Ophthalmic compositions and methods of use therefor

Abstract

To provide ophthalmic compositions that may be used for various conditions of the eye, and particularly, conditions of the cornea, and also provide methods that utilise these compositions and kits that include these compositions.SOLUTION: A composition includes a TGFβ3 polypeptide or a variant or fragment thereof, and dexamethasone or a derivative or related steroid agent. The composition is used for (i) treating or preventing a condition associated with the thinning or irregularity of a cornea; (ii) treating or preventing damage or injury of the cornea; or (iii) treating or preventing a refractive error of the eye.SELECTED DRAWING: Figure 13

Description

[Technical field]

[0001] Related Applications This application claims the benefit of New Zealand Provisional Patent Application No. NZ705727, filed March 5, 2015, the entire contents of which are incorporated herein by reference.

[0002] Technical Field The present disclosure relates to compositions and methods useful for the treatment and / or prevention of ophthalmic conditions. In particular, the present disclosure relates to compositions and methods that can be used to strengthen and regenerate the cornea and correct refractive errors of the eye. [Background technology]

[0003] Previously, it was believed that differentiated cells no longer had the ability to revert to their previous state. However, this insight has been challenged by the induction of pluripotent stem cells (cell reprogramming) and evidence showing that differentiated cells can be converted to another phenotype (Takahashi & Yamanaka 2006; Wernig et al. 2007; Yamanaka & Blau 2010; Gurdon & Melton 2008; Peran et al. 2011). Furthermore, it is now believed that the cellular microenvironment, including surrounding cells, extracellular matrix, and growth and differentiation factors, plays an important role in effecting the redirection of cell differentiation (Hakelien and Collas 2002). Based on this information, researchers have begun to develop therapies using cell reprogramming and stem cell technologies.

[0004] The cornea of ​​the eye accounts for more than two-thirds of the eye's total refractive power (focusing power). Even slight changes in the shape of the cornea can dramatically affect the sharpness of the image focused on the retina. The corneal stroma (the transparent anterior surface of the eye) comprises the majority of the corneal tissue and consists of highly organized lamellae composed of densely packed collagen fibrils, mostly collagen types I and V (Marshall et al. 1993). The unique structure of the stroma, resulting from the uniformly aligned collagen fibrils, gives the cornea its properties of toughness and transparency (Funderburgh 2000).

[0005] When stromal cells (keratocytes) are removed from the cornea and cultured in monolayers, they display fibroblastic morphology and transform from stellate cells to multinucleated spindle-shaped cells (Funderburgh et al. 2001). Another commonly observed keratocyte phenotype is the myofibroblastic morphology seen in the cornea after injury (Jester et al. 1987). Changes in exogenous growth factors and cytokines are thought to mediate these phenotypic changes (Funderburgh et al. 2001).

[0006] The TGFβ family of growth factors has been shown to be the most potent inducers of chondrocyte (cartilage) differentiation (Heng, Cao, & Lee 2004; Johnstone et al. 1998; Menetrey et al. 2000). TGFβ1 stimulates collagen and fibronectin synthesis by chick embryo fibroblasts (Ignotz and Massague 1986). In keratocytes, TGFβ1 and TGFβ2 have been shown to induce the deposition of scar-associated ECM, possibly due to the conversion of keratocytes to a myofibroblast phenotype (Funderburgh, Mann, Funderburgh, Corpuz, & Roth 2001). In contrast, TGFβ3 has been shown to induce corneal fibroblasts to deposit an ECM composed of type I collagen without causing fibrosis or scarring (Karamichos, Hutcheon, & Zieske 2011). Certain nonproteinaceous compounds, such as dexamethasone (Johnstone, Hering, Caplan, Goldberg, & Yoo 1998), ascorbic acid (Farquharson, Berry, Barbara Mawer, Seawright, & Whitehead 1998), and ethanol (Kulyk & Hoffman 1996), have also been shown to promote chondrocyte differentiation in vitro.

[0007] There are numerous conditions that affect the cornea, including various defects, injuries, diseases, and degenerative disorders. Myopia is caused by excessive curvature of the cornea, which causes light entering the eye to be focused in front of the retina. It is the most common vision disorder worldwide, affecting the vision of 70-90% of people in some Asian countries and 30-40% of people in Western countries (Frederick 2002). In most cases, myopia first occurs in school-age children and progresses until about age 20. It is also associated with the prevalence of macular degeneration, retinal detachment, and glaucoma in adults (Ebenstein & Pruitt 2006).

[0008] Myopia is most commonly corrected by the use of prescription glasses or contact lenses. However, these devices do not provide a permanent treatment for the condition and are not suitable for use during certain activities. Contact lenses are also associated with eye infections and more serious conditions, including corneal abrasions and ulcers. In certain circumstances, refractive surgery or orthocorneal treatment is indicated for myopia. Nevertheless, these treatments only provide a temporary correction for mild to moderate myopia: they are not a permanent treatment and are not suitable for severe cases.

[0009] Keratoconus is a malformed corneal degeneration associated with thinning of the corneal stroma and disruption of a portion of the cornea known as Bowman's layer. Progressive thinning of the corneal stroma typically occurs over decades, resulting in a conical shape of the cornea. This causes vision loss due to irregular astigmatism and myopia. The cause of keratoconus remains unknown, but has been linked to factors such as constant eye rubbing and contact lens wear (Krachmer, Feder, & Belin 1984; Sherwin & Brookes 2004). It can appear as early as adolescence and progress into the 20s or 30s.

[0010] The prevalence of keratoconus is estimated at approximately 1 in 2000 in the general population worldwide (Rabinowitz 1998), with no gender bias. Because the onset of keratoconus is typically in early adulthood and continues into the prime working and child-rearing years, the loss of quality of life and the economic burden of treating keratoconus represent a significant public health concern. Keratoconus is the leading indication for corneal transplantation in the Western world, with researchers finding it accounts for 28.8% of corneal transplants in France (Legeais et al. 2001) and 11.4%–15.4% in the United States (Cosar et al. 2002; Dobbins et al. 2000). New Zealand has an unusually high prevalence of keratoconus, with disproportionately high incidence in Pasifika and Maori populations (Patel et al. 2005; Patel & McGhee 2013). Approximately 50% of all corneal transplants performed in New Zealand are for keratoconus (Edwards et al. 2002).

[0011] Despite several studies on keratoconus, the underlying biochemical processes remain poorly understood. The familial occurrence of keratoconus suggests that one of the etiologies is genetic (Ihalainen 1985). The condition is also associated with certain biochemical and biomechanical factors. For example, it has been shown that corneal thinning in keratoconus is due to the loss of extracellular matrix (ECM) components. However, this may be due to their destruction, their impaired formation, or a combination of both (Klintworth & Damms 1995; Klintworth 1999; Jhanji et al. 2011). In the corneal stroma, changes associated with keratoconus include a decrease in the number of lamellae and keratocytes (Ku, Niederer, Patel, Sherwin, & McGhee 2008; Sherwin & Brookes 2004), as well as changes in the organization of lamellae and the distribution of collagen fibril mass (Meek et al. 2005).

[0012] It is believed that the degradation of the stromal layer may be due to abnormal proteolytic enzyme activity (Fukuchi, Yue, Sugar, & Lam 1994). Keratoconic corneas have been found to have low levels of enzyme inhibitors and high levels of degradative enzymes (Kenney & Brown 2003). Biomechanical factors include corneal thinning and reduced stiffness due to oxidative damage caused by UV exposure and mechanical trauma (Kenney & Brown 2003). Biomechanical studies have shown that keratoconic corneas exhibit reduced elasticity and stiffness, the reasons for which remain unclear (Edmund 1988). It has been proposed that reduced collagen cross-linking may be responsible (Wollensak & Buddecke 1990). Currently, there are no satisfactory animal models of keratoconus, and studies are mainly limited to the ex vivo environment.

[0013] Depending on the severity of the condition, attempts to slow the progression of keratoconus include the use of special glasses and contact lenses. Severe cases may require corneal implants, intrastromal corneal rings, or corneal transplants (Jhanji, Sharma, & Vajpayee 2011). Penetrating keratoplasty, a procedure in which the entire thickness of the cornea is removed and replaced with donor corneal tissue, is the most commonly used surgical procedure to treat severe cases of keratoconus (Rabinowitz 1998). Keratoconus is the main indication for corneal transplant surgery worldwide, with approximately 12-20% of people affected by keratoconus requiring a corneal transplant (Pramanik, Musch, Sutphin, & Farjo 2006).

[0014] Early treatment options for keratoconus, such as specially fabricated gas-permeable lenses known as Rose K lenses, have focused on improving vision. Newer treatments aim to slow the progression of the disease. A treatment known as corneal collagen crosslinking (CXL) focuses on increasing the stiffness and biomechanical stability of the cornea. In this procedure, the epithelium is removed, topical riboflavin eye drops are administered, and the cornea is exposed to 370 nm ultraviolet A light for approximately 30 minutes (Ashwin & McDonnell 2010; G. Wollensak, Spoerl, & Seiler 2003). It is believed that the UVA light activates riboflavin, thereby generating reactive oxygen species that induce the formation of covalent bonds between collagen molecules in the corneal stroma (Spoerl, Huhle, & Seiler 1998; G. Wollensak et al. 2003). However, this procedure is not suitable for treating corneas thinner than 400 μm due to the possibility of endothelial cell damage. Although this treatment results in a stiffer cornea, it does not address the problem of corneal thinning.

[0015] Thus, there is a continuing need for therapeutic compositions and methods for addressing ocular conditions, including conditions affecting the cornea. In particular, there is a need for therapeutic agents that are relatively minimally invasive and easy to administer. Summary of the Invention

[0016] The present inventors have developed compositions and methods for modifying corneal cells to alter collagen expression and extracellular matrix formation in corneal tissue, which compositions and methods are useful for regenerating and / or strengthening the cornea, thereby treating and / or preventing various conditions of corneal and ocular refractive error.

[0017] In one embodiment, the invention includes a method of treating or preventing a condition associated with corneal thinning or irregular shape abnormality, comprising contacting the cornea with a composition comprising a TGFβ3 polypeptide or a variant or fragment thereof, and dexamethasone or a derivative or related steroidal agent, thereby treating or preventing the condition.

[0018] In various embodiments, The TGFβ3 polypeptide consists of the amino acid sequence of SEQ ID NO:1. Dexamethasone is dexamethasone phosphate.

[0019] The composition contains 10 to 100 ng / ml of TGFβ3 polypeptide. The composition contains 40 to 4000 ng / ml of dexamethasone. The composition is formulated as an eye drop.

[0020] The composition is formulated with gellan gum. The compositions are administered once daily or twice daily. The composition is co-administered with one or more additional ophthalmic agents.

[0021] The one or more additional ophthalmic agents are selected from the group consisting of anesthetics, anti-inflammatory agents, antibacterial agents, and lubricants. The composition is administered in conjunction with the use of a contact lens, a corneal insert, a corneal implant, or an intrastromal corneal ring.

[0022] The contact lens, corneal insert, corneal implant, or intrastromal corneal ring is adapted to shape or maintain the shape of the cornea during and / or after treatment with the composition.

[0023] The contact lens, corneal insert, corneal implant, or intrastromal corneal ring is adapted to act as a carrier for the composition or as a composition eluting device. The composition is administered in conjunction with corneal collagen crosslinking.

[0024] Administration of the composition may occur before and / or after crosslinking. The condition is selected from the group consisting of keratoconus, myopia, and astigmatism. In an alternative embodiment, the method comprises the co-administration of a composition comprising a TGFβ3 polypeptide or a variant or fragment thereof, and a composition comprising dexamethasone or a derivative thereof or a related steroidal agent.

[0025] In a further aspect, the present invention includes a method of treating or preventing corneal damage or injury comprising contacting the cornea with a composition comprising a TGFβ3 polypeptide or a variant or fragment thereof and dexamethasone or a derivative or related steroidal agent, thereby treating or preventing corneal damage or injury.

[0026] In various embodiments, The TGFβ3 polypeptide consists of the amino acid sequence of SEQ ID NO:1. Dexamethasone is dexamethasone phosphate.

[0027] The composition contains 10 to 100 ng / ml of TGFβ3 polypeptide. The composition contains 40 to 4000 ng / ml of dexamethasone. The composition is formulated as an eye drop.

[0028] The composition is formulated with gellan gum. The compositions are administered once daily or twice daily. The composition is co-administered with one or more additional ophthalmic agents.

[0029] The one or more additional ophthalmic agents are selected from the group consisting of anesthetics, anti-inflammatory agents, antibacterial agents, and lubricants. The composition is administered in conjunction with the use of a contact lens, a corneal insert, a corneal implant, or an intrastromal corneal ring.

[0030] The contact lens, corneal insert, corneal implant, or intrastromal corneal ring is adapted to shape or maintain the shape of the cornea during and / or after treatment with the composition.

[0031] The contact lens, corneal insert, corneal implant, or intrastromal corneal ring is adapted to act as a carrier for the composition or as a composition eluting device. The corneal injury or damage is associated with one or more of an abrasion, laceration, ulcer, burn, puncture, or surgery.

[0032] In an alternative embodiment, the method comprises the co-administration of a composition comprising a TGFβ3 polypeptide or a variant or fragment thereof, and a composition comprising dexamethasone or a derivative thereof or a related steroidal agent.

[0033] In a further aspect, the invention includes a method of treating or preventing ocular refractive errors comprising contacting the eye with a composition comprising a TGFβ3 polypeptide or a variant or fragment thereof and dexamethasone or a derivative or related steroidal agent, thereby treating or preventing ocular refractive errors.

[0034] In various embodiments: The TGFβ3 polypeptide consists of the amino acid sequence of SEQ ID NO:1. Dexamethasone is dexamethasone phosphate.

[0035] The composition contains 10 to 100 ng / ml of TGFβ3 polypeptide. The composition contains 40 to 4000 ng / ml of dexamethasone. The composition is formulated as an eye drop.

[0036] The composition is formulated with gellan gum. The compositions are administered once daily or twice daily. The composition is co-administered with one or more additional ophthalmic agents.

[0037] The one or more additional ophthalmic agents are selected from the group consisting of anesthetics, anti-inflammatory agents, antibacterial agents, and lubricants. The compositions are administered for use in conjunction with a contact lens, a corneal insert, a corneal implant, or an intrastromal corneal ring.

[0038] The contact lens, corneal insert, corneal implant, or intrastromal corneal ring is adapted to shape or maintain the shape of the cornea during and / or after treatment with the composition.

[0039] The contact lens, corneal insert, corneal implant, or intrastromal corneal ring is adapted to act as a carrier for the composition or as a composition eluting device. The method may be performed before or after refractive surgery.

[0040] Refractive errors of the eye relate to one or more of myopia, hyperopia, astigmatism, and presbyopia. In an alternative embodiment, the method comprises the co-administration of a composition comprising a TGFβ3 polypeptide or a variant or fragment thereof, and a composition comprising dexamethasone or a derivative thereof or a related steroidal agent.

[0041] In a still further aspect, the present invention provides a method for producing a method for treating a cancer cell comprising the steps of: a composition comprising a TGFβ3 polypeptide or a variant or fragment thereof, and dexamethasone or a derivative or related steroid agent; and one or more contact lenses.

[0042] In various embodiments, The contact lens, corneal insert, corneal implant, or intrastromal corneal ring is adapted to shape or maintain the shape of the cornea during and / or after treatment with the composition.

[0043] The contact lens, corneal insert, corneal implant, or intrastromal corneal ring acts as a carrier for the composition or as a composition eluting device. The TGFβ3 polypeptide consists of the amino acid sequence of SEQ ID NO:1.

[0044] Dexamethasone is dexamethasone phosphate. The composition contains 10 to 100 ng / ml of TGFβ3 polypeptide. The composition contains 40 to 4000 ng / ml of dexamethasone.

[0045] The composition is formulated as an eye drop. The composition is formulated with gellan gum. The compositions are formulated for once-daily or twice-daily administration.

[0046] The composition is co-formulated with one or more additional ophthalmic agents. The kit may include one or more additional ophthalmic agents. The one or more additional ophthalmic agents are selected from the group consisting of anesthetics, anti-inflammatory agents, antibacterial agents, and lubricants.

[0047] The kit includes a contact lens solution. The kit includes instructions for use. The kit is used for the treatment or prevention of refractive errors of the eye.

[0048] The kit is used for the treatment or prevention of a corneal condition selected from the group consisting of keratoconus, myopia, hyperopia, astigmatism, presbyopia, and corneal stromal dystrophy. The kit is used for the treatment of a corneal condition selected from the group consisting of an abrasion, a laceration, an ulcer, a burn, a puncture, a corneal melt, and a surgical injury.

[0049] In an alternative embodiment, the kit comprises, as separate components, a composition comprising a TGFβ3 polypeptide or a variant or fragment thereof, and a composition comprising dexamethasone or a derivative or related steroidal agent.

[0050] In a further aspect, the present invention includes a method for inducing expression of type II collagen in corneal stromal cells, comprising contacting the corneal stromal cells with a composition comprising a TGFβ3 polypeptide or a variant or fragment thereof, and dexamethasone or a derivative or related steroid agent, thereby inducing expression of type II collagen in the corneal stromal cells.

[0051] In various embodiments, The TGFβ3 polypeptide consists of the amino acid sequence of SEQ ID NO:1. Dexamethasone is dexamethasone phosphate.

[0052] The composition contains 10 to 100 ng / ml of TGFβ3 polypeptide. The composition contains 40 to 4000 ng / ml of dexamethasone. The composition is formulated for administration via a contact lens, a corneal insert, a corneal implant, or an intrastromal corneal ring.

[0053] The composition is formulated for administration as a solution, gel, cream, or emulsion. The method is carried out in vivo.

[0054] The method is carried out ex vivo. In an alternative embodiment, the method comprises the co-administration of a composition comprising a TGFβ3 polypeptide or a variant or fragment thereof, and a composition comprising dexamethasone or a derivative thereof or a related steroidal agent.

[0055] The above summary broadly describes the features and technical advantages of certain embodiments of the present invention. Further technical advantages are described in more detail in the following detailed description of the invention and examples.

[0056] The novel features believed to be characteristic of the present invention will be better understood from the detailed description of the invention when considered in conjunction with the accompanying drawings and examples. However, the drawings and examples provided herein are intended to serve to explain the invention or to aid in a better understanding of the invention, and are not intended to limit the scope of the invention. [Brief description of the drawings]

[0057] [Figure 1A] FIG. 1 is a schematic diagram illustrating that light entering the eye is not focused on the retina due to myopia caused by increased curvature of the cornea. [Figure 1B] Normal (A) and keratoconus (B) corneas, respectively. (C) Scheimpflug image of severe keratoconus, showing significant central corneal thinning. [Diagram 2] Setting up organotypic slice cultures. [Diagram 3] Growth factor eye drops are instilled into the eyes of adult male Wistar rats. [Figure 4] A Phoenix Micron IV in vivo ocular imaging system specifically configured to image rat eyes. [Diagram 5] (A) Schematic of the nanoindenter system, (B) typical load-displacement curves obtained during the indentation process used to calculate the corneal elasticity and hysteresis. Pmax = maximum load applied, hmax = indentation depth, hc = contact depth (contact height between tip and sample), hf = final depth, S = unloading stiffness. [Figure 6]Nanoindentation equipment designed to hold a human corneal button (A) and a rat eyeball (B) and then position the central portion of the cornea using a microscope (C) and then position the indenter probe (D). [Figure 7] Keratocytes seeded in chondrogenic differentiation medium. Keratocytes cultured for 3 weeks in chondrogenic differentiation medium containing TGFβ3 and dexamethasone formed spheres that were labeled with nestin (A) at the periphery of the spheres and with type II collagen (B) within the core. The culture medium was then replaced with serum containing fibroblast growth medium for 1 week, allowing the cells to spread from the spheres and aggregate in the dish (C). Cells in the monolayer were negative for type II collagen, whereas cell clusters remained positive for type II collagen (D). [Figure 8] Keratocytes seeded in serum containing fibroblast growth medium. Keratocytes cultured for 3 weeks in control fibroblast growth medium were negative for nestin (A) and type II collagen (B). Confluent fibroblasts were then cultured for 3 weeks in chondrogenic differentiation medium containing TGFβ3 and dexamethasone (C). Cells remained negative for type II collagen (D). (E) Pellet culture of confluent fibroblasts in chondrogenic differentiation medium. After 3 weeks of culture, cell pellets were sectioned and positively labeled for the keratocyte marker keratocan (F) and negative for chondrocyte-specific type II collagen (G). [Figure 9] Human corneal sections cultured for 2 weeks in control medium (A) and (D) were negative for type II collagen and positive for type I collagen, respectively. Human corneal sections cultured in chondrogenic differentiation medium for 1 week (B) and (E) and for 2 weeks (C) and (F) were labeled for type II collagen (B) and (C) and type I (E) and (F). Strong labeling for type II collagen was seen in corneal sections treated for 2 weeks, whereas sections treated for only 1 week were negative for type II collagen. Sections cultured for both periods in chondrogenic differentiation medium were less strongly labeled compared to control-treated sections, but were positive for type I collagen in untreated corneas. [Figure 10] Human corneal slices were cultured in (A) control medium and (B) chondrogenic differentiation medium for 2 weeks and labeled for type II collagen. Similar results were obtained as shown by FIG. 9. In vivo experiments showing (C) untreated corneas and (D) and (E) treated corneas with widespread labeling of type II collagen in corneas from rats treated with TGFβ3 and dexamethasone. Stronger labeling was seen in the anterior (superior) portion of the cornea (D). Type II collagen appeared fibrous and was evenly distributed throughout the ECM. [Figure 11] Keratoconus corneal buttons cultured in vitro for 2 weeks in control medium (A), (C), and (E), and in chondrogenic differentiation medium (B), (D), and (F), respectively, and labeled for type II collagen (A) and (B), and vimentin (C)-(F). Labeling for type II collagen in treated keratoconus corneas (B) was weaker compared to labeling in normal human corneas. However, deposition of type II collagen was similar to patterns previously seen after in vitro and in vivo treatment of normal human and rat corneas. Compared to untreated halves of keratoconus corneas (C) and (E), the fibroblast population in treated halves of keratoconus buttons (D) and (F) was increased in number, and keratocytes appeared healthier and more intact, with numerous long cellular processes (F). [Figure 12] Ex vivo cultured human keratoconus corneas were cultured for 3 weeks in control medium (A) and (C) and in chondrogenic differentiation medium (B) and (D) and were labeled for alpha smooth muscle actin (αSMA) (A) and (B) and type III collagen (C) and (D). Compared to corneas cultured in chondrogenic differentiation medium, there was stronger labeling for αSMA in the stromal layer of corneas cultured in control medium (A). Corneas cultured in either of the two media did not positively label for type III collagen. [Figure 13]Corneal transparency of in vivo treated corneas. Treated corneas (A) and (C) and untreated corneas (B) and (D). After 3 weeks, the treated and untreated corneas were indistinguishable from one another. Frontal views of corneas (A) and (B) reveal clear corneas through which light passes easily and the blood vessels at the back of the eye are very clearly visible. In vivo images of corneal cross sections at 8 weeks reveal clear, transparent corneas through which light passes easily. No signs of corneal opacity or scarring were observed. [Figure 14] Quantitative gene expression of collagen type II (A) and collagen type I (B) in in vivo treated corneas. After 1 week of treatment, there was an initial increase in collagen type II expression. When treatment was discontinued, there was a significant decrease in collagen type II expression (A). Expression of native corneal collagen type I was also initially upregulated, but after long-term treatment (up to 7 weeks), its expression was comparable to that of untreated control corneas (B). [Figure 15] Comparison of treated and untreated corneas in vivo for 1 week showed no significant difference in the reduction in hardness (H) and elastic modulus (Er). [Figure 16] Load-deformation curves obtained for treated and untreated corneas from two rats after 3 weeks in vivo. The corresponding graphs with plotted values ​​clearly show an increase in elastic modulus (Er) and hardness (H) in the treated corneas. [Figure 17] Comparison of elastic modulus and hardness between 8 week treated and control human keratoconus corneas reveals a significant increase in both parameters in treated corneas. [Figure 18] The cornea of ​​a sheep eye is reshaped by combining in vivo cell reprogramming with a rigid contact lens to maintain the desired corneal shape during treatment. [Figure 19](A) Phoenix Micron IV ocular imaging system. This imaging system allows for measurements of corneal thickness, curvature, and transparency. (B) The OCT attachment allows for visualization of the anterior segment, as well as measurements of corneal thickness and integrity, similar to the images shown herein. (C) Nanoindenter used to assess corneal biomechanics ex vivo in sheep, and (D) schematic of its setup. The output can be shown as a load-displacement curve and analyzed to obtain elastic Young's modulus, and to obtain a measure of hardness. In large animals such as sheep, corneal thickness (E) is shown in microns, and corneal curvature measurements (F) are obtained using a handheld Pentacam®. For corneal curvature (F), widely spaced color contours signify large radii of curvature, while narrower contours signify areas of steeper curvature. Numbers indicate the radius of curvature at each point. [Figure 20] Only TGFβ3 in combination with dexamethasone resulted in the deposition of collagen type II. Sheep corneal tissue was cultured in (A) BMP6, (B) BMP6 + hydrocortisone, (C) TGFβ3 + hydrocortisone, (D) BMP6 + dexamethasone, (E) TGFβ3 + prednisone, (F) TGFβ3 + Triesense®, (G) and (H) TGFβ3 + dexamethasone at 20× and 60× magnification, respectively, and labeled for cartilage-specific collagen type II. [Figure 21] Dose-response study of TGFβ3 in combination with dexamethasone. Sheep corneas were cultured for 3 weeks and labeled for type II collagen. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0058] The following description sets forth numerous example configurations, parameters, etc. However, it should be recognized that such description is not intended to be a limitation on the scope of the invention, but instead is provided as a description of example embodiments.

[0059] definition In each of the examples herein in the description, aspects, embodiments, and examples of the present invention, the terms "comprising", "including", and the like are to be understood inclusively, without limitation. Thus, unless the context clearly requires otherwise, throughout the specification and claims, the terms "comprising", "including", and the like are to be interpreted in an inclusive sense, i.e., "including, but not limited to", rather than an exclusive sense.

[0060] As used herein, "enhancement" refers to a method of increasing one or more of the thickness, hardness, modulus of elasticity, tensile strength, and regularity of the cornea, including the corneal tissue (e.g., the stromal layer). Enhancing may be used to impart a particular shape, i.e., corneal curvature, to the cornea. Enhancing methods may be performed in the presence or absence of particular ocular or corneal conditions. Enhancing may include increasing components (e.g., type II collagen) in the extracellular matrix of the cornea. Enhancing may also include increasing the number of cells (e.g., keratocytes) of the cornea. The number of cells may be increased, for example, by changing the proliferation state of such cells from a quiescent state to an active state.

[0061] "Co-administration" or "co-administering" refers to the combination of agents, e.g., ophthalmic therapeutic agents, and includes administration of co-formulations (i.e., combined formulations) and simultaneous or sequential administration of separate formulations. Similarly, "in conjunction with" refers to the combination of a therapeutic composition with a therapeutic device / procedure. This can include use of a composition prior to, simultaneous with, and / or after use of the device / procedure.

[0062] A "condition" of the cornea refers to a state of disease, defect, injury, damage, degeneration, or dysfunction of the cornea. The condition may affect corneal tissue (e.g., the stromal layer) or corneal cells (e.g., keratocytes). The condition may be an acute condition, such as an abrasion or ulcer, or may be a chronic condition, such as keratoconus or myopia.

[0063] As used herein, "cornea" refers to the clear anterior portion of the eye that covers the iris, pupil, and anterior chamber. It includes the corneal epithelium, Bowman's layer, corneal stroma, Descemet's membrane, and corneal epithelium. Of particular interest is the stromal layer of the cornea (also called the substantia propria), which contains an extracellular matrix of regularly arranged collagen fibers together with keratocytes.

[0064] "Derivative," in reference to chemical derivatives, refers to a compound that has been chemically modified. The present disclosure encompasses each of the compounds described herein as well as any derivatives thereof, including chemically modified forms such as salts, hydrides, esters, and other modifications of the parent compound.

[0065] "Isolated" as used herein with particular reference to polypeptides refers to a molecule that has been separated from its natural environment. Isolated molecules can be obtained by any method or combination of methods known and used in the art, including biochemical, recombinant, and synthetic techniques. To obtain an isolated component, the polypeptide may be prepared by at least one purification or enrichment step. Of particular interest are polypeptides and peptides obtained by artificial means, i.e., non-natural means. This includes, but is not limited to, synthetic chemistry, recombinant techniques, purification protocols, and the like. Polypeptides isolated from natural, recombinant, or synthetic sources are included. Also included are polypeptides produced by chemical synthesis or by plasmids, vectors, or other expression constructs that can be introduced into cellular or cell-free translation systems. Such polypeptides are clearly distinguished from naturally occurring polypeptides without human intervention.

[0066] The terms "protein" or "polypeptide" (e.g., SEQ ID NO:1), and other such terms, refer to the molecules described herein for brevity. Such terms are not meant to provide a complete characterization of these molecules. Thus, although a protein or polypeptide may be characterized herein as having a particular amino acid sequence, a particular two-dimensional representation of the structure, it is understood that the actual molecule claimed has other characteristics, including three-dimensional structure, mobility for certain bonds, and other properties of the molecule as a whole. What is encompassed in this disclosure are the molecules themselves and their properties as a whole. The terms "protein" and "polypeptide" are used interchangeably herein.

[0067] TGFβ3 "polypeptide" refers to a polypeptide obtained from any source, such as an isolated naturally occurring polypeptide, a recombinant polypeptide, and a synthetic polypeptide, and includes not only a polypeptide having a naturally occurring amino acid sequence, but also a polypeptide having a mutant amino acid sequence and a fragment of such a sequence, as detailed herein. TGFβ3 may also be referred to in the art as transforming growth factor-β3, TGFB3, ARVD, and FLJ16571.

[0068] "Sequence identity" of amino acids refers to an amino acid-to-amino acid comparison of two or more polypeptides. A test sequence may be identical to a reference sequence (i.e., share 100% identity) or may contain one or more amino acid substitutions. In preferred embodiments, the amino acid substitutions may have similar chemical and / or physical properties, such as charge or hydrophobicity, compared to the reference amino acid. Sequence identity may typically be determined by sequence alignment in the regions of highest homology. Sequence alignment algorithms, such as BLAST, (登録商標) Sequence alignment programs are well known and widely used in the art. Based on the sequence alignment, the percent identity between the compared polypeptide sequences can be determined.

[0069] As used herein, "refractive error" refers to a defect in the focusing of light by the eye. Refractive errors can include spherical and cylindrical errors. Both low-order and high-order aberrations are included. Included as refractive errors in particular are the eye conditions known as myopia, hyperopia, astigmatism, anisometropia, and presbyopia.

[0070] "Regeneration" in reference to the cornea refers to the restoration of one or more of the shape, thickness, regularity, hardness, elasticity, and tensile strength of the cornea (including the corneal tissue (e.g., stromal layer)). Methods of regeneration may be used to impart a particular shape, i.e., corneal curvature, to the cornea. Regeneration methods may be performed in the treatment of particular conditions of the eye or cornea. Regeneration may include an increase in components (e.g., type II collagen) in the extracellular matrix of the cornea. Regeneration may also include increasing the number of cells (e.g., keratocytes) of the cornea. The number of cells may be increased, for example, by changing the proliferative state of such cells from a quiescent state to an active state.

[0071] For example, with respect to corneal cells (e.g., keratocytes), "reprogramming" of a cell refers to a change in differentiation state. Reprogramming is associated with one or more changes in cell morphology, cell gene expression (e.g., collagen expression, including expression of collagen type I and / or collagen type II), or cell proliferation status (e.g., quiescent or active state).

[0072] The term "subject" refers to a human or non-human animal. "Preventing" refers to halting or delaying the onset of a condition, for example, an eye condition, or specifically a corneal condition, such as a corneal disorder or other defect. A preventative measure results in the halting or delay of one or more symptoms of the condition, or reduces such symptoms if they occur. Prevention of a corneal condition can include strengthening the cornea, as detailed herein.

[0073] "Treating" refers to the alleviation, improvement, or elimination of a condition, for example, an ocular condition, or specifically a corneal condition, such as a corneal disorder or other defect. Treatment results in the alleviation, improvement, or elimination of one or more symptoms of the condition. Treatment of a corneal condition may include corneal regeneration, as detailed herein. The compositions and methods of the present invention may be used to treat, prevent, or both treat and prevent a variety of conditions, as detailed herein.

[0074] Cell and tissue regeneration Cell and tissue regeneration techniques hold great promise in therapeutic treatment. As disclosed herein, the inventors have developed compositions and methods for modifying cells using in situ cell reprogramming to affect the expression of type II collagen and the deposition of extracellular matrix (ECM) in corneal tissue. This in turn is used to strengthen and / or reinforce the cornea of ​​the eye. In so doing, the inventors provide a unique approach for the in situ / in vivo regeneration and reinforcement of the corneal stromal matrix.

[0075] Thus, the disclosed methods may be used for in vivo tissue engineering treatments for various corneal conditions, including myopia and keratoconus. As previously mentioned, myopia is characterized by excessive curvature of the cornea (FIG. 1A), and keratoconus is a progressive ectatic corneal dystrophy that leads to a characteristic pattern of corneal thinning (FIG. 1B; image taken from Romero-Jimenez, Santodomingo-Rubido, & Wolffsohn 2010).

[0076] Keratocytes are relatively quiescent and only produce large amounts of extracellular matrix (ECM) when they transform into a fibroblast or myofibroblast phenotype. The deposition of ECM associated with these phenotypes usually leads to corneal fibrosis and loss of transparency (Kadler, Baldock, Bella, & Boot-Handford 2007). Chondrocytes, the cells that compose cartilage, secrete type II collagen, a fibrillar collagen similar to type I found in the cornea. Type II collagen is also expressed by keratocytes during the development of the chick cornea, but this is later replaced by type I in the mature chick corneal stroma (Linsenmayer et al. 1990).

[0077] We have previously shown that stromal cells from adult human and rat corneas can be reprogrammed to produce neuron-specific proteins when treated with neural lineage specifying growth factors (Greene et al. 2013). This data indicates that adult cell populations can be reprogrammed simply by modulating the growth factor environment both in vitro and in vivo.

[0078] Next, as demonstrated herein, the inventors show that corneal stromal cells can be induced in vitro and ex vivo to produce type II collagen, a cartilage-specific fibrous collagen, by treating cells with transforming growth factor β3 (TGFβ3) and dexamethasone (Examples 8 and 9). Specifically, the inventors demonstrated that in corneal biopsies of human keratoconus, corneal stromal cells express type II collagen when treated with these two compounds (Example 8). Furthermore, using animal studies, the inventors demonstrated that two compounds, TGFβ3 and dexamethasone, can be delivered in vivo using eye drops to stimulate the deposition of type II collagen (Example 9). In particular, the deposition of type II collagen was uniform, without fibrosis or scarring, and improved corneal biomechanics without affecting corneal transparency (Examples 11 and 13).

[0079] Without wishing to be bound by theory, it is hypothesized that collagen deposition results from reprogramming cells in the corneal stroma to a chondrocyte phenotype. Chondrocytes have been shown to secrete type II collagen, which is expressed during chick cornea development as well as fibrillar collagen similar to type I found in the cornea (Linsenmayer et al. 1990), which is later replaced by type I collagen in the mature corneal stroma (Linsenmayer et al. 1990).

[0080] In the results described herein, when keratocytes are treated with TGFβ3 and dexamethasone, an initial increase in the expression of type I collagen is observed (Example 12).However, the inventor believes that the observed level of collagen I deposition is insufficient to harden / reform the cornea.Furthermore, collagen II deposition is considered to be more feasible as a therapeutic strategy.It should be noted that collagen II is less susceptible to enzymatic degradation by the enzymes present in the cornea of ​​keratoconus, for example.

[0081] According to the inventor's results, it is possible to use reprogramming of keratocytes to produce new ECM molecules as an effective treatment to improve the biomechanical characteristics of the cornea. This approach is considered advantageous because it reduces the susceptibility of the cornea to enzymatic degradation as mentioned above. The disclosed treatment module is not only intended to stabilize the cornea, but also to provide therapeutic support for various corneal conditions and ocular conditions, including ocular refractive errors. Thus, the method of the present invention can be used, for example, for the treatment of keratocytes of keratoconus in ectatic corneas. Furthermore, the method of the present invention can be used for the treatment of myopia and various other corneal conditions, as detailed herein.

[0082] Conditions affecting the eyes and cornea The compositions described herein are particularly useful in regenerating or strengthening the cornea (e.g., stromal layer) and corneal cells (e.g., keratocytes). The compositions may be used to address corneal thinning, weakening, cell loss, tissue loss, matrix loss, collagen loss, and / or irregularities. Thus, the compositions described herein may be used for a variety of conditions affecting the eye, including defects, diseases, injuries, damage, and / or degeneration of the cornea, as well as conditions involving refractive errors of the eye.

[0083] In a specific embodiment, the present invention includes a method for treating corneal defects. In certain circumstances, the method of the present invention can also be used to prevent corneal defects. The defects may be associated with certain corneal conditions. Exemplary conditions include keratoconus as detailed herein, and related conditions including corneal ectasia, such as keratoconus globulosa, pellucid marginal degeneration, and posterior keratoconus (see, for example, Arffa 1997; Krachmer et al. 1984; Rabonitz 2004; Jinabhai et al. 2010). Myopia, presbyopia, and astigmatism, including regular and irregular astigmatism, are also specifically included as defects. Congenital defects of the cornea are also included. These include cornea planus and microcornea, the latter of which may be associated with fetal alcohol syndrome, Turner syndrome, Ehlers-Danlos syndrome, Weill-Marchesani syndrome, Waardenburg syndrome, Nance-Horan syndrome, and Cornelia de Lange syndrome, as well as cornea globus (discussed above), which may be associated with Ehlers-Danlos syndrome type IV.

[0084] In a further aspect, the present invention includes a method for treating corneal damage or degeneration. In certain circumstances, the method of the present invention can be used to prevent corneal damage. Damage or degeneration can be associated with certain conditions of the cornea. Particularly included is corneal melting, for example, corneal melting associated with inflammatory diseases such as rheumatoid arthritis. Other exemplary conditions include keratitis, for example, marginal keratitis, keratostromal keratitis, lagophthalmos keratitis, neurotrophic keratitis, filamentous keratitis, rosacea keratitis, viral keratitis (including herpes keratitis), fungal keratitis, protozoal keratitis, and other infectious keratitis, for example, syphilitic keratitis, microsporidia keratitis, Thygeson's keratitis, and infectious crystalline keratopathy. Also included are ulcerative keratitis, also called peripheral ulcerative keratitis (PUK), which includes ulcerative keratitis associated with systemic diseases such as rheumatoid arthritis, Wegener's granulomatosis, systemic lupus erythematosus, relapsing polychondritis, and polyarteritis nodosa. Also included are endophthalmitis. Also included are chronic corneal edema, Mooren's ulcer, corneal limbal pit, phlyctenosis, Therrien's peripheral degeneration, Salzmann's degeneration, globular degeneration, and Fuchs' dystrophy. Such conditions are well known and well characterized in the art. For example, see Jackson 2008; Denniston 2009; and Willshaw et al. 2000. Further included are corneal stromal dystrophies, such as lattice corneal stromal dystrophy (e.g., types 1 and 2), granular corneal dystrophy (e.g., types 1 and 2), macular corneal dystrophy, Schneiderian corneal dystrophy, congenital corneal stromal dystrophy, and spotty corneal dystrophy.

[0085] In a further aspect, the present invention includes a method for treating injury to the cornea. Injury caused by physical injury, chemical injury, radiation injury, and / or injury caused by certain drugs is included. Injury may be related to corneal abrasion, corneal erosion, corneal puncture, membrane rupture, corneal scar, or corneal ulcer (including melting ulcer, painless ulcer, and superficial ulcer). Also included are injury and other injury related to eye surgery, including surgical wounds, corneal injury after radial keratotomy, and acute problems after keratoplasty, including persistent epithelial defects. In addition, included are injury related to corneal melt, for example, corneal melt after eye surgery or other treatment (e.g., topical NSAID administration). Corneal melt may be due to infectious, inflammatory, or nutritional causes. Also included are age-related corneal injuries and damage.

[0086] In a further aspect, the present invention includes a method for treating or preventing ocular refractive errors.Such refractive errors may be related to certain conditions, including myopia, hyperopia, presbyopia, anisometropia, high-order aberrations, and various astigmatisms.High-order aberrations include, but are not limited to, coma, trefoil, quadrafoil, spherical aberration, and aberrations that are specified by mathematical expressions (e.g., Zernike polynomials).

[0087] Corneal conditions can be diagnosed by various methods, including fluorescein staining, which can include Seidel test, specular microscopy, corneal topography, isometric tomography, corneal thickness measurement, ultrasound, slit lamp, corneal scraping cytology, and biopsy. Diagnosis can also include evaluation of visual acuity and / or opacity. Corneal conditions can be associated with one or more symptoms of pain, photophobia, foreign body sensation, decreased visual acuity, edema, leukocyte infiltration, fluorescein uptake, angiogenesis, redness, and systemic symptoms such as headache, nausea, and fatigue. Similarly, symptoms of refractive errors may include, but are not limited to, reduced vision, as well as blurred vision, double vision, blurred vision, eye strain, foreign body sensation, problematic glare or halos, floaters, ghost images, impaired night vision, strabismus, excessive staring, excessive blinking, headaches, eye rubbing, eye fatigue, dry ocular surface, eye irritation, redness, and eye twitching.

[0088] therapeutic composition As mentioned above, the compositions described herein can be used to treat and / or prevent various eye conditions, including conditions that affect the cornea and ocular refractive errors. The compositions may include a TGFβ3 polypeptide or a variant or fragment thereof, together with dexamethasone or a derivative or related steroid agent.

[0089] In various embodiments, the composition may be formulated to include a combination of the components described (TGFβ3 polypeptide (or variant or fragment thereof) and dexamethasone (or derivative or related steroidal agent)) or may be formulated to include a first component (TGFβ3 polypeptide (or variant or fragment thereof) or alternatively dexamethasone (or derivative or related steroidal agent)) and a second component that is added prior to administration. Alternatively, the composition may be formulated to include a first component (TGFβ3 polypeptide (or variant or fragment thereof) or alternatively dexamethasone (or derivative or related steroidal agent)) for use in simultaneous or sequential administration with a formulation that includes the second component.

[0090] In one embodiment, the TGFβ3 polypeptide may comprise at least the following amino acid sequence: ALDTNYCFRN LEENCCVRPL YIDFRQDLGW KWVHEPKGYY ANFCSGPCPY LRSADTTHST VLGLYNTLNP EASASPCCVP QDLEPLTILY YVGRTPKVEQ LSNMVVKSCK CS (SEQ ID NO: 1) (GenBank Reference No. CAR70088.1). The TGFβ3 polypeptide may comprise at least the 112 amino acids shown above and may have a molecular weight of 25.5 kDa. Alternatively, the TGFβ3 polypeptide may be derived from amino acids 644-850 (207 amino acids) of the precursor polypeptide sequence identified in GenBank Reference No. CAA33024.1, GenBank Accession No. CAA33024, or NCBI Reference Sequence NP_003230.1.

[0091] In other embodiments, variants or fragments of TGFβ3 may be used.For example, variants or fragments may show at least 75% sequence identity with SEQ ID NO:1, preferably at least 80% identity, more preferably at least 85%, and most preferably at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% sequence identity with SEQ ID NO:1 described herein.TGFβ3 variants are particularly interesting when they show biological activity, for example, activity that is similar or improved compared to non-mutated polypeptide.

[0092] In further embodiments, specific fragments may be used.For example, TGFβ3 fragments may comprise at least 80 amino acids, at least 85 amino acids of SEQ ID NO:1, more preferably at least 90 amino acids, at least 91 amino acids, at least 92 amino acids, at least 93 amino acids, at least 94 amino acids, at least 95 amino acids, at least 96 amino acids, at least 97 amino acids, at least 98 amino acids, at least 99 amino acids of SEQ ID NO:1, most preferably at least 100 amino acids, at least 101 amino acids, at least 102 amino acids, at least 103 amino acids, at least 104 amino acids, at least 105 amino acids, at least 106 amino acids, 107 amino acids, 108 amino acids, 109 amino acids, 110 amino acids, or 111 amino acids of SEQ ID NO:1.Of particular interest are functional fragments of TGFβ3, such as fragments that exhibit biological activity, such as similar or improved activity compared to reference polypeptide.

[0093] In certain embodiments, the TGFβ3 polypeptide or its variant or fragment may be provided as a recombinant polypeptide. For example, the polypeptide may be expressed in cells or cell-free expression systems, as is widely known and used in the art. These include bacterial, fungal, plant, and mammalian expression systems. Expression systems using E.coli cells, CHO cells, HEK cells, and Nicotiana benthamiana cells are specifically included. The TGFβ3 polypeptide or its variant or fragment may be provided as a human recombinant polypeptide expressed in a human or non-human expression system. The TGFβ3 polypeptide or its variant or fragment may be provided as a disulfide-bonded homodimeric non-glycosylated polypeptide chain according to known methods.

[0094] TGFβ3 polypeptide or variants or fragments thereof may be isolated from recombinant expression systems by standard methods, including well-known chromatography techniques. TGFβ3 polypeptide or variants or fragments thereof may include sequence tags to facilitate cleavage, isolation, and / or localization of the polypeptide. According to the present invention, TGFβ3 polypeptide may be obtained from various commercial sources. For example, recombinant human TGFβ3 may be obtained from R&D Systems (catalog number 243-B3-002; 243-B3-010), BioVision, Inc. (catalog number 4344-500; 4344-50; 4344-5), or Prospec Protein Specialists (catalog number CYT-113; CYT-319).

[0095] The biological activity of a TGFβ3 polypeptide or a variant or fragment thereof may be measured according to widely known and used methods. For example, biological activity may be measured in culture by the ability of the polypeptide to inhibit the proliferation of mink lung epithelial cells (Mv1Lu) (see, for example, Premaraj et al. 2006). Exemplary activity by this measurement is an ED of ≦50 ng / ml. 50 Alternatively, biological activity may be measured by dose-dependent inhibition of IL-4-induced proliferation of mouse HT-2 cells (BALB / c spleen activated by sheep erythrocytes in the presence of IL-2) (see, e.g., Tsang et al. 1995). Exemplary activities by this measurement are typically 0.1-0.5 ng / ml. Alternatively, compositions containing the combinations of agents described herein may be measured for biological activity using the methods described below. For example, induction of type II collagen (e.g., type II collagen, α1) in keratocytes may be assessed by one or more of immunohistochemistry assays, protein assays, Western blot analysis, polymerase chain reaction (PCR) analysis, and quantitative PCR techniques.

[0096] As described herein, the compositions may also include dexamethasone, its derivatives, and / or related steroidal agents. Dexamethasone is characterized as having the following chemical structure:

[0097] [ka]

[0098] Trade names for dexamethasone include Decadron. (登録商標) , Dexasone (登録商標) , Diodex (登録商標) , Hexadrol (登録商標) , Maxidex (登録商標) , and Minims (登録商標) Includes.

[0099] In various embodiments, derivatives of dexamethasone may be used, including any of its esters and salts. Exemplary derivatives include, but are not limited to, dexamethasone-17-acetate (CAS Reference Number: 1177-87-3), dexamethasone disodium phosphate (CAS Reference Number: 2392-39-4), dexamethasone valerate (CAS Reference Number: 14899-36-6), dexamethasone-21-isonicotinate (CAS Reference Number: 2265-64-7), dexamethasone palmitate (CAS Reference Number: 33755-46-3), dexamethasone propionate (CAS Reference Number: 55541-30-5), dexamethasone aceflate (CAS Reference Number: 83880-70-0), dexamethasone-21-galactoside (CAS Reference Number: 92901-23-0), dexamethasone 21 -thiopivalate, dexamethasone 21-thiopentanoate, dexamethasone 21-thiol-2-methyl-butanoate, dexamethasone 21-thiol-3-methyl-butanoate, dexamethasone 21-thiohexanoate, dexamethasone 21-thiol-4-methyl-pentanoate, dexamethasone 21-thiol-3,3-dimethyl-butanoate, dexamethasone 21-thiol-2-ethyl-butanoate, dexamethasone 21-thiooctanoate, dexamethasone 21-thiol-2-ethyl-hexanoate, dexamethasone 21-thiononanoate, dexamethasone 21-thiodecanoate, dexamethasone 21-p-fluorothiobenzoate or combinations thereof. Particularly included are dexamethasone alcohol and dexamethasone sodium phosphate. Also included are dexamethasone derivatives, as described in US4177268.

[0100] The composition may contain related steroids instead of or in addition to dexamethasone. For example, other corticoid steroids may be used instead of or together with dexamethasone. Preferred for use as related steroids are group C steroids according to Coopman classification, including betamethasone-type steroids, such as dexamethasone, dexamethasone sodium phosphate, betamethasone, betamethasone sodium phosphate, and fluocortolone. Other related steroids include, but are not limited to, fluoromethalone, lotoprendol, medrysone, prednisolone, prednisone, rimexolone, hydrocortisone, lodoxamide, or any derivative or combination thereof. Particularly included are fluoromethalone acetate, fluoromethalone alcohol, prednisolone acetate, prednisolone sodium phosphate, lotoprendol etabonate, hydrocortisone acetate, and lodoxamide tromethamine. With respect to any of the chemical entities of this disclosure, it is understood that the chemical entities may be in various modified forms, such as acetate and sodium phosphate forms, sodium salts, and the like.

[0101] The composition may comprise, for example, 0.04ng / ml to 4ng / ml, or 0.04ng / ml to 0.4ng / ml, or 0.4ng / ml to 4ng / ml, or 4 to 40ng / ml, or 40ng / ml to 400ng / ml, or 40ng / ml to 4000ng / ml of dexamethasone or a derivative or related steroid, or about 0.04ng / ml, about 0.08ng / ml, about 0.12ng / ml, about 0.4ng / ml, about 0. The composition may comprise about 8 ng / ml, about 1.2 ng / ml, about 4 ng / ml, about 12 ng / ml, about 24 ng / ml, about 40 ng / ml, about 80 ng / ml, about 120 ng / ml, about 240 ng / ml, about 400 ng / ml, about 800 ng / ml, about 1000 ng / ml, about 1600 ng / ml, about 2000 ng / ml, about 2400 ng / ml, about 3200 ng / ml, or about 4000 ng / ml of dexamethasone or a derivative or related steroid agent.

[0102] By way of further example, the composition may comprise 0.4 μg / ml to 40 μg / ml, or 0.4 μg / ml to 4 μg / ml, or 4 μg / ml to 40 μg / ml of dexamethasone or a derivative or related steroidal agent, or about 0.4 μg / ml, about 0.8 μg / ml, about 1 μg / ml, about 1.2 μg / ml, about 2 μg / ml, about 4 μg / ml, about 8 μg / ml, about 12 μg / ml, about 20 μg / ml, or about 40 μg / ml of dexamethasone or a derivative or related steroidal agent.

[0103] By way of further example, the composition may comprise 0.1 mg / ml to 1 mg / ml, or 0.5 mg / ml to 5 mg / ml, or 1 mg / ml to 10 mg / ml of dexamethasone or a derivative or related steroidal agent, or about 0.1 mg / ml, about 0.5 mg / ml, about 1 mg / ml, about 2 mg / ml, about 5 mg / ml, or about 10 mg / ml of dexamethasone or a derivative or related steroidal agent.

[0104] The composition may, for example, comprise 1 ng / ml to 1 μg / ml, or 1 ng / ml to 10 ng / ml, or 10 ng / ml to 100 ng / ml, or 100 ng / ml to 1 μg / ml of TGFβ3 polypeptide or variant or fragment thereof, or about 1 ng / ml, about 5 ng / ml, about 10 ng / ml, about 20 ng / ml, about 50 ng / ml, about 100 ng / ml, about 200 ng / ml, about 500 ng / ml, about 800 ng / ml, or about 1 μg / ml of TGFβ3 polypeptide or variant or fragment thereof. In certain embodiments, the composition may comprise at least 4 ng / ml of TGFβ3 polypeptide or variant or fragment thereof together with at least 4 ng / ml of dexamethasone or a derivative or related steroidal agent.

[0105] The composition may also include one or more anti-inflammatory drugs. Exemplary anti-inflammatory drugs include at least ketotifen fumarate, diclofenac sodium, flurbiprofen sodium, ketollac tromethamine, suprofen, celecoxib, naproxen, rofecoxib, or any derivative or combination thereof. Nonsteroidal anti-inflammatory drugs (NSAIDs) are specifically included. The composition may further include one or more anesthetic drugs. Exemplary anesthetic drugs include at least local anesthetics, such as proparacaine, lidocaine, and tetracaine, and any derivative or combination thereof. Other ophthalmic drugs may be selected to be included in the composition: these can be selected by those skilled in the art based on the condition and needs of the subject to be treated.

[0106] The compositions described herein can be formulated for topical administration as described herein and according to known methods. In certain circumstances, intraocular administration may be desirable. The compositions can be provided in any form suitable for administration to the eye. Exemplary formulations include at least a solution, suspension, emulsion (dispersion), gel, cream, or ointment in a suitable ophthalmic vehicle. For example, the compositions can be provided in the form of eye drops, semi-solid gel, or spray. In certain embodiments, molding contact lenses or other inserts / implants can be impregnated with the compositions of the present invention. In this way, the composition can be delivered to the cornea continuously and in a sustained release manner when the subject wears the contact lenses.

[0107] For topical administration to the eye, the composition may be formulated in a pH range of 5.0 to 8.0. This pH range may be achieved by the addition of a buffer to the solution. The formulation is preferably stable in a buffer solution. That is, there is no adverse interaction between the buffer and the active agent that would destabilize the composition, for example, by precipitation or aggregation. The composition may be hypertonic (5% to 40%, preferably about 10, 20, 30, or 40%) or hypotonic (0% to 5%, preferably about 1, 2, 3, or 4%) depending on the needs of the subject (e.g., work needs, rest time, sleep, etc.). Hypertonic compositions (e.g., 40%) may be used when combined with molding contact lenses, as detailed herein.

[0108] The composition may include one or more suitable preservatives as optional ingredients. Suitable preservatives may be added to prevent contamination, e.g., bacterial contamination. Such agents include, but are not limited to, benzalkonium chloride, thimerosal, chlorobutanol, methylparaben, propylparaben, phenylethyl alcohol, EDTA, sorbic acid, Onamer, tetrahydrofuran ... (登録商標) M, and other agents known to those of skill in the art, or any combination thereof. Such preservatives may typically be employed at a level of from 0.001% to 1.0% by weight of the composition.

[0109] The composition may contain an optional co-solvent. The solubility of the components of the composition of the present invention may be enhanced by a surfactant or other suitable co-solvent in the composition. Such co-solvents / surfactants include, for example, polysorbate 20, 60, and 80, polyoxyethylene / polyoxypropylene surfactants (e.g., Pluronic (登録商標) F-68, F-84, and P-103), cyclodextrins, tyloxapol, and other agents known to those skilled in the art, and any combination thereof. Such co-solvents may be used at levels typically between 0.01% and 2% by weight of the composition.

[0110] Penetration enhancers may be used to increase the uptake of the composition into the eye.Exemplary agents include at least cetylpyridinium chloride, ionophores such as lasalocid, benzalkonium chloride, parabens, Tween 20, saponin, Brij 35, Brij 78, Brij 98, ethylenediaminetetraacetic acid, bile salts and bile acids (such as sodium cholate, sodium taurocholate, sodium glycodeoxycholate, sodium taurodeoxycholate, taurocholic acid, chenodeoxycholic acid, and ursodeoxycholic acid), capric acid, azone, fusidic acid hexamethylene lauric acid amide, saponin, hexamethylene octanamide, and decyl methyl sulfoxide.

[0111] In addition, bioadhesive polymers may be used to adhere to the mucin membranes covering the eye to prolong the contact of the composition with the eye. The bioadhesive polymer may be a macromolecular hydrocolloid with multiple hydrophilic functional groups, such as carboxy-, hydroxy-, amide, and sulfate, which can establish electrostatic interactions. Exemplary agents include at least polyacrylic acid (e.g., Carbopol, Carbophil, and polycarbophil) and carboxymethylcellulose.

[0112] Controlled release systems may also be used: such systems may include in situ gels, colloidal particles, nanoparticles, and / or niosomes. Other drug delivery systems include, but are not limited to, erodible ophthalmic inserts, erodible ophthalmic inserts, hydrogels, collagen shields, liposomes, drug-loaded films (e.g., NOD (登録商標) ), and iontophoresis.

[0113] The composition may also include an optional agent for increasing viscosity. In order to enhance intraocular absorption of the active compound, to reduce variability in dispensing the formulation, to reduce physical separation of the suspension or emulsion components of the formulation, and / or to otherwise improve the ophthalmic formulation, increased viscosity beyond that of a simple aqueous solution may be desirable. Such viscosity-increasing agents include, by way of example, polyvinyl alcohol, polyvinylpyrrolidone, methylcellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose, carboxymethylcellulose, hydroxypropylcellulose, other agents known to those skilled in the art, or combinations thereof. Such agents may typically be used at levels of 0.01% to 2% by weight of the composition.

[0114] In certain embodiments, the composition includes a gelling agent, e.g., a water-soluble polymeric polysaccharide such as gellan gum. Gellan gum can be obtained from a variety of commercial sources, e.g., Kelcogel (登録商標) It is sold under the trade name Kelcogel. (登録商標) LT100 can be used as a fine mesh high acyl gellan that forms soft, elastic, non-brittle gels. In a specific embodiment, the gellan gum based composition is formulated as a 0.5% eye drop.

[0115] Other agents may be used to further stabilize or otherwise enhance the composition. For example, one or more of EDTA, sodium chloride, tyloxapol, sodium sulfate, and / or hydroxyethylcellulose may have the additional beneficial effect of further stabilizing the composition.

[0116] Treatment method As noted above, the compositions described herein are particularly useful in regenerating or strengthening the cornea (e.g., stromal layer) and corneal cells (e.g., keratocytes). Specifically, the compositions can be used to improve the shape, thickness, regularity, hardness, modulus, tensile strength, or function (e.g., refraction) of the cornea. Thus, the compositions described herein may be used to address various corneal conditions and ocular refractive errors, and may be used as adjunctive treatments to other ocular treatments.

[0117] As mentioned above, the composition can be formulated in any suitable way for administration to the eye.Formulations include eye drops, creams, emulsions, ointments, and gels.Particular attention should be paid to the formulations made as eye drops.In certain embodiments, the composition can be administered as eye drops using any of the many types of commercially available eye drop dispensers.By way of example, the container for the composition of the present invention can be transparent, translucent, and opaque, and can include other characteristics or combinations of characteristics, such as glass lining, tamper-proof, packaged in single or multiple dose aliquots, and any combination thereof.

[0118] The composition may be administered to a subject in a therapeutically effective amount to achieve a desired medical outcome. Specifically, the composition may be administered in an amount to address the ocular conditions described herein or to at least alleviate one or more symptoms of such conditions. The exact dosage (i.e., amount and schedule) of the composition may be determined by the clinician based on the subject and the condition presented. Exemplary formulations (e.g., eye drops) may be administered 1-24 times per day, or 1-12 times per day, or 1-6 times per day, or 1-4 times per day, or 1-3 times per day, or 1-2 times per day, or 1, 2, 3, 4, 6, 8, 12, 18, or 24 times per day. The composition may be applied topically as eye drops by placing one drop in each eye to be treated. Alternatively, 2-3 drops may be applied to each eye.

[0119] For the described compositions, dosage ranges may be, for example, 0.2 pg to 2.4 ng, or 2 pg to 2.4 ng of dexamethasone or a derivative or related steroid, or about 0.2 pg, about 0.4 pg, about 0.6 pg, about 0.8 pg, about 1.2 pg, about 2.4 pg, about 2 pg, about 4 pg, about 6 pg, about 8 pg, about 12 pg, about 18 pg, about 24 pg, about 0.2 ng, about 0.26 ng, about 0.4 ng, about 0.6 ng, about 0.8 ng, about 1.2 ng, about 1.8 ng, or about 2.4 ng of dexamethasone or a derivative or related steroid, per eye per dose.

[0120] As another example, dosage ranges include 12 ng to 1.3 μg, or 6 ng to 600 ng of dexamethasone or a derivative or related steroid per eye per dose, or 6 ng, about 8 ng, about 12 ng, about 16 ng, about 18 ng, about 24 ng, about 26 ng, about 30 ng, about 36 ng, about 40 ng, about 48 ng, about 52 ng, about 54 ng, about 60 ng, about 72 ng, about 78 ng, or about 80 ng of dexamethasone or a derivative or related steroid per eye per dose. g, about 80 ng, about 90 ng, about 120 ng, about 130 ng, about 160 ng, about 180 ng, about 240 ng, about 260 ng, about 300 ng, about 360 ng, about 400 ng, about 480 ng, about 520 ng, about 540 ng, about 600 ng, about 720 ng, about 780 ng, about 900 ng, about 1.2 μg, or about 1.3 μg of dexamethasone or a derivative or related steroid agent.

[0121] As yet another example, dosage ranges include 1.5 μg to 150 μg, 2.6 μg to 260 μg, or 6.5 μg to 650 μg of dexamethasone or a derivative or related steroid per eye per dose, or about 1.5 μg, about 2 μg, about 3 μg, about 4.5 μg, about 6 μg, about 6.5 μg, about 7.5 μg, about 10 μg, about 15 μg, about 22.5 μg, about 32.5 μg, about 20 μg, about 26 μg, or about 32.5 μg. , about 30 μg, about 40 μg, about 45 μg, about 60 μg, about 65 μg, about 75 μg, about 80 μg, about 90 μg, about 100 μg, about 120 μg, about 130 μg, about 150 μg, about 180 μg, about 225 μg, about 240 μg, about 260 μg, about 200 μg, about 300 μg, about 325 μg, about 450 μg, about 600 μg, or about 650 μg of dexamethasone or a derivative or related steroid agent. It will be appreciated that certain formulations of dexamethasone are commercially available and may be used according to accepted dosages and dosing schedules.

[0122] Any of the above doses of dexamethasone may be, for example, 5 pg to 65 ng, or 0.5 ng to 65 ng of TGFβ3 polypeptide or a variant or fragment thereof per eye per dose, or about 5 pg, about 10 pg, about 15 pg, about 20 pg, about 30 pg, about 45 pg, about 60 pg, about 0.05 ng, about 0.1 ng, about 0.15 ng, about 0.2 ng, about 0.3 ng, about 0.45 ng, about 0.5 ng, about 0.6 ng, about 0.65 ng, about 1 ng, about 1.5 In some embodiments, the TGFβ3 polypeptide or a variant or fragment thereof may be administered in a dose range of about 10 ng, about 2 ng, about 3 ng, about 4 ng, about 4.5 ng, about 6 ng, about 6.5 ng, about 7.5 ng, about 9 ng, about 10 ng, about 12 ng, about 13 ng, about 15 ng, about 16 ng, about 20 ng, about 22.5 ng, about 24 ng, about 30 ng, about 32.5 ng, about 36 ng, about 40 ng, about 45 ng, about 48 ng, about 50 ng, about 52 ng, about 60 ng, or about 65 ng of a TGFβ3 polypeptide or a variant or fragment thereof.

[0123] The dosage per eye may be about one drop of the disclosed composition. A drop of the composition may be 10 μl to 200 μl, 20 μl to 120 μl, or 50 μl to 80 μl, or any value therebetween. For example, a dispenser such as a pipettor may dispense drops of 1 μl to 300 μl and any value therebetween. Preferably, the dispenser measures about 15 μl, about 20 μl, about 30 μl, about 45 μl, about 60 μl, or about 65 μl per drop of the disclosed composition.

[0124] If the composition is administered via a contact lens or another insert / implant device, the contact lens or insert / implant may contain, for example, 0.01 mg to 10 mg of dexamethasone or a derivative or related steroid agent, or about 0.01 mg, about 0.1 mg, about 0.5 mg, about 0.7 mg, about 1 mg, about 5 mg, or about 10 mg of dexamethasone or a derivative or related steroid agent. Alternatively, the contact lens or insert / implant may contain 10 ng to 100 ng of dexamethasone or a derivative or related steroid agent, or about 1 ng, about 5 ng, about 10 ng, about 20 ng, about 50 ng, about 80 ng, or about 100 ng of dexamethasone, or a derivative or related steroid agent. As a further example, the contact lens or insert / implant may contain about 10 ng to 1 μg of TGFβ3 polypeptide or a variant or fragment thereof, or about 10 ng, about 50 ng, about 100 ng, about 200 ng, about 500 ng, about 800 ng, or about 1 μg of TGFβ3 polypeptide or a variant or fragment thereof.

[0125] The compositions described herein may be used in conjunction with various surgical procedures or other treatments. For example, the compositions may be used in conjunction with surgical and non-surgical methods for refractive correction of the eye. Exemplary methods include, but are not limited to, radial keratotomy (RK), including mini asymmetric radial keratotomy (MARK), hexagonal keratotomy (HK), laser photorefractive keratectomy (PRK), keratomileusis, laser in situ keratomileusis (LASIK), e.g., intraLASIK. (登録商標) , Laser epithelial keratomileusis (LASEK), e.g., Epi-LASEK, Automated lamellar keratomileusis (ALK), Laser thermal keratomileusis (LTK), Conductive keratomileusis (CK), Limbal relaxing incision (LRI), Astigmatic keratomileusis (AK), Epikeratophakia, Anterior ciliary sclerotomy (ACS), Scleral reinforcement, Presbyopia correction, Laser recovery for presbyopia (LRP), Intracorneal ring therapy (ICR), Intrastromal ring therapy (e.g., INTACTS) (登録商標) ), implantable contact lenses, scleral expansion bands (SEBs), and Kamra (商標) Also included are thermal keratoplasty, orthokeratology, enzymatic orthokeratology, and chemical orthokeratology.

[0126] The composition may be used in conjunction with the surgical correction of non-refractive conditions, such as the surgical correction of corneal lacerations.In certain embodiments, the composition described herein may be used in conjunction with certain surgical procedures performed on the cornea.Exemplary procedures include, but are not limited to, corneal transplantation surgery, penetrating keratoplasty (PK), therapeutic lamellar keratectomy (PTK), pterygium removal, corneal staining, artificial corneal insertion (e.g., KPro or Dohlman-Doane), and tooth-root-based artificial corneal insertion (OOKP).

[0127] The compositions may be used in conjunction with corneal collagen crosslinking. Corneal crosslinking typically involves the use of a riboflavin solution activated by exposure to UVA light. Known crosslinking methods include, but are not limited to, corneal crosslinking in which the epithelium is removed (Dresden Protocol, or Epi-Off), transepithelial crosslinking (Epi-On), and accelerated crosslinking. Crosslinking procedures are commonly available and include, among others, CXL, C3-R (登録商標) CCL (登録商標) , and KXL (登録商標) It is sold as corneal crosslinking. Administration of the composition may be before and / or after the crosslinking procedure. It is proposed that the disclosed composition can be used to avoid or counteract the deleterious effects of crosslinking procedures, such as corneal stroma clouding and cell loss (described in more detail below). Furthermore, corneal regeneration using the disclosed composition can allow crosslinking to be performed on subjects who were previously unsuitable for such procedures, for example, subjects with corneal thicknesses less than 400 μm. Furthermore, the disclosed composition can be used to slow or stop progressive corneal thinning that is not addressed by the use of crosslinking alone.

[0128] The compositions described herein may be co-administered with one or more additional ophthalmic agents. In various embodiments, co-administration may be by simultaneous or subsequent administration with such agents, or by co-formulation with such agents. Depending on the condition to be treated or prevented, the compositions described herein may be co-administered with one or more agents, including, but not limited to, antihistamines, sympathomimetics, beta-receptor blockers, parasympathomimetics, parasympatholytics, prostaglandins, nutrients, vasoconstrictors, lubricants, antibacterial agents, and anesthetics. Particularly included are various anti-inflammatory agents, including nonsteroidal anti-inflammatory drugs (NSAIDs). The compositions may also be co-administered with ocular lubricants and artificial tears.

[0129] Non-limiting examples of anesthetics include benzocaine, bupivacaine, cocaine, etidocaine, lidocaine, mepivacaine, pramoxine, prilocaine, chloroprocaine, procaine, proparacaine, ropicaine, and tetracaine. Non-limiting examples of anti-inflammatory drugs include aspirin, acetaminophen, indomethacin, sulfasalazine, olsalazine, sodium salicylate, choline magnesium trisalicylate, salsalate, diflunisal, salicylsalicylic acid, sulindac, etodolac, tolmetin, diclofenac, ketorolac, ibuprofen, naproxen, flurbiprofen, ketoprofen, fenoprofen, suprofen, oxaproxycin, mefenamic acid, meclofenamic acid, oxicam, piroxicam, tenoxicam, pyrazolidinedione, phenylbutazone, oxyphentatrazone, pheniramine, antazoline, nabumetone, COX-2 inhibitors (Celebrex (登録商標) ), apazone, nimesulide, and zileuton. Glucocorticoids such as hydrocortisone, prednisolone, fluorometholone, and dexamethasone may also be used as anti-inflammatory agents.

[0130] Exemplary antibacterial agents include, but are not limited to, zinc bacitracin, chloramphenicol, chlorotetracycline, ciprofloxacin, erythromycin, gentamicin, norfloxacin, sulfacetamide, sulfisoxazole, polymyxin B, tetracycline, tobramycin, idoxuridine, trifluridine, vidarabine, acyclovir, foscarnet, ganciclovir, natamycin, amphotericin B, clotrimazole, econazole, fluconazole, ketoconazole, miconazole, flucytosine, clindamycin, pyrimethamine, folinic acid, sulfadiazine, and trimethoprim-sulfamethoxazole. Exemplary vasoconstrictors include, but are not limited to, dipivefrin (Propine (登録商標)), epinephrine, phenylephrine, apraclonidine, cocaine, hydroxyamphetamine, naphazoline, tetrahydrozoline, dapiprazole, betaxolol, carteolol, levobunolol, metipranolol, and timolol. Nutrients include vitamins, minerals, and other beneficial substances, such as vitamin A, vitamin B1, vitamin B6, vitamin B 12 , vitamin C (ascorbic acid), vitamin E, vitamin K, and zinc.

[0131] In specific embodiments, the compositions described herein are formulated as eye drops, which are used in conjunction with other eye drop formulations, including, but not limited to, irrigation / lubrication eye drops, dry eye medications, steroid and antibiotic eye drops, glaucoma eye drops, allergy / anti-inflammatory eye drops, and conjunctivitis eye drops.

[0132] The composition may be used in conjunction with contact lenses, corneal inserts, corneal implants, or intrastromal rings to help support or reshape the subject's cornea. Corneal inserts include corneal inlays and corneal onlay devices. For example, contact lenses, corneal intrastromal rings, or other inserts / implants may be used to shape or maintain the shape of the cornea before, during, and / or after treatment with the composition. It should be noted that a corneal "insert" typically refers to a device that is temporarily inserted into the cornea, whereas a corneal "implant" typically refers to a more permanent device. However, in the art, many well-known devices are described interchangeably as implants / inserts. Thus, the term "insert / implant" as used herein should not be considered strictly limiting based on the time of use.

[0133] Contact lenses, corneal inserts, corneal implants, or intrastromal rings may be used with the disclosed compositions to treat corneal defects, diseases, injuries, damage, and / or degeneration, and ocular refractive errors. In various embodiments, contact lenses, corneal inserts, corneal implants, or intrastromal rings may act as carriers for compositions or as composition-eluting devices. In other embodiments, contact lenses, corneal inserts, or other corneal inserts / implants may be used with compositions suitable for ocular administration, such as eye drops, as detailed herein. In certain embodiments, computer software may be used to determine the most suitable contact lenses, corneal inserts, corneal implants, or intrastromal rings for a subject and / or to determine the composition formulation. In certain embodiments, treatment with contact lenses, corneal inserts, corneal implants, or intrastromal rings with the compositions described herein is used before or after ocular surgery, such as refractive surgery or transplant surgery.

[0134] Treatment involves evaluating the subject (e.g., the subject's age, occupational needs, ocular abnormalities or disorders, etc.), prescribing the use of a molding contact lens, corneal insert, corneal implant, or intrastromal corneal ring to aid in the necessary change in the radius of curvature of the anterior surface of the cornea, and prescribing a composition described herein for use in conjunction with the contact lens or implant / insert. The contact lens or implant / insert that is prescribed and used by the subject can be used to exert a mechanical force on the cornea, thereby inducing a change in shape, i.e., refractive power, of the cornea.

[0135] In certain preferred embodiments, the cornea can be supported or shaped by the use of a corneal insert, corneal implant, or intrastromal corneal ring in conjunction with the disclosed compositions. An example of a commercially available device is INTACS (登録商標)and KeraRing corneal intrastromal ring. In another embodiment, molding contact lenses may be used in conjunction with the disclosed compositions. The contact lenses may be hard or rigid, or may be soft lenses. Alternatively, the contact lenses may comprise both hard and soft portions. When soft contact lenses are used, a greater positive or negative curvature may be induced in the cornea, and discomfort in the subject's eye will decrease as the subject adapts to the contact lenses. When hard contact lenses are used, greater mechanical pressure may be exerted on the cornea. The contact lenses may be gas permeable. The molding contact lenses may be obtained from commercial sources. Examples of commercially available lenses include at least the lenses of DreamLite, OK Lens, EyeDream, MiracLens, DreamLens, i-GO OVC, GOV, Wake and See, CRT, Fargo / iSee, Emerald, and Wave Contact Lens System.

[0136] Once the contact lens, corneal insert, corneal implant, or intrastromal corneal ring is placed on / in the subject's eye, the composition described herein (e.g., eye drops) may be administered to the eye. In certain circumstances, it may be desirable to pre-administer the composition prior to the placement of the contact lens, corneal insert, corneal implant, or intrastromal corneal ring. Advantageously, the contact lens, corneal intrastromal ring, or other insert / implant and the composition may be used in conjunction to effect a change in the shape of the cornea, thereby producing refractive power. The composition may be administered more frequently to allow the cornea to accommodate the desired shape change. In certain embodiments, the composition is administered at least every 24, 12, or 8 hours. In other embodiments, the composition is administered every 6 hours. In certain other embodiments, the composition is administered about every 3 hours. In still other embodiments, the composition is administered about every 2 hours. In still other embodiments, the composition is administered every hour.

[0137] Without wishing to be bound by any particular theory, the combination of contact lens, corneal insert, corneal implant or corneal intrastromal ring with the composition described herein may include changes in the molecular structure of the cornea, and may induce changes in the cells and proteins found in the corneal stroma, such as collagen (e.g., type II collagen).This makes the surface of the cornea more uniform.By reducing the irregularity of the surface of the cornea, the quality and clarity of all images (i.e., vision) are improved.

[0138] The flattest keratometric measurement is used for the calculation of the molding contact lens. The skilled person can also use the more tilted keratometric measurement or an average of both, and based on this corneal curvature, perform the necessary calculations to flatten or tilt the radius of curvature of the anterior surface of the cornea, thus correcting the refractive defect of the eye. The base curve of the molding contact lens can be calculated separately based on the change in refractive power of each eye. In certain embodiments, the base curve of the molding contact lens can start to be calculated at 1 to 4 flatter or more tilted diopters, more preferably 1 to 3 flatter or more tilted diopters, even more preferably 1 to 2 flatter or more tilted diopters, depending on the refractive error required. The peripheral base curve depends on the accommodation of the molding contact lens and is calculated to be 0.5 mm in diameter larger than the central zone, but can vary depending on the design.

[0139] The diameter of the molding contact lenses used according to the present invention may be between 8.0 mm and 18.0 mm. Commercially available lenses are manufactured with such diameters. In certain embodiments, the molding contact lenses may be hard contact lenses having a diameter ranging from 8.0 mm to 12.0 mm. In other embodiments, the molding contact lenses may be soft contact lenses having a diameter ranging from 13.0 mm to 15.0 mm. The soft contact lenses may cover the entire cornea and extend from sclera to sclera. In still other embodiments, the molding contact lenses may be composed of hard and soft materials. The contact lenses may be hard in the center and may be about 12.0 mm, 13.0 mm, 14.0 mm, or up to 15.0 mm, and the periphery may be soft and may be up to 16.0 mm, 17.0 mm, and up to 18.0 mm. Larger contact lenses, preferably soft contact lenses, may be used at night as molding contact lenses.

[0140] The power of the molding contact lens can be determined to be as close as possible to the refractive power required for the subject to see comfortably. If during the molding contact lens adaptation process, the visual acuity is not sufficient to meet the needs of the subject, the subject is prescribed glasses during treatment. When the cornea is remodeled or has been remodeled, various optometric measurements are repeated to ensure that the treatment is proceeding as planned and is appropriate. Such measurements can include visual acuity for near and far vision, orthotype, keratometry, objective and subjective retinoscopy, adaptation diagram of the molding contact lens, movement of the molding contact lens, and comfort of the molding contact lens.

[0141] After measurements are taken, changes may be made to the treatment program based on these measurements. At each evaluation, a decision may be made as to whether to continue the same molding contact lens or to use new contact lenses. Furthermore, the same decision may be made regarding the composition used with the molding contact lens. Changes in molding contact lens and / or composition may be made to induce the desired reshaping of the cornea over a period of several weeks. In certain embodiments, weekly regular corrections are made during the first 8 weeks after treatment begins.

[0142] The compositions described herein cause changes in the collagen content (e.g., type II collagen) of the cornea. Other aspects of the corneal anatomy, histology, and physiology can also be affected by the compositions. In certain embodiments, the compositions can be hypertonic or hypotonic to cause changes in the corneal hydration. In other embodiments, the compositions can be used to modify the molecular structure (e.g., extracellular matrix) of the cornea, thus strengthening or repairing the cornea or reshaping it to a desired curvature.

[0143] When reshaping the cornea, it may be desirable to co-administer one or more enzymes to soften the cornea. Exemplary enzymes include, but are not limited to, hyaluronidase, chondroitinase ABC, chondroitinase AC, keratinase, and stromelysin, which have been shown to act on various proteoglycan components of the cornea. Also included are the enzymes collagenase, matrix metalloproteinase 1 (interstitial collagenase), and matrix metalloproteinase 2 (gelatinase). When the composition is co-administered with such enzymes, it may be desirable to include a vehicle such as a polymer in the composition (e.g., methylcellulose, polyvinyl alcohol, cellulose, etc.) to enhance the action of such enzymes. Additional agents may be included to activate metalloproteinase enzymes, such as interleukin-1α, tumor necrosis factor α / β and any subtypes thereof, sodium urate monohydrate, 4-aminophenylmercuric acetate, human serum amyloid A, human B2 microglobulin, and copper chloride. Carbamide (urea) may also be included. Also, any combination of these agents may be used.

[0144] The composition may also be co-administered with one or more enzymes that break down other sugars or proteins found in the cornea. The composition may also be co-administered with one or more anesthetics used to reduce irritation of molding contact lenses or any corneal inserts / corneal implants. The composition may also be co-administered with one or more lubricants to improve the subject's comfort during treatment. In other embodiments, the composition may be co-administered with one or more antimicrobial agents, such as antibacterial agents, antiviral agents, and / or antifungal agents. The composition may also be co-administered with one or more vasoconstrictors. One skilled in the art can determine the appropriate agents for co-administration to a subject based on the condition being treated.

[0145] In certain embodiments, the composition may be provided in a kit. The kit may include one or more of molding contact lenses, lubricating eye drops, cleaning solution or other solution for contact lenses, contact lens carrying case, spare pair of contact lenses, and instructions for wearing contact lenses and using the composition. The composition provided with the kit may be formulated to include a combination of components (TGFβ3 polypeptide (or variant or fragment thereof) and dexamethasone (or derivative or related steroid agent)), or the kit may include the components as separate formulations that are mixed together before administration or administered together, i.e., by simultaneous or sequential administration. EXAMPLES

[0146] The examples described herein are provided for the purpose of illustrating certain embodiments and aspects and are not intended to limit the invention in any way. Those skilled in the art can use the disclosure and teachings described herein to create other embodiments, aspects, and variations without undue experimentation. All such embodiments, aspects, and variations are considered to be part of the present invention.

[0147] Example 1: Experimental Overview In previous experiments, the inventors have shown that it is possible to induce keratocytes to differentiate into a neural lineage. The experiments described herein aimed to investigate the possibility of converting keratocytes into chondrocyte-like cells secreting cartilage-specific collagen type II. This type of cartilage is thought to be expressed during development (Linsenmayer et al. 1990). A further objective was to ascertain whether it was possible to induce collagen type II deposition in vivo in living rat corneas and whether this treatment had a favorable effect on the optical properties of the cornea. A further objective was to determine whether keratocytes in keratoconus tissue could be applicable to this method of cell reprogramming and the subsequent production of collagen type II-rich ECM. Finally, the experiments aimed to evaluate the effect of collagen type II deposition on the biomechanical properties of in vivo and ex vivo treated corneas using nanoindentation testing, a bioengineering approach that allows the analysis of hardness and elastic modulus.

[0148] Example 2: Tissue Samples Human tissue Cadaveric whole human corneas, keratoconus corneas obtained at transplantation surgery, human limbal peripheries, and surgeon-excised DSEK caps (excess stromal tissue from corneal endothelial transplantation) were obtained from donors sourced from the New Zealand National Eye Bank (Auckland, New Zealand). Human limbal peripheries were harvested after the central corneal button was removed for corneal transplantation surgery leaving a 2 mm limbus from the corneal limbal junction. Research ethics approval and consent were obtained from the Northern X Regional Human Ethics Committee before using the tissues. All tissues were stored in New Zealand Eye Bank medium (2% FCS, 2 mM L-glutamine, 1x Anti-Anti in Eagle's MEM medium) until use and transported in New Zealand Eye Bank transport medium (Eye Bank medium supplemented with 5% dextran).

[0149] animal tissue Ethical approval for animal studies was obtained from the University of Auckland Animal Ethics Committee (approval number R856). Eyes and cartilage were obtained from 6-8 week-old adult male Wistar rats after euthanasia using a carbon dioxide chamber. All eyes were removed from the animals and the cornea was carefully excised using surgical scissors under a dissecting microscope. The xiphoid process, a part of the zygomatic bone containing a thin, wide cartilage plate at its end, was excised using a scalpel blade. Animal tissues were washed with povidone-iodine (PVP-I) and sodium thiosulfate. Excess fat and tissue overlying the cartilage were scraped off with a blade. Freshly collected eyes and cartilage were stored in phosphate-buffered saline for a minimal time until use.

[0150] Example 3: Histological Analysis Tissue preparation and cryosectioning Corneal and cartilage pieces (2 mm × 2 mm) were embedded in Optimal Cutting Temperature (OCT) compound (Tissue-Tek, Sakura, The Netherlands) before being flash frozen in liquid nitrogen. Sections 10–15 μm thick were cut using a Microm HM550 Cryostat (Thermo-Scientific, USA) and stained with SuperFrost (商標) Plus electrostatic slides (Menzel-Glenser, Germany). Frozen sections were stored at -20°C until further use.

[0151] Cell and tissue culture Tissue digestion and cell preparation from human and rat corneas The corneal limbus was excised and the corneal stroma was isolated from the sclera in a class II laminar flow hood. The corneal epithelium and endothelium were then gently scraped with a keratome and discarded. The DSEK cap was also gently scraped with a keratome to remove the epithelium. The remaining corneal stromal tissue was then soaked in 37°C Hanks Balanced Salt Solution (GIBCO) with gentle mixing on a rotary shaker. (登録商標)The tissue was digested with 0.4% type II collagenase (Sigma-Aldrich) in 0.5% CO (Life Technologies). Various digestion times were used, but 5 h was required for optimal tissue digestion and cell viability.

[0152] After tissue digestion was complete, cells were pelleted by centrifugation at 1200 rpm for 7 minutes. Cells were then resuspended in a minimal amount of appropriate cell culture medium and purified using a Leica DM Counting was performed using an IL benchtop inverted microscope and a Neubauer hemocytometer. Cell suspensions in a 1:1 ratio with trypan blue solution (0.04% trypan blue stock in PBS) were used with a minimum of 3 counts per sample and the average was taken.

[0153] Keratocyte cell culture Cell manipulations were performed using sterile technique in a class II laminar flow hood. Isolated keratocytes were cultured in 12-well or 24-well cluster plates (Falcon) on plastic or glass coverslips in 2–3 ml of cell culture medium. Cells were maintained in a humidified incubator at 37°C with 5% CO2. Culture medium was changed after 24 h and then every 2 days or more frequently if necessary. Cells were observed daily using a Leica DM IL tabletop inverted microscope. For cell pellet culture, cells were freshly obtained after pelleting the tissue by centrifugation at 300 g for 7 min at 20°C in plastic conical tubes. The appropriate culture medium was added to the tube. After 24 h of incubation at 37°C, the cells shrank and formed a pellet that did not adhere to the tube wall. Pellets were cultured for 3 weeks in 2 ml of medium in a humidified atmosphere of 5% CO2 at 37°C. Medium was changed every other day.

[0154] Organotypic section culture Human and rat corneal and cartilage tissues were sectioned (1–2 mm) in the anteroposterior plane using a blade, and the sections were placed in an organotypic air / liquid interface culture system (Figure 2). Briefly, explants of normal tissues were cultured on cell culture inserts with 0.4 μm pore size (Millicell, France) at the interface of culture medium and a CO2-enriched environment. Corneal slices were placed epithelial side up on cell culture plates containing 3 ml of culture medium. The culture medium was changed every other day.

[0155] Example 3: In vitro reprogramming Culture medium Several custom made media were used, as described in the table below.

[0156] [Table 1-1]

[0157] [Table 1-2]

[0158] Chondrogenic reprogramming of keratocytes Tissue explants were cultured in chondrogenic differentiation medium for various time intervals to determine the optimal time required for growth factor treatment. Samples were harvested at each time point (Table 2). To obtain a monolayer of cells, 15 × 10 cells were plated on a glass coverslip. 4 / cm 2 Keratocytes were seeded at a density of 1000 x 1000 μg / ml. Cells were allowed to attach to the coverslips for 24 hours and the culture medium was changed every other day. Cells were maintained for up to 3 weeks.

[0159] [Table 2]

[0160] Example 4: In vivo reprogramming Gel eye drop formulations for growth factor delivery Gellan gum, a water-soluble polysaccharide produced by the bacterium Pseudomonas elodea, was used to formulate eye drops. The use of a gel-based formulation allows for extended corneal residence time and increased ocular availability of therapeutic agents. High molecular weight gellan gum is an anionic polymer, so it is suitable for the treatment of ocular diseases such as glaucoma, glaucoma, and glaucoma. 2+ , Mg 2+ , K + , and Na + It undergoes in situ gelation in the presence of monovalent and divalent cations such as α- and β-catenin (Bakliwal, & Pawar 2010). Electrolytes present in tears cause the polymer to gel when instilled in the eye, which in turn results in longer residence times and increased bioavailability of the drug (Ludwig 2005). Based on previous formulation studies, the polymer formulation is non-irritating and safe for use in vivo (Rupenthal, Green, & Alany 2011).

[0161] First, distilled water was heated to 80°C, and then gellan gum (Kelcogel (商標) A 0.5% solution was prepared by adding 100 mg of TGFβ3 (USA) with constant stirring. Once the powder was completely dissolved, the solution was cooled and stored at 4°C. The appropriate amount of growth factor was added to the fluid gel with constant stirring. A 10-fold higher concentration of growth factor than that used in the culture medium was used to supplement the drug lost through nasolacrimal drainage and blinking. The eye drop gel contained a final concentration of 100 ng / ml TGFβ3 and approximately 4 μg / ml dexamethasone.

[0162] Treatment with neurogenic and chondrogenic factors The animals were manually restrained and approximately 15 μL of eye drops were instilled into the right eye (Figure 3). The contralateral eye served as a control. Eye drops were administered three times daily for up to 5 days for neuronal specification and up to 8 weeks for chondrogenic specification.

[0163] Example 5: Immunohistochemistry (IHC) analysis Tissue collection and processing At the end of the treatment, the animals were euthanized using a carbon dioxide chamber. The eyes were harvested and washed with phosphate-buffered saline. The corneas were then carefully excised, fixed in 4% paraformaldehyde (PFA) for 1 h, and treated with sucrose solution to cryoprotect the tissue before freezing and sectioning. Sucrose as cryoprotectant is a dehydrating agent that prevents the formation of ice crystal artifacts in frozen tissue sections. Cryoprotection is particularly important in the case of slow freezing of tissues.

[0164] Briefly, corneas were immersed in a 20% sucrose solution at 4°C for 5 hours, then transferred to a 30% sucrose solution and kept at 4°C until the tissue sank (usually overnight). Corneas were then embedded in OCT compound and immersed in liquid nitrogen to induce rapid freezing. Frozen tissue blocks were stored at -80°C until further use. Cryostat sections of approximately 10-15 μm thickness were cut using SuperFrost (商標) Plus slides and the slides were stored at -80°C until required. For cell culture, cells grown on coverslips were rinsed with PBS and fixed with 4% PFA for 15 min. Coverslips were immersed in PBS until further use.

[0165] immunohistochemistry For frozen tissue sections, slides were kept at room temperature for 15–20 min before immunohistochemistry was performed. PBS was used to wash off OCT and the peripheral zone of tissue was demarcated using a wax pen. Tissue sections were first incubated with a blocking solution of 10% normal goat serum for 1 h, followed by incubation with primary antibodies at the appropriate dilution overnight at 4°C. Slides were then rinsed three times in PBS before incubation with secondary antibodies at the appropriate dilution. Secondary antibodies were left at room temperature for 2 h. Sections were counterstained with the nuclear marker 4',6'-diamidino-2-phenylindole (DAPI) and embedded in Citifluor antifade agent (ProSciTech, Australia). Olympus FluoView (商標)Imaging was performed using an FV-1000 confocal laser scanning microscope (405 nm, 473 nm, and 559 nm wavelength lasers) and a Leica DMRA fluorescence microscope.

[0166] [Table 3]

[0167] Example 6: Gene Expression Analysis RNA isolation and cDNA synthesis Extract mRNA from samples using PureLink (登録商標) Briefly, the tissue samples were diluted with 0.75 ml of TRIzol. (登録商標) and carrier RNA and homogenized using a handheld homogenizer (PRO Scientific, Inc.). The sample was then incubated with 0.2 ml of chloroform, followed by centrifugation at 12000 rpm and 4° C. for 15 minutes. The upper phase was separated, mixed with ethanol, and then transferred to a collection column tube.

[0168] The RNA was collected by centrifugation at 12000 rpm for 1 min. The flow-through was discarded and the extracted RNA was treated with DNAse. The column was washed several times with the provided buffer and finally the RNA was dispersed in RNAse-free water. NanoDrop (登録商標) (Thermo Scientific) was used to determine the concentration, and the mRNA was stored at −80°C.

[0169] SuperScript (登録商標) VILO (商標) cDNA Synthesis Kit (Invitrogen (商標) cDNA was prepared using the VILO PCR kit (Life Technologies, Inc.). (商標) Reaction Mix, SuperScript (登録商標)Enzyme Mix and RNAse-free water were incubated for 10 minutes at 25° C. Samples were then incubated for 120 minutes at 42° C., followed by a 5 minute incubation at 85° C. cDNA was stored at −20° C.

[0170] TaqMan (登録商標) Quantitative PCR using gene expression assays TaqMan for your gene of interest (登録商標) Gene expression assays were obtained. In the PCR step, 10 μL of TaqMan (登録商標) Universal Master Mix II was combined with 1 μL of assay sample, approximately 25 ng of cDNA, and 9 μL of water to a volume of 20 μL. The tubes were vortexed and briefly centrifuged to pellet the contents. Each cDNA sample was prepared in triplicate and pipetted into a 384-well plate. 20 μL of each reaction mixture was pipetted into a MicroAmp (登録商標) Each well of a MicroAmp Optical 384-Well Reaction Plate (Applied Biosystems) was then filled with 100% ethanol. (登録商標) After covering with Optical Adhesive Film (Applied Biosystems), the plate was briefly centrifuged to remove air bubbles. The plate was transferred to a 7900HT Fast Real-Time PCR System and run using the following thermal cycling parameters: 50°C for 2 min, 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Results were analyzed as described in the previous section.

[0171] [Table 4]

[0172] Example 7: Examination of biomechanical and optical properties following in situ corneal stromal ECM protein deposition Experiments on the preocular segment (anterior structure) of rodents Corneal biomechanics has been shown to be relevant in the diagnosis and treatment of various corneal diseases, providing insight into the structure of the cornea and its relationship to physiological function. Corneas that have been treated to induce deposition of ECM proteins should also be examined for corneal opacity, as loss of transparency is undesirable.

[0173] The corneas of treated rats were examined using a Phoenix Micron IV rodent eye imaging system (Phoenix Research Labs). (登録商標) Rats were sedated using an intraperitoneal injection of Antisedan (3:2). The slit lamp attachment of a Micron IV imaging system was used to closely examine the layers of the cornea to confirm corneal integrity and transparency. Retinal imaging was also performed to confirm corneal transparency. After imaging, rats were given Antisedan for recovery from sedation. (登録商標) (atipamezole) was administered.

[0174] Nanoindentation measurements of in vitro and in vivo treated corneas Nanoindentation applies a small force perpendicular to the specimen surface and provides a mechanical measurement of the material of interest through the measurement of the resulting indentation on the specimen (Dias & Ziebarth 2013). Nanoindentation has recently emerged as a powerful tool for measuring nano- and micro-scale mechanical properties in tissues and other biomaterials (Ebenstein & Pruitt 2006). More recently in With the development of in situ scanning probe microscope (SPM) imaging, a nanoindenter tip is simultaneously used as a 3D imaging device combined with nanoindentation, leading to a new wave of novel materials research (Dickinson & Schirer 2009). Force, displacement, and time are simultaneously recorded while the nanoindentation tip is pressed into corneal tissue under a controlled load. The force applied during nanoindentation can be as small as a few nanonewtons or as large as several newtons, allowing testing over a wide range of size scales. Nanoindentation tests are output as load-displacement curves that can be analyzed using well-defined equations to calculate mechanical properties related to the stiffness, integrity, and elasticity of the cornea.

[0175] Human keratoconus corneas were placed in organotypic culture in control medium or in medium containing specific ECM proteins that induce reprogramming factors. Nanoindentation measurements were then taken at the end of the treatment period. For in vivo studies, animals were manually restrained and approximately 15 μL of a gel eye drop formulation containing the reprogramming factors was instilled into the right eye of each Wistar rat. The contralateral eye served as the control eye. The eye drops were administered three times daily for up to seven weeks. Nanoindentation measurements were recorded after the first, third, or seventh week of the treatment period on the isolated eyes.

[0176] Nanoindentation testing was performed in the Chemical and Materials Engineering lab at the University of Auckland. To test the cornea in its natural position, a mould was required for nanoindentation. Previous testing had used polystyrene and Blu-Tack to hold the cornea in place. The effect of the mould deforming under load is a potential source of error, so a rigid mould was decided upon for testing. The first material used to create the mould was traditional clay. This was formed to the exact shape and curvature of the human corneal specimen (Figure 6(A)). The clay was then left to harden for 2 days before being used for testing. Testing of the rat eye was slightly different as the entire eyeball was used. To hold the eyeball in place, a petri dish with a small depression to hold the eyeball, filled with resin, was used (Figure 6(B)). PBS was used to keep the specimen from drying out.

[0177] As the samples are very soft biological samples, a cone-spherical fluid tip was used for all nanoindentation tests. The indentation load used for human samples was 50 μN. For rat eyes, a load range of 3–5 μN was used. The fiber optic light was turned on and the sample was placed directly under the light beam from the microscope. The central part of the cornea was placed directly under the light beam as accurately as possible (Figure 6(C)). The sample was focused by adjusting the Z-axis slider until the surface of the cornea could be observed with good resolution. To ensure that the highest point in the center of the corneal sample was in focus, the field of view was moved in the X- and Y-axis directions to observe how the focus changed.

[0178] Once the data collection point was focused on the center of the cornea, the sample boundaries were defined and a rapid approach was taken. Before indentation, the load function had to be set precisely. The actual indentation process was carried out using a Hysitron Triboindenter. (登録商標)The indentation was automated by a 3D scanner (Figure 6(D)). A predefined load was placed on the indenter tip, which penetrated the sample until a defined limit was reached. The tip was then held for 10 seconds before being removed from the sample. The hardness of the sample was determined by the area of ​​the indentation (Ar) after the tip was removed.

[0179]

number

[0180] P max is the contact area between the cone-spherical tip and the sample, where is the maximum indentation load. The low elastic modulus represents the elastic modulus of both the sample and the indenter tip, as shown by the following equation:

[0181]

number

[0182] where i refers to the indenter and m refers to the sample material. The low modulus gives an idea of ​​how elastic the sample is. Since the same indenter tip is used for each test, the low modulus can be used to compare the elasticity of each sample tested.

[0183] Example 8: Treatment of adult human corneal keratocytes with exogenous TGFβ3 and dexamethasone produces cartilage-specific type II collagen It has been found that one growth factor may act on several cell types with the same or different effects, while more than one growth factor may share similar biological functions.When selecting growth factors, cytokines, and chemicals that can cause collagen deposition in corneal stroma, it is important to consider the known effects of certain exogenous factors.In the present experiment, the combined treatment of TGFβ3 and dexamethasone is used.

[0184] Most of the evidence for the effects of TGFβ3 and dexamethasone comes from studies that have been conducted on their effects on stem / progenitor cells (Schuldiner, Yanuka, Itskovitz-Eldor, Melton, & Benvenisty 2000; Worster, Nixon, Brower-Toland, & Williams 2000). The combination of TGFβ and dexamethasone has previously been used to induce progenitor cells to differentiate into chondrocytes in vitro (Diekman, Rowland, Lennon, Caplan, & Guilak 2009; Johnstone et al. 1998; Kolambkar, Peister, Soker, Atala, & Guldberg 2007; Winter et al. 2003). In addition, dexamethasone, a synthetic steroid drug, has been used to treat inflammatory eye diseases. Therefore, a combination of TGFβ3 and dexamethasone was used in the chondrogenic differentiation medium to promote keratocyte differentiation towards a chondrocyte phenotype.

[0185] In the present experiment, the expression of collagen types I and II is noteworthy. Fibrillar collagens such as types I and II have been shown to self-assemble and cross-link to form highly crystalline fibers that exhibit very high stiffness, low extensibility, and elastic energy storage capacity (Wells 2003). It is the cross-links that contribute to the stiffness and tensile strength of the fibers.

[0186] The extracellular matrix (ECM) of the corneal stroma consists of densely packed heterotypic collagen fibrils, composed mostly of collagen types I and V. Like corneal fibrils, cartilage fibrils are heterotypic (composed of types II and XI) and have a uniform diameter of 25 nm (slightly smaller than corneal fibrils) (Mendler, Eich-Bender, Vaughan, Winterhalter, & Bruckner 1989). Type II collagen is the major fibril component of collagen and is similar to type I collagen in that its molecule consists essentially of a single uninterrupted helical domain, 300 nm in length. Due to their similarity, types II and XI collagen are considered to be the cartilage analogues of collagen types I and V in other tissues (corneal stromal collagen).

[0187] In the present experiments, keratocytes from mature corneas were seeded in either chondrogenic differentiation medium containing TGFβ3 and dexamethasone, or in standard fibroblast growth medium. Within 2-3 days, keratocytes seeded in chondrogenic differentiation medium formed cell aggregates / spheres of approximately 50-100 μm in diameter (Figure 7(A)). The center of the sphere was labeled for chondrocyte-specific collagen type II and the periphery for nestin (Figure 7(B)). Furthermore, once the sphere was placed in fibroblast growth medium, cells from the sphere began to spread outward (Figure 7(C)) and aggregated in the culture dish, forming a monolayer of cells. The areas where the cells had once aggregated were labeled for collagen type II, whereas the cells in the monolayer were not labeled (Figure 7(D)).

[0188] Keratocytes seeded in fibroblast growth medium formed a uniform monolayer of fibroblast-like cells (Figure 8(A)), but did not label for either nestin or type II collagen (Figure 8(B)). When the medium was replaced with chondrogenic differentiation medium, no change in the appearance of the cultures was observed, and the cells remained negative for type II collagen. These results suggest that cell aggregation appears to be important for the production of cartilage-like ECM. Keratocytes seeded in fibroblast growth medium were unable to form the required cell aggregation. Therefore, to form fibroblast clusters, confluent fibroblasts were dissociated from the culture dish, pelleted, and grown for an additional 3 weeks as pellet cultures in chondrogenic differentiation medium. The cell pellets were positively labeled for the keratocyte-specific ECM protein keratocan, but negative for the cartilage-specific ECM protein type II collagen (Figure 8(F) and (G)).

[0189] Example 9: Keratocytes from adult human cornea and adult rat cornea treated with TGFβ3 and dexamethasone secrete type II collagen containing ECM Slices of adult human cornea were placed in organotypic slice cultures in control medium or chondrogenic differentiation medium for two weeks. The tissue slices were then labeled for the chondrocyte-specific ECM protein collagen type II and native cornea collagen type I. Positive labeling was only seen in corneas treated with TGFβ3 and dexamethasone (Figures 9(C) and 10(B)). It was found that a two-week treatment period resulted in the deposition of collagen type II within the stromal ECM of treated corneas (Figure 9(C)). One week of treatment did not result in visible deposition of collagen type II in the stromal ECM (Figure 9(B)).

[0190] The amount and pattern of native type I collagen appeared to be slightly altered in the treated corneas. In general, the intensity of labeling was similar, but the distribution was more widespread and the amount of labeling was higher in the untreated corneas (Figure 9(D)). Furthermore, the newly produced type II collagen was deposited uniformly and regularly in the ECM without forming any large clumps or aggregates. The labeling was clearly seen along the existing collagen structure of the corneal stroma and was distributed throughout the entire thickness of the stromal layer.

[0191] The in vitro human corneal tissue experiments were then extended to in vivo rodent studies, where the right corneas of male Wistar rats were treated for two weeks with 15 μl of a gellan gum-based eye drop formulation of TGFβ3 and dexamethasone administered three times a day. After two weeks, the rats were euthanized and the corneas processed for immunohistochemistry. Only the treated corneas were positively labeled for type II collagen, with higher levels of deposition observed in the anterior part of the cornea (Figure 10(D) and (E)). Thus, only corneal slices cultured in chondrogenic differentiation medium were positive for type II collagen. Furthermore, type II collagen was deposited as a uniform layer along the existing collagen structure of the corneal stroma.

[0192] Example 10: Induction of type II collagen deposition in the cornea of ​​keratoconus The inventors next attempted to confirm that the in vivo reprogramming observed in their study could be utilized for the treatment of keratoconus. Experiments were performed to confirm that keratocytes in keratoconus corneas were applicable to induce collagen type II deposition. Corneal buttons from keratoconus obtained after corneal transplant surgery were placed in culture medium as soon as they were obtained. Half of each button was placed in control medium and the other half in chondrogenic differentiation medium and maintained for 2 weeks. After 2 weeks, the tissues were processed for either immunohistochemistry or mRNA extraction. The corneal stromal ECM of only half of the treated corneas was positive for collagen type II (Figure 11(B)). Compared to normal corneal tissue, the intensity of the labeling was lower in keratoconus tissue, but the labeling pattern was similar and followed an ordered arrangement along the existing collagen lamellar structure.

[0193] Vimentin labeling revealed clear differences in keratocytes between untreated and treated keratoconus corneas. In general, the density of keratocytes was lower in untreated corneas, with fewer cells in the posterior part of the cornea (Figure 11(C)). Also, keratocytes in treated corneas appeared more filamentous and had a more mature morphology compared to keratocytes in untreated corneas (Figures 11(E) and (F)). Compared to keratocytes in untreated corneas, keratocytes in treated corneas were longer and had more cell processes that were strongly labeled for vimentin.

[0194] Example 11: Treatment with TGFβ3 and dexamethasone does not induce deposition of fibrotic proteins and causes corneal opacity Human corneas cultured in chondrogenic differentiation medium for up to 3 weeks were labeled for collagen III and αSMA, which are associated with fibrosis and scarring (Gabbiani 2003; Karamichos et al. 2012). There was no evidence of any fibrotic matrix deposition, whereas a higher degree of αSMA labeling was observed in control tissue (Figure 12). These results support previous findings that, unlike TGFβ1 and TGFβ2, TGFβ3 does not induce differentiation of keratocytes into myofibroblasts.

[0195] Slit lamp examinations were performed on live rats throughout the study period. At the time of examination, treated and untreated corneas were indistinguishable without signs of scarring or opacity. Images of the back of the eye to show blood vessels showed a clear cornea that did not block the passage of light (Figures 13(A) and (B)), and in vivo cross-sectional images of rat corneas using Micron IV lenses revealed clear corneas through which light easily passed (Figures 13(C) and (D)). There was no sign of any corneal opacity or cloudiness that would lead to obstruction of the passage of light through the cornea.

[0196] Example 12: Changes in mRNA expression of collagen types II and I upon in vivo treatment Corneas from rats treated in vivo for 1, 7, and 3 weeks, followed by a 4-week untreated period, were subjected to quantitative gene expression analysis. The aim was to determine whether expression of type II collagen decreased again and / or ceased permanently after growth factor treatment was discontinued. The effect of treatment on type II collagen in native corneas was also examined.

[0197] Compared to corneas treated for 7 weeks, corneas treated for 1 week expressed significantly higher levels of collagen type II. When treatment was stopped, the expression levels dropped significantly, as shown by the first graph in Figure 35. In the case of collagen type I expression, corneas treated for 1 week and 7 weeks were compared to their untreated corneas, respectively. After 1 week of treatment, an initial spike in collagen type I expression was observed, but by week 7, collagen type I expression was significantly lower and comparable to its expression in untreated corneas (Figure 14(B)).

[0198] Example 13: Changes in the biomechanical properties of corneas treated in vitro and in vivo We hypothesized that the deposition of type II collagen would affect corneal stiffness and elasticity. To assess these changes, in vivo rat corneas and ex vivo treated human corneas, as well as their matched controls, were subjected to nanoindentation testing.

[0199] Compared to untreated controls, rat corneas treated in vivo for 1 week did not show a significant increase in either hardness or elasticity (Figure 15). Clear differences were observed between treated and control eyes in corneas treated in vivo for 3 weeks. Each cornea was tested up to 8 times, and the resulting load-deformation graphs showed good reproducibility (Figure 16). Both hardness and the decrease in elastic modulus were significantly higher in the right eye exposed to growth factor treatment. Corneal pairs with corresponding spheroid corneas cultured ex vivo for 6 weeks in either control medium or chondrogenic differentiation medium were also subjected to the same biomechanical tests. Again, the tests revealed a significant increase in hardness and elastic modulus of the treated corneas (Figure 17).

[0200] Example 14: Comparative combinations of growth factors and steroids Ex vivo studies on sheep corneas were performed to investigate the efficacy of combinations of other growth factors and steroids in the chondrogenic differentiation of keratocytes.

[0201] Fresh sheep eyes were obtained from Auckland Meat Processors. Corneas were immediately excised and washed with povidone-iodine (PVP-I) and sodium thiosulfate solution. A punch was then used to excise 8 mm disks of sheep corneal tissue. The disks were placed in each culture condition (outlined in Table 5) for 3 weeks. The disks were then placed in an organotypic air / liquid interface culture system.

[0202] Briefly, explants of normal tissue were cultured on cell culture inserts with 0.4 μm pore size (Millicell, France) at the interface of culture medium and CO2-enriched environment. Corneal slices were placed epithelial side up on cell culture plates containing 3 ml of culture medium. Culture medium was changed every other day. The basal medium used was 1% Anti-Anti (antibiotic-antimycotic solution) and 1% GlutaMAX. (商標) (GIBCO (登録商標) At the end of the 3 weeks, each corneal disc was fixed in 4% paraformaldehyde (PFA) for 1 h and treated with sucrose solution to cryoprotect the tissue before freezing and sectioning.

[0203] Briefly, corneas were immersed in a 20% sucrose solution at 4°C for 5 h, then transferred to a 30% sucrose solution and kept at 4°C until the tissue sank (usually overnight). Corneas were then embedded in OCT (optimal cutting temperature) compound and immersed in liquid nitrogen to induce rapid freezing. Frozen tissue blocks were stored at -80°C until further use. Approximately 4-6 40 μm thick cryostat sections were cut using SuperFrost (商標) Plus slides and the slides were stored at -80°C until needed. Corneal sections were then labeled for type II collagen.

[0204] For immunohistochemistry, slides were kept at room temperature for 15-20 min. OCT was washed off using PBS and the peripheral zone of tissue was demarcated using a wax pen. Tissue sections were first incubated for 1 h with a blocking solution of 10% normal goat serum and then incubated overnight at 4 °C with mouse anti-type II collagen antibody (Millipore / MAB8887). Appropriate dilutions of goat anti-mouse Alexa Fluor (登録商標) 488 Secondary Antibody (Molecular Probes (登録商標) The sections were then incubated with 100 mM NaCl (A-11001) and the secondary antibody was incubated for 2 hours at room temperature. Sections were counterstained with the nuclear marker 4',6'-diamidino-2-phenylindole (DAPI) and embedded in Citifluor antifade agent (ProSciTech, Australia). An Olympus FluoView (商標) Labeling was visualized using an FV-1000 confocal laser scanning microscope (405 nm, 473 nm, and 559 nm wavelength lasers) and a Leica DMRA fluorescence microscope.

[0205] Table 5 presents the findings from this study. Figure 20 shows representative images of collagen type II labeling in corneal sections from each condition.

[0206] [Table 5]

[0207] The results confirmed that the combination of TGFβ3 and dexamethasone was the only combination tested that elicited the desired response from the target cells (Figure 20, Panels G-H). No other growth factor-steroid combinations were able to produce the desired changes in collagen type II in keratocytes (Figure 20, Panels A-F). The results also confirmed the reprogramming of keratocytes in sheep corneas (Figure 20, Panels G-H).

[0208] Previous studies have shown that other growth factors and other steroid compounds are inappropriate for corneal treatment and repair. Both TGFβ1 and TGFβ2 produce fibrotic scars (Carrington, Albon et al. 2006; Desmouliere, Chaponnier et al. 2005; Jester, Huang et al. 2002; Cowin et al. 2001; Shah et al. 1995). EGF negatively regulates chondrogenesis (Yoon 2000). Estrogen also negatively regulates chondrogenesis (Kato & Gospodarowicz 1985). Hydrocortisone has been shown to promote adipogenesis rather than chondrocyte differentiation (Ghoniem et al. 2015; Lee, Kuo et al. 2004). These initial studies demonstrate the significance of the present findings regarding TGFβ3 and dexamethasone acting together to promote chondrocyte differentiation of keratocytes and scar-free corneal healing.

[0209] Example 15: Comparative Dosages of TGFβ3 and Dexamethasone Prior to in vivo testing, experiments were performed to identify different effective doses for ex vivo treatment. Dose-ranging studies were performed for TGFβ3 and dexamethasone by culturing sheep corneas in culture medium containing different concentrations of these two factors.

[0210] Fresh sheep eyes were obtained and the corneas were excised and processed as described in Example 14. One sheep corneal disk was placed in each of the 16 culture conditions for three weeks (FIG. 21). The disks were cultured and then subjected to immunohistochemical and microscopic analysis as described in Example 14. FIG. 21 shows representative images of type II collagen labeling in corneal sections from each condition.

[0211] This study revealed that lower concentrations of TGFβ3 (2-4 ng / mL) and dexamethasone (1-10 nM) had lower ex vivo efficacy (Figure 21, rows 1 and 2). Higher doses, i.e., 8-10 ng / mL TGFβ3 and 100-1000 nM dexamethasone, were efficient in inducing collagen type II deposition (Figure 21, rows 3 and 4).

[0212] These results support the use of 100 nM dexamethasone and 10 ng / mL TGFβ3 as effective concentrations (Figure 21, line 3). Higher concentrations of dexamethasone (1000 nM, i.e., 400 ng / mL) were also shown to be effective (Figure 21, line 4). Note that the dexamethasone concentrations tested in this study are significantly lower than those used in commercial eye drops (i.e., 1 mg / mL dexamethasone).

[0213] Example 16: Summary of Experimental Observations and Results The combination of TGFβ1 and dexamethasone has been used previously to induce precursor cells to differentiate into chondrocytes in vitro (Diekman et al. 2009; Johnstone et al. 1998; Kolambkar et al. 2007; Winter et al. 2003). Other studies have shown that a side population of keratocytes produces a matrix composed of cartilage-specific type II collagen under similar chondrocyte differentiation conditions (Du, Funderburgh, Mann, Sundar Raj, & Funderburgh 2005). It has also been reported that scleral cells expressed cartilage-specific markers including aggrecan and type II collagen after 4 weeks in chondrogenic differentiation medium containing TGFβ1 and BMP2. Furthermore, human scleral cells have been shown to retain chondrogenic potential in vivo after transplantation into rat cartilage defects (Seko et al. 2008). Scleral fibroblasts and corneal stroma have been found to share a common developmental origin.

[0214] As shown herein, keratocytes seeded in culture medium containing TGFβ3 and dexamethasone but in the absence of serum spontaneously formed cell spheroids within 2–3 days by cell aggregation, and by the third week, these cell clusters were positively labeled for cartilage-specific type II collagen. When initially treated with TGFβ3 and dexamethasone, expression of type I collagen was also increased. When the medium was replaced with control medium containing fetal bovine serum, the cell clusters dispersed into a monolayer of cells. Cells growing in a monolayer no longer expressed type II collagen. These results suggest that cell aggregation and environment may be important in the induction of type II collagen.

[0215] Notably, keratocytes initially grown as fibroblasts in serum-containing medium did not secrete type II collagen when the medium was switched to chondrogenic differentiation medium containing TGFβ3 and dexamethasone, suggesting that once grown as fibroblasts, the cells lose the ability to differentiate along the chondrogenic pathway. In addition, fibroblasts grown in 3D culture as pellets in chondrogenic differentiation medium also failed to express cartilage-specific type II collagen. These results suggest that the quiescent keratocyte phenotype and cell aggregation are important for chondrocyte differentiation.

[0216] It is shown herein that ex vivo culture of normal and keratoconus corneas in chondrogenic differentiation medium revealed uniform deposition of type II collagen along the lamellae of the corneal stroma. Every keratocyte in the stroma was associated with type II collagen labeling, again suggesting that reprogramming to a chondrogenic phenotype is stochastic and supporting that the results obtained from in vitro cell culture were not the result of proliferation of a side population of progenitor cells. Furthermore, in vivo treatment of corneas in rats also led to deposition of type II collagen in a similar manner as seen in ex vivo culture. However, more intense immunolabeling of type II collagen was seen in the anterior part of the cornea when treated in vivo, most likely reflecting easier diffusion of growth factors from the ocular surface to the anterior layer of the corneal stroma.

[0217] Studies investigating density differences in keratocytes in keratoconus have reported an overall decrease in cell density. The results herein support this. However, unlike other studies that have reported a significant decrease in cell density in the anterior stroma (Hollingsworth, Efron, & Tullo 2005; Ku et al. 2008; Mencucci et al. 2010; Niederer et al. 2008), the results herein also show a significant decrease in keratocyte density in the posterior stroma of untreated keratoconus corneas. There is an overall thinning of the cornea in keratocytes. However, it is not known whether this is due to keratocyte apoptosis and subsequent decreased production of ECM, or whether keratocyte apoptosis is secondary to the corneal thinning process.

[0218] As shown herein, the treated halves of keratoconus corneas cultured in chondrogenic medium containing TGFβ3 and dexamethasone showed increased keratocyte density compared to controls. Furthermore, the posterior part of the stroma appeared to be repopulated with keratocytes. The keratocytes in the treated halves also appeared to look healthier, with large prominent nuclei and some cell processes. This suggests that treatment with the two factors may have caused keratocyte proliferation and repopulation of the stroma in certain posterior parts that were devoid of keratocytes.

[0219] Collagen cross-linking, one of the current treatments for keratoconus, resulted in early keratocyte apoptosis in the anterior part of the corneal stroma. This was then followed by a period of stromal repopulation by keratocytes. Keratocyte cell death is usually seen as a response to injury and, in the case of cross-linking, is understood to be the result of UVA-induced cell damage. This apoptotic response is thought to have occurred to protect the cornea from further inflammation (Wilson, Netto, & Ambrosio 2003).

[0220] Corneal stroma haze was also observed after crosslinking treatment, which could persist for up to several months. This is due to an increase in collagen diameter and interfibril spaces resulting in changes in the corneal microstructure. Most studies report a reduction in corneal haze 6-12 months after treatment (Greenstein, Fry, Bhatt, & Hersh 2010; Mazzotta et al. 2008). Although several clinical observations of the cornea have been performed after crosslinking treatment, ambiguity exists regarding the cause of corneal haze and other possible downstream effects of the treatment. The fact that it takes several months for the cornea to reassemble and become transparent suggests that crosslinking may induce a wound healing response in the corneal stroma.

[0221] In this study, no evidence of corneal opacification was observed, even during prolonged (up to 8 weeks) in vitro and in vivo treatment. This is likely due to the deposition of uniform, laminar collagen type II along pre-existing collagen lamellae. Deposition of collagen type III (associated with fibrosis) and alpha-smooth muscle actin (in forming myofibroblasts) leads to opacification and scarring, both of which are found in corneal wounds. None of these proteins were expressed in treated corneas, suggesting that a wound-healing cascade that could lead to scarring was not induced.

[0222] As described herein, quantitative measurements of collagen II mRNA expression showed that its expression was significantly reduced when TGFβ3 and dexamethasone were withdrawn, suggesting that keratocyte reprogramming is not irreversible and that the subsequent deposition of collagen II in the ECM can potentially be controlled. This is important for the development of therapeutic approaches, as it is undesirable to induce irreversible ECM deposition.

[0223] Nanoindentation has been used to evaluate post-surgical therapies such as cross-linking for keratoconus (corneal dystrophy) and ocular ectasia after LASIK. A study performed on human cadaveric corneas revealed that collagen cross-linking caused a two-fold increase in elastic modulus in the anterior stroma, whereas the posterior stroma was unaffected by the treatment (Dias, Diakonis, Kankariya, Yoo, & Ziebarth 2013). In this study, the elasticity of the anterior cornea was measured. Furthermore, the results of this study show that the keratocyte density of the posterior stroma was altered in corneas treated with TGFβ3 and dexamethasone.

[0224] Nanoindentation does not measure the properties of individual collagen fibrils, but it can measure changes in the intrinsic elastic properties of the cornea, which change with the deposition of collagen type II and increase later in collagen crosslinking. Differences in structure within the corneal stroma are reflected in corresponding differences in biomechanical properties. The results herein show that a nearly three-fold increase in elastic modulus and hardness was observed in rat corneas treated with growth factors. These results suggest that the treatment results in stiffer corneas with higher elasticity. Elastic modulus is a measure of the resistance of a material to be elastically deformed, and therefore a higher elastic modulus means that it is more difficult to deform the material. In this study, the significant increase in hardness and elastic modulus in corneas treated for 3 weeks compared to corneas treated for 1 week is consistent with the results of immunohistochemical labeling, which show that at least 2-3 weeks of treatment are required to deposit a detectable layer of collagen type II.

[0225] The results of immunohistochemical labeling, coupled with gene expression and biomechanical tests, indicate that keratocytes in intact corneas are amenable to reprogramming along the chondrogenic pathway by treatment with TGFβ3 and dexamethasone. Reprogramming by combined treatment with TGFβ3 and dexamethasone is stochastic and can be controlled by adjusting the duration of growth factor treatment to result in a stiffer, more elastic cornea. In particular, administration of both drugs is necessary: ​​no production of type II collagen is observed in keratocytes when TGFβ3 and dexamethasone are tested separately. A novel treatment is therefore proposed for keratoconus and other eye diseases using in vivo tissue engineering by administration of TGFβ3 and dexamethasone, as described herein.

[0226] Example 17: Large animal model to study corneal remodeling Reshaping the cornea while achieving optimal treatment planning in a sheep model Further experiments will be performed to use a large animal model to demonstrate corneal reshaping. In these experiments, a large animal model was used to allow for the placement of prescription contact lenses. Sheep were used as model animals because the sheep eye is comparable in size and physiology to the human eye. In addition, housing facilities were available at Lincoln University, Christchurch. It is noted that sheep have a calm temperament and are amenable to handling.

[0227] The sheep were sedated according to standard operating procedures in the vivarium. An eye drop formulation (with volumetric calibration) with optimal TGFβ3 and dexamethasone concentrations based on the rodent dose optimization study was instilled into the right eye, followed by corneal INTACS to maintain the desired corneal curvature during collagen deposition. (登録商標) (or a similar scleral ring) was placed (FIG. 18). Eye drops were administered continuously for a period of 3 weeks, either once daily or twice daily (as determined in rodent optimization studies). INTACS (登録商標) The mice were then removed and kept for an additional 3 weeks or 6 months.

[0228] Before and after processing (INTACS (登録商標) Corneal thickness and curvature measurements were performed at 100 nm (when the corneal septum was removed). A portable corneal pachymeter was used to detect in vivo changes in corneal thickness of treated corneas versus the contralateral control corneas. A portable Pentacam (登録商標) The corneal curvature and corneal thickness of the sheep's eyes were measured before and after treatment using the Pentacam (Figure 19 (E) and (F)). (登録商標)Keratometry is repeated 3 weeks after lens removal, using measurements of 0.1 μg / mL for corneal keratometry. As previously described for rodent corneas, these are performed after sacrificing the animals but before removing the eyes for immunohistological and biomechanical analysis. In the unlikely event that a sheep does not tolerate the hard contact lens (signs of infection, inflammation, or irritation), the study will continue without the lens, allowing completion of key parameters such as type II collagen deposition and distribution, and biomechanical properties.

[0229] Given these results, it is proposed to use in vivo tissue engineering as detailed herein in combination with a gas permeable rigid OrthoK contact lens (or similar) to permanently reshape and stabilize the cornea and provide treatment for common corneal defects, including myopia.

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Accordingly, the present invention is intended to include within its scope modifications, alternatives, and variations to the processes, products, compositions, compounds, means, methods, and / or steps disclosed herein.

[0231] All references cited herein, including patents and patent applications, are hereby incorporated by reference. No admission is made that any reference constitutes prior art, nor is any admission that the discussion of any reference forms part of the common general knowledge in the art in New Zealand or any other country.

Claims

[Claim 1] A method for (i) treating or preventing a condition associated with thinning or irregularity of the cornea, (ii) treating or preventing corneal damage or injury, or (iii) treating or preventing ocular refractive error, comprising contacting the cornea or the eye with a composition comprising a TGFβ3 polypeptide or a variant or fragment thereof, and dexamethasone or a derivative or related steroidal agent, thereby treating or preventing the condition, damage, injury, or refractive error.

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