. The net change in intraocular pressure was determined in untreated hypertensive rats (n=9) and in rats that received eye drop formulations of the shear-reducing hydrogel composition of the present invention, prepared in gellan, twice daily.

[0364] result The results are shown in Figure 36, where treated rats are shown by a dashed line and untreated controls are shown by a solid black line. The results were analyzed using two-way ANOVA and the Sidak multiple comparison test, and it was shown that the composition of the present invention (p<0.05) significantly reduced intraocular pressure in intraocularly hypertensive rats to D28 compared to the control.

[0365] conclusion The results achieved demonstrate that the shear-reducing hydrogel formulation of the present invention is capable of reducing intraocular hypertension (demonstrating the ability to prevent or treat glaucoma). Surprisingly, this activity was observed even when the composition was formulated without an active agent.

[0366] Throughout this description and claims, the words “comprise” and “contain,” and their variations, mean “including but not limited to,” and are not intended (and will not exclude) other parts, additions, components, numbers, or processes. Throughout this description and claims, unless the context otherwise requires, singular forms include plural forms. In particular, where the indefinite article is used, this specification should be understood to intend both plural and singular forms unless the context otherwise requires.

[0367] Any properties, numbers, features, compounds, chemical parts, or groups described in conjunction with specific aspects, embodiments, or examples of the present invention should be understood to be applicable to any other aspects, embodiments, or examples described herein, unless otherwise incompatible. All of the features disclosed herein (including the appended claims, abstract, and drawings) and / or all of the steps of any method or process so so disclosed can be combined in any combination, except for any combination in which at least some of such features and / or steps are mutually exclusive. The present invention is not limited to the details of any prior embodiments. The present invention extends to any novel one or any novel combination of the features disclosed herein (including the appended claims, abstract, and drawings), or any novel one or any novel combination of the steps of any method or process so so disclosed.

[0368] The reader's attention is directed to all pages and documents filed concurrently with or prior to this specification in connection with this application and made publicly available together with this specification, the contents of all such pages and documents being incorporated herein by reference.

[0369] (References) TIFF0007835559000014.tif237170TIFF0007835559000015.tif240170TIFF0007835559000016.tif245170TIFF0007835559000017.tif106170

Claims

1. A shear-reducing hydrogel composition comprising microgel particles dispersed in an aqueous vehicle, wherein the microgel particles are (i) A microgel particle-forming polymer in an amount of 0.5 to 1.35 wt%, which is gellan; and (ii) Monovalent metal ion salts of 5-20 mM as crosslinking agents; Includes, A composition having a pH in the range of 3 to 8, a viscosity of 5 Pa.s or more at 20°C when exposed to zero shear, a viscosity that decreases to less than 1 Pa.s when the hydrogel composition is subjected to shear, and an elastic modulus (G') of 5 Pa to 200 Pa at zero shear.

2. A shear-reducing hydrogel composition according to claim 1, which does not contain collagen and / or fibrin.

3. A shear-reducing hydrogel composition according to claim 1 or 2, comprising 0.8 to 1.0 wt% (e.g., 0.9 wt%) of a microgel particle-forming polymer.

4. A shear-reducing hydrogel composition according to any one of claims 1 to 3, wherein the microgel particles do not contain decorin.

5. A shear-reducing hydrogel composition according to any one of claims 1 to 4, comprising a 5-15 mM monovalent metal ion salt as a crosslinking agent.

6. A shear-reducing hydrogel composition according to any one of claims 1 to 5, comprising an 8-12 mM monovalent metal ion salt as a crosslinking agent.

7. A shear-reducing hydrogel composition according to any one of claims 1 to 6, comprising a 10 mM monovalent metal ion salt as a crosslinking agent.

8. A shear-reducing hydrogel composition according to any one of claims 1 to 7, having a pH in the range of 6 to 8 or 6.5 to 8.

9. A shear-reducing hydrogel composition according to any one of claims 1 to 8, having a pH in the range of 7 to 7.5 (e.g., pH 7.4).

10. The shear-reducing hydrogel composition according to any one of claims 1 to 9, wherein the monovalent metal ion salt is NaCl.

11. A shear-reducing hydrogel composition according to any one of claims 1 to 10, having a viscosity of 5 Pa.s to 200 Pa.s at 20°C when exposed to zero shear.

12. The shear-reducing hydrogel composition according to claim 11, having a viscosity of 10 Pa.s to 50 Pa.s at 20°C when exposed to zero shear.

13. A shear-reducing hydrogel composition according to any one of claims 1 to 12, having an elastic modulus (G') of 5 Pa to 40 Pa at zero shear.

14. A shear-reducing hydrogel composition according to any one of claims 1 to 13, further comprising one or more pharmacologically active agents.

15. The shear-reducing hydrogel composition according to claim 14, comprising one or more pharmacologically active agents selected from the group consisting of antifibrotic agents (e.g., decorin); anti-infective agents; analgesics; anti-inflammatory agents; anti-proliferative agents; keratolytic agents; extracellular matrix modifiers; intercellular junction modifiers; basement membrane modifiers; biological lubricants; and pigmentation modifiers.

16. A shear-reducing hydrogel composition according to claim 14 or claim 15, comprising decorin at a concentration between approximately 0.1 μg / mL and 0.5 μg / mL.

17. A topical gel composition suitable for local administration, which is a shear-thinning gel composition according to any one of claims 1 to 16.

18. An ophthalmic gel composition suitable for administration to the eye, which is a shear-thinning gel composition according to any one of claims 1 to 16.

19. A method for producing the shear-reducing gel composition described in claim 1, a) A step of dissolving gellan in an aqueous vehicle to form a gellan solution; b) A step of mixing the gellan solution formed in step (a) with an aqueous solution of a monovalent metal ion salt at a temperature exceeding the gelling temperature of gellan; and c) A step of cooling the mixture obtained from step b) to a temperature below the gelling temperature of gellan to form the composition according to any one of claims 1 to 13. Methods that include...

20. The method according to claim 19, wherein step (a) includes heating and stirring the aqueous vehicle to promote the dissolution of gellan.

21. The method according to claim 20, wherein the aqueous vehicle is heated to a temperature exceeding the gelling temperature of gellan at 50°C.

22. The method according to any one of claims 19 to 21, wherein in step b), the mixing of the gellan solution formed in step (a) and the aqueous solution of the monovalent metal ion salt occurs at an elevated temperature along with shear mixing.

23. The method according to claim 22, wherein in step b), the mixing of the gellan solution formed in step (a) and the aqueous solution of the monovalent metal ion salt occurs at a temperature greater than 25°C.

24. The method according to any one of claims 19 to 23, wherein in step c), the mixture from step b) is cooled with continuous mixing at a rate of 0.1 to 5°C / min.

25. The method according to any one of claims 19 to 24, wherein in step c), the mixture from step b) is cooled at a rate of 0.5 to 2°C / min with continuous mixing.

26. The method according to any one of claims 19 to 25, wherein in step c), the mixture from step b) is cooled with continuous mixing at a rate of 0.5 to 1.5°C / min (e.g., 1°C / min).

27. The method according to any one of claims 19 to 26, wherein in 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.

28. A shear-reducing hydrogel composition according to any one of claims 1 to 18, for use in therapeutic purposes.

29. A shear-reducing hydrogel composition for use according to claim 28, which is intended for topical administration.

30. A shear-reducing hydrogel composition for use according to claim 28 or claim 29 in inhibiting scarring.

31. A shear-reducing hydrogel composition for use according to claim 30, comprising an antifibrotic agent.

32. The shear-reducing hydrogel composition for use according to claim 31, wherein the antifibrotic agent is selected from the group consisting of antifibrotic extracellular matrix (ECM) components; antifibrotic growth factors; and inhibitors of the antifibrotic agent.

33. A shear-reducing hydrogel composition for use according to any one of claims 30 to 32, comprising decorin, an anti-fibrotic ECM component.

34. The shear-reducing hydrogel composition for use according to claim 33, wherein decorin is present at a concentration between approximately 0.1 μg / mL and 0.5 μg / mL.

35. A shear-reducing hydrogel composition for use according to any one of claims 28 to 34, for administration to the surface of the eye.

36. A shear-reducing hydrogel composition for use according to claim 35, comprising modified dextran sulfate.

37. A shear-reducing hydrogel composition for use according to claim 35, for use in the treatment of bacterial keratitis.

38. A shear-thinning hydrogel composition for use according to claim 37, for use in combination with one or more agents selected from the group consisting of steroids and antibacterial agents.

39. A shear-reducing hydrogel composition for use according to claim 38, for use in combination with one or more agents selected from the group consisting of prednisolone and gentamicin.

40. A shear-reducing hydrogel composition for use according to claim 39, comprising decorin, prednisolone, and gentamicin.

41. A shear-thinning hydrogel composition for use according to claim 35, for use in the prevention and / or treatment of glaucoma.

42. A shear-reducing hydrogel composition for use in the prevention and / or treatment of glaucoma, free of active agents, as described in claim 41.

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